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Sample records for actin depolymerizing factor

  1. Arabidopsis actin-depolymerizing factor7 severs actin filaments and regulates actin cable turnover to promote normal pollen tube growth.

    Science.gov (United States)

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-09-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana actin-depolymerizing factor7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7-enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.

  2. Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics.

    Directory of Open Access Journals (Sweden)

    Katie Porter

    Full Text Available The primary role of Actin-Depolymerizing Factors (ADFs is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1. These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.

  3. Green fluorescent protein-mtalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

    NARCIS (Netherlands)

    Ketelaar, T.; Anthony, R.G.; Hussey, P.J.

    2004-01-01

    Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that

  4. Synthetic peptides that cause F-actin bundling and block actin depolymerization

    Science.gov (United States)

    Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

    2011-10-18

    Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

  5. Comprehensive analysis of differentially expressed rice actin depolymerizing factor gene family and heterologous overexpression of OsADF3 confers Arabidopsis Thaliana drought tolerance.

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    Huang, Ya-Chen; Huang, Wen-Lii; Hong, Chwan-Yang; Lur, Hur-Shen; Chang, Men-Chi

    2012-11-27

    Actin depolymerizing factors (ADFs) are small actin-binding proteins. Many higher-plant ADFs has been known to involve in plant growth, development and pathogen defense. However, in rice the temporal and spatial expression of OsADF gene family and their relationship with abiotic stresses tolerance is still unknown. Here we reported the first comprehensive gene expression profile analysis of OsADF gene family. The OsADF genes showed distinct and overlapping gene expression patterns at different growth stages, tissues and abiotic stresses. We also demonstrated that both OsADF1 and OsADF3 proteins were localized in the nucleus. OsADF1 and OsADF3 were preferentially expressed in vascular tissues. Under ABA or abiotic stress treatments, OsADF3::GUS activity was enhanced in lateral roots and root tips. Ectopically overexpressed OsADF3 conferred the mannitol- and drought-stress tolerance of transgenic Arabidopsis seedlings by increasing germination rate, primary root length and survival. Several drought-tolerance responsive genes (RD22, ABF4, DREB2A, RD29A, PIP1; 4 and PIP2; 6) were upregulated in transgenic Arabidopsis under drought stress. These results suggested that OsADF gene family may participate in plant abiotic stresses response or tolerance and would facilitate functional validation of other OsADF genes.

  6. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  7. Ca2+ bound to the high affinity divalent cation-binding site of actin enhances actophorin-induced depolymerization of muscle F-actin but inhibits actophorin-induced depolymerization of Acanthamoeba F-actin.

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    Mossakowska, M; Korn, E D

    1996-08-01

    The cation tightly bound to actin, Mg2+ or Ca2+, affects the ability of actophorin to accelerate depolymerization of filaments and bind to monomers of actin prepared from rabbit skeletal muscle and Acanthamoeba castellanii. Actophorin interacted similarly with muscle and Acanthamoeba Mg2(+)-F-actin but depolymerized muscle Mg2(+)-F-actin more efficiently. Muscle Ca2(+)-F-actin depolymerized about 5 times more rapidly than Mg2(+)-F-actin in the presence of actophorin but Acanthamoeba Ca2(+)-F-actin was highly resistant to actophorin. Muscle actin subunits dissociated more rapidly than Acanthamoeba actin subunits from copolymers of muscle and Acanthamoeba Ca2(+)-actin upon addition of actophorin although Acanthamoeba actin dissociated much more rapidly from copolymers than from its homopolymer. The Kd of the 1:1 complex between actophorin and monomeric actin was somewhat lower for muscle Mg2(+)-ATP-G-actin than for both Acanthamoeba Mg2(+)-ATP-G-actin and muscle Ca2(+)-ATP-G-actin. The data for the interactions of actophorin with Acanthamoeba Ca2(+)-ATP-G-actin or muscle and amoeba Mg2(+)- and Ca2(+)-ADP-G-actin were incompatible with the formation of 1:1 actin: actophorin complexes and, thus, Kd values could not be calculated. While it may not be surprising that actophorin would interact differently with Mg2(+)- and Ca2(+)-actin, it is unexpected that the nature of the tightly bound cation would have such dramatically opposite effects on the ability of actophorin to depolymerize muscle and Acanthamoeba F-actin. Differential severing by actophorin, with Acanthamoeba Ca2(+)-actin being almost totally resistant, is sufficient to explain the results but other possibilities cannot be ruled out.

  8. The rpg4-mediated resistance to wheat stem rust (Puccinia graminis) in barley (Hordeum vulgare) requires Rpg5, a second NBS-LRR gene, and an actin depolymerization factor.

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    Wang, X; Richards, J; Gross, T; Druka, A; Kleinhofs, A; Steffenson, B; Acevedo, M; Brueggeman, R

    2013-04-01

    The rpg4 gene confers recessive resistance to several races of wheat stem rust (Puccinia graminis f. sp. tritici) and Rpg5 provides dominant resistance against isolates of the rye stem rust (P. graminis f. sp. secalis) in barley. The rpg4 and Rpg5 genes are tightly linked on chromosome 5H, and positional cloning using high-resolution populations clearly separated the genes, unambiguously identifying Rpg5; however, the identity of rpg4 remained unclear. High-resolution genotyping of critical recombinants at the rpg4/Rpg5 locus, designated here as rpg4-mediated resistance locus (RMRL) delimited two distinct yet tightly linked loci required for resistance, designated as RMRL1 and RMRL2. Utilizing virus-induced gene silencing, each gene at RMRL1, i.e., HvRga1 (a nucleotide-binding site leucine-rich repeat [NBS-LRR] domain gene), Rpg5 (an NBS-LRR-protein kinase domain gene), and HvAdf3 (an actin depolymerizing factor-like gene), was individually silenced followed by inoculation with P. graminis f. sp. tritici race QCCJ. Silencing each gene changed the reaction type from incompatible to compatible, indicating that all three genes are required for rpg4-mediated resistance. This stem rust resistance mechanism in barley follows the emerging theme of unrelated pairs of genetically linked NBS-LRR genes required for specific pathogen recognition and resistance. It also appears that actin cytoskeleton dynamics may play an important role in determining resistance against several races of stem rust in barley.

  9. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte...... differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating h...

  10. Angiotensin II type 2 receptor stimulation increases the rate of NG108-15 cell migration via actin depolymerization.

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    Kilian, Peter; Campbell, Shirley; Bilodeau, Lyne; Guimond, Marie-Odile; Roberge, Claude; Gallo-Payet, Nicole; Payet, Marcel Daniel

    2008-06-01

    Angiotensin II (Ang II) has been reported to induce migration in neuronal cell types. Using time-lapse microscopy, we show here that Ang II induces acceleration in NG108-15 cell migration. This effect was antagonized by PD123319, a selective AT2 receptor antagonist, but not by DUP753, a selective AT1 receptor antagonist, and was mimicked by the specific AT2 receptor agonist CGP42112. This Ang II-induced acceleration was not sensitive to the inhibition of previously described signaling pathways of the AT2 receptor, guanylyl cyclase/cyclic GMP or p42/p44 mapk cascades, but was abolished by pertussis toxin treatment and involved PP2A activation. Immunofluorescence studies indicate that Ang II or CGP42112 decreased the amount of filamentous actin at the leading edge of the cells. This decrease was accompanied by a concomitant increase in globular actin levels. Regulation of actin turnover in actin-based motile systems is known to be mainly under the control of the actin depolymerizing factor and cofilin. Basal migration speed decreased by 77.2% in cofilin-1 small interfering RNA-transfected NG108-15 cells, along with suppression of the effect of Ang II. In addition, the Ang II-induced increase in cell velocity was abrogated in serum-free medium as well as by genistein or okadaic acid treatment in a serum-containing medium. Such results indicate that the AT2 receptor increases the migration speed of NG108-15 cells and involves a tyrosine kinase activity, followed by phosphatase activation, which may be of the PP2A type. Therefore, the present study identifies actin depolymerization and cofilin as new targets of AT2 receptor action, in the context of cellular migration.

  11. Selective, retrieval-independent disruption of methamphetamine-associated memory by actin depolymerization.

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    Young, Erica J; Aceti, Massimiliano; Griggs, Erica M; Fuchs, Rita A; Zigmond, Zachary; Rumbaugh, Gavin; Miller, Courtney A

    2014-01-15

    Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder. There is a growing consensus that memory is supported by structural and functional plasticity driven by F-actin polymerization in postsynaptic dendritic spines at excitatory synapses. However, the mechanisms responsible for the long-term maintenance of memories, after consolidation has occurred, are largely unknown. Conditioned place preference (n = 112) and context-induced reinstatement of self-administration (n = 19) were used to assess the role of F-actin polymerization and myosin II, a molecular motor that drives memory-promoting dendritic spine actin polymerization, in the maintenance of METH-associated memories and related structural plasticity. Memories formed through association with METH but not associations with foot shock or food reward were disrupted by a highly-specific actin cycling inhibitor when infused into the amygdala during the postconsolidation maintenance phase. This selective effect of depolymerization on METH-associated memory was immediate, persistent, and did not depend upon retrieval or strength of the association. Inhibition of non-muscle myosin II also resulted in a disruption of METH-associated memory. Thus, drug-associated memories seem to be actively maintained by a unique form of cycling F-actin driven by myosin II. This finding provides a potential therapeutic approach for the selective treatment of unwanted memories associated with psychiatric disorders that is both selective and does not rely on retrieval of the memory. The results further suggest that memory maintenance depends upon the preservation of polymerized actin. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  12. Plasma Gelsolin Levels Decrease in Diabetic State and Increase upon Treatment with F-Actin Depolymerizing Versions of Gelsolin

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    Neeraj Khatri

    2014-01-01

    Full Text Available The study aims to map plasma gelsolin (pGSN levels in diabetic humans and mice models of type II diabetes and to evaluate the efficacy of gelsolin therapy in improvement of diabetes in mice. We report that pGSN values decrease by a factor of 0.45 to 0.5 in the blood of type II diabetic humans and mice models. Oral glucose tolerance test in mice models showed that subcutaneous administration of recombinant pGSN and its F-actin depolymerizing competent versions brought down blood sugar levels comparable to Sitagliptin, a drug used to manage hyperglycemic condition. Further, daily dose of pGSN or its truncated versions to diabetic mice for a week kept sugar levels close to normal values. Also, diabetic mice treated with Sitagliptin for 7 days, showed increase in their pGSN values with the decrease in blood glucose as compared to their levels at the start of treatment. Gelsolin helped in improving glycemic control in diabetic mice. We propose that gelsolin level monitoring and replacement of F-actin severing capable gelsolin(s should be considered in diabetic care.

  13. depolymerizing factor gene in Hevea brasiliensis

    African Journals Online (AJOL)

    Yomi

    2010-11-08

    Nov 8, 2010 ... In Arabidopsis, the ancient subclasses of ADF genes exhibited novel and differential expression profiles. (Ruzicka et al., 2007). These data point to the differential regulation of plant ADFs, and also provide a framework for a model where the differentially expressed actins and. ABPs co-evolved in specific ...

  14. Human MCF10A mammary epithelial cells undergo apoptosis following actin depolymerization that is independent of attachment and rescued by Bcl-2.

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    Martin, S S; Leder, P

    2001-10-01

    Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton.

  15. Difference in F-Actin Depolymerization Induced by Toxin B from the Clostridium difficile Strain VPI 10463 and Toxin B from the Variant Clostridium difficile Serotype F Strain 1470

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    Harald Genth

    2013-01-01

    Full Text Available Clostridium difficile toxin A (TcdA and toxin B (TcdB are the causative agent of the C. difficile-associated diarrhea (CDAD and its severe form, the pseudomembranous colitis (PMC. TcdB from the C. difficile strain VPI10463 mono-glucosylates (thereby inactivates the small GTPases Rho, Rac, and Cdc42, while Toxin B from the variant C. difficile strain serotype F 1470 (TcdBF specifically mono-glucosylates Rac but not Rho(A/B/C. TcdBF is related to lethal toxin from C. sordellii (TcsL that glucosylates Rac1 but not Rho(A/B/C. In this study, the effects of Rho-inactivating toxins on the concentrations of cellular F-actin were investigated using the rhodamine-phalloidin-based F-actin ELISA. TcdB induces F-actin depolymerization comparable to the RhoA-inactivating exoenzyme C3 from C. limosum (C3-lim. In contrast, the Rac-glucosylating toxins TcdBF and TcsL did not cause F-actin depolymerization. These observations led to the conclusion that F-actin depolymerization depends on the toxin’s capability of glucosylating RhoA. Furthermore, the integrity of focal adhesions (FAs was analyzed using paxillin and p21-activated kinase (PAK as FA marker proteins. Paxillin dephosphorylation was observed upon treatment of cells with TcdB, TcdBF, or C3-lim. In conclusion, the Rho-inactivating toxins induce loss of cell shape by either F-actin depolymerization (upon RhoA inactivation or the disassembly of FAs (upon Rac1 inactivation.

  16. Specification of Architecture and Function of Actin Structures by Actin Nucleation Factors.

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    Skau, Colleen T; Waterman, Clare M

    2015-01-01

    The actin cytoskeleton is essential for diverse processes in mammalian cells; these processes range from establishing cell polarity to powering cell migration to driving cytokinesis to positioning intracellular organelles. How these many functions are carried out in a spatiotemporally regulated manner in a single cytoplasm has been the subject of much study in the cytoskeleton field. Recent work has identified a host of actin nucleation factors that can build architecturally diverse actin structures. The biochemical properties of these factors, coupled with their cellular location, likely define the functional properties of actin structures. In this article, we describe how recent advances in cell biology and biochemistry have begun to elucidate the role of individual actin nucleation factors in generating distinct cellular structures. We also consider how the localization and orientation of actin nucleation factors, in addition to their kinetic properties, are critical to their ability to build a functional actin cytoskeleton.

  17. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking.

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    Christopher Arnette

    Full Text Available The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.

  18. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

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    Morita, Tsuyoshi; Hayashi, Ken'ichiro

    2013-08-02

    Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Plant pathogenic bacteria target the actin microfilament network involved in the trafficking of disease defense components

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    Jelenska, Joanna; Kang, Yongsung; Greenberg, Jean T

    2014-01-01

    Cells of infected organisms transport disease defense-related molecules along actin filaments to deliver them to their sites of action to combat the pathogen. To accommodate higher demand for intracellular traffic, plant F-actin density increases transiently during infection or treatment of Arabidopsis with pathogen-associated molecules. Many animal and plant pathogens interfere with actin polymerization and depolymerization to avoid immune responses. Pseudomonas syringae, a plant extracellular pathogen, injects HopW1 effector into host cells to disrupt the actin cytoskeleton and reduce vesicle movement in order to elude defense responses. In some Arabidopsis accessions, however, HopW1 is recognized and causes resistance via an actin-independent mechanism. HopW1 targets isoform 7 of vegetative actin (ACT7) that is regulated by phytohormones and environmental factors. We hypothesize that dynamic changes of ACT7 filaments are involved in plant immunity. PMID:25551177

  20. Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity

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    Alon Kalo

    2015-04-01

    Full Text Available The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

  1. Histones bundle F-actin filaments and affect actin structure.

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    Edna Blotnick

    Full Text Available Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  2. Histones bundle F-actin filaments and affect actin structure.

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    Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

    2017-01-01

    Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  3. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

    International Nuclear Information System (INIS)

    Morita, Tsuyoshi; Hayashi, Ken’ichiro

    2013-01-01

    Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction

  4. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Tsuyoshi, E-mail: tsuyo@nbiochem.med.osaka-u.ac.jp; Hayashi, Ken’ichiro

    2013-08-02

    Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction.

  5. Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip

    Directory of Open Access Journals (Sweden)

    Katsch Kristin

    2012-03-01

    Full Text Available Abstract Serum response factor (SRF acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs or myocardin-related transcription factors (MRTFs. Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE. Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.

  6. Thymosin beta4 sequesters actin in cystic fibrosis sputum and decreases sputum cohesivity in vitro

    NARCIS (Netherlands)

    Rubin, Bruce K.; Kater, Arnon P.; Goldstein, Allan L.

    2006-01-01

    Filamentous actin (F-actin) forms polymers that contribute to the abnormal biophysical properties of sputum. Thymosin beta4 (Tbeta4) is the major monomeric actin-sequestering peptide in cells and can depolymerize F-actin. Tbeta4 could potentially decrease sputum viscoelasticity and adhesivity and

  7. Hydroxide catalysts for lignin depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Beckham, Gregg T; Biddy, Mary J.; Kruger, Jacob S.; Chmely, Stephen C.; Sturgeon, Matthew

    2017-10-17

    Solid base catalysts and their use for the base-catalyzed depolymerization (BCD) of lignin to compounds such as aromatics are presented herein. Exemplary catalysts include layered double hydroxides (LDHs) as recyclable, heterogeneous catalysts for BCD of lignin.

  8. Hydroxide catalysts for lignin depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Beckham, Gregg T.; Biddy, Mary J.; Chmely, Stephen C.; Sturgeon, Matthew

    2017-04-25

    Solid base catalysts and their use for the base-catalyzed depolymerization (BCD) of lignin to compounds such as aromatics are presented herein. Exemplary catalysts include layered double hydroxides (LDHs) as recyclable, heterogeneous catalysts for BCD of lignin.

  9. Actin dynamics and the elasticity of cytoskeletal networks

    Directory of Open Access Journals (Sweden)

    2009-09-01

    Full Text Available The structural integrity of a cell depends on its cytoskeleton, which includes an actin network. This network is transient and depends upon the continual polymerization and depolymerization of actin. The degradation of an actin network, and a corresponding reduction in cell stiffness, can indicate the presence of disease. Numerical simulations will be invaluable for understanding the physics of these systems and the correlation between actin dynamics and elasticity. Here we develop a model that is capable of generating actin network structures. In particular, we develop a model of actin dynamics which considers the polymerization, depolymerization, nucleation, severing, and capping of actin filaments. The structures obtained are then fed directly into a mechanical model. This allows us to qualitatively assess the effects of changing various parameters associated with actin dynamics on the elasticity of the material.

  10. Mechanochemical depolymerization of inulin.

    Science.gov (United States)

    Xing, Haoran; Yaylayan, Varoujan A

    2018-02-19

    Although chemical reactions driven by mechanical force is emerging as a promising tool in the field of physical sciences, its applications in the area of food sciences are not reported. In this paper, we propose ball milling as an efficient tool for the controlled generation of fructooligosaccharide (FOS) mixtures from inulin with a degree of polymerization (dp) ranging between 4 and 7. The addition of catalytic amounts of AlCl 3 together with ball milling (30 min, at 30 Hz) generated mixtures rich in dehydrated disaccharides such as di-D-fructose dianhydrides. Based on anion exchange chromatography in conjunction with ESI/qTOF/MS/MS analysis, catalysis increased the overall content of mono-, di-, and tri-saccharides by around 30 fold compared to un-catalyzed milling. In addition, dialysis results of the untreated and treated samples have indicated that under catalysis the percent of depolymerization (dp inulin to value-added food ingredients. Copyright © 2018. Published by Elsevier Ltd.

  11. Increased actin polymerization and stabilization interferes with neuronal function and survival in the AMPKγ mutant Loechrig.

    Directory of Open Access Journals (Sweden)

    Mandy Cook

    Full Text Available loechrig (loe mutant flies are characterized by progressive neuronal degeneration, behavioral deficits, and early death. The mutation is due to a P-element insertion in the gene for the γ-subunit of the trimeric AMP-activated protein kinase (AMPK complex, whereby the insertion affects only one of several alternative transcripts encoding a unique neuronal isoform. AMPK is a cellular energy sensor that regulates a plethora of signaling pathways, including cholesterol and isoprenoid synthesis via its downstream target hydroxy-methylglutaryl (HMG-CoA reductase. We recently showed that loe interferes with isoprenoid synthesis and increases the prenylation and thereby activation of RhoA. During development, RhoA plays an important role in neuronal outgrowth by activating a signaling cascade that regulates actin dynamics. Here we show that the effect of loe/AMPKγ on RhoA prenylation leads to a hyperactivation of this signaling pathway, causing increased phosphorylation of the actin depolymerizating factor cofilin and accumulation of filamentous actin. Furthermore, our results show that the resulting cytoskeletal changes in loe interfere with neuronal growth and disrupt axonal integrity. Surprisingly, these phenotypes were enhanced by expressing the Slingshot (SSH phosphatase, which during development promotes actin depolymerization by dephosphorylating cofilin. However, our studies suggest that in the adult SSH promotes actin polymerization, supporting in vitro studies using human SSH1 that suggested that SSH can also stabilize and bundle filamentous actin. Together with the observed increase in SSH levels in the loe mutant, our experiments suggest that in mature neurons SSH may function as a stabilization factor for filamentous actin instead of promoting actin depolymerization.

  12. Spatiotemporal dynamics of actin remodeling and endomembrane trafficking in alveolar epithelial type I cell wound healing.

    Science.gov (United States)

    Godin, Lindsay M; Vergen, Jorge; Prakash, Y S; Pagano, Richard E; Hubmayr, Rolf D

    2011-04-01

    Alveolar epithelial type I cell (ATI) wounding is prevalent in ventilator-injured lungs and likely contributes to pathogenesis of "barotrauma" and "biotrauma." In experimental models most wounded alveolar cells repair plasma membrane (PM) defects and survive insults. Considering the force balance between edge energy at the PM wound margins and adhesive interactions of the lipid bilayer with the underlying cytoskeleton (CSK), we tested the hypothesis that subcortical actin depolymerization is a key facilitator of PM repair. Using real-time fluorescence imaging of primary rat ATI transfected with a live cell actin-green fluorescent protein construct (Lifeact-GFP) and loaded with N-rhodamine phosphatidylethanolamine (PE), we examined the spatial and temporal coordination between cytoskeletal remodeling and PM repair following micropuncture. Membrane integrity was inferred from the fluorescence intensity profiles of the cytosolic label calcein AM. Wounding led to rapid depolymerization of the actin CSK near the wound site, concurrent with accumulation of endomembrane-derived N-rhodamine PE. Both responses were sustained until PM integrity was reestablished, which typically occurs between ∼10 and 40 s after micropuncture. Only thereafter did the actin CSK near the wound begin to repolymerize, while the rate of endomembrane lipid accumulation decreased. Between 60 and 90 s after successful PM repair, after translocation of the actin nucleation factor cortactin, a dense actin fiber network formed. In cells that did not survive micropuncture injury, actin remodeling did not occur. These novel results highlight the importance of actin remodeling in ATI cell repair and suggest molecular targets for modulating the repair process.

  13. Sound attenuation of polymerizing actin reflects supramolecular structures: viscoelastic properties of actin gels modified by cytochalasin D, profilin and alpha-actinin.

    Science.gov (United States)

    Wagner, O; Schüler, H; Hofmann, P; Langer, D; Dancker, P; Bereiter-Hahn, J

    2001-05-01

    Polymerization and depolymerization of cytoskeletal elements maintaining cytoplasmic stiffness are key factors in the control of cell crawling. Rheometry is a significant tool in determining the mechanical properties of the single elements in vitro. Viscoelasticity of gels formed by these polymers strongly depends on both the length and the associations of the filaments (e.g. entanglements, annealings and side-by-side associations). Ultrasound attenuation is related to viscosity, sound velocity and supramolecular structures in the sample. In combination with a small glass fibre (2 mm x 50 microm), serving as a viscosity sensor, an acoustic microscope was used to measure the elasticity and acoustic attenuation of actin solutions. Changes in acoustic attenuation of polymerizing actin by far exceed the values expected from calculations based on changes in viscosity and sound velocity. During the lag-phase of actin polymerization, attenuation slightly decreases, depending on actin concentration. After the half-maximum viscosity is accomplished and elasticity turns into steady state, attenuation distinctly rises. Changes in ultrasound attenuation depend on actin concentration, and they are modulated by the addition of alpha-actinin, cytochalasin D and profilin. Thus absorption and scattering of sound on the polymerization of actin is related to the packing density of the actin net, entanglements and the length of the actin filaments. Shortening of actin filaments by cytochalasin D was also confirmed by electron micrographs and falling-ball viscosimetry. In addition to viscosity and elasticity, the attenuation of sound proved to be a valuable parameter in characterizing actin polymerization and the supramolecular associations of F-actin.

  14. Unconventional actin conformations localize on intermediate filaments in mitosis

    International Nuclear Information System (INIS)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-01

    Research highlights: → Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. → These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). → Mitotic unconventional actin cables are independent of filamentous actin or microtubules. → Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  15. F-actin cytoskeleton reorganization is associated with hepatic stellate cell activation

    Science.gov (United States)

    CUI, XIAODONG; ZHANG, XIAOYUN; YIN, QINGLING; MENG, AIXIA; SU, SHAOJUAN; JING, XU; LI, HONG; GUAN, XIUMEI; LI, XIN; LIU, SHUNMEI; CHENG, MIN

    2014-01-01

    The activation of hepatic stellate cells (HSCs) is involved in the development of hepatic fibrosis. Previous studies have indicated that the acquisition of certain properties by activated HSCs is highly dependent on the reorganization of the actin cytoskeleton. However, direct evidence showing that the reorganization of the actin cytoskeleton is responsible for HSC activation is lacking. The aim of the present study was to investigate the role of cytoskeletal reorganization during HSC activation and to clarify the underlying mechanism. HSC-T6 cells were treated either with the F-actin stabilizer jasplakinolide (Jas) or the depolymerizer cytochalasin D (Cyto D). The actin cytoskeleton was evaluated via assessment of stress fiber formation. Furthermore, the activation properties of HSCs, including proliferation, adhesion, migration and the expression of α-smooth muscle actin (α-SMA) and collagen 1, were investigated in vitro. The results showed that Jas and Cyto D affected the actin distribution in HSC-T6 cells. Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells. In parallel, polymerization of actin microfilaments induced by Jas upregulated the expression of α-SMA and collagen 1, and also enhanced the migration and adhesion properties of HSC-T6 cells. Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway. In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process. The inhibition of F-actin reorganization may thus be a potential key factor or molecular target for the control of liver fibrosis or cirrhosis. PMID:24626324

  16. F‑actin cytoskeleton reorganization is associated with hepatic stellate cell activation.

    Science.gov (United States)

    Cui, Xiaodong; Zhang, Xiaoyun; Yin, Qingling; Meng, Aixia; Su, Shaojuan; Jing, Xu; Li, Hong; Guan, Xiumei; Li, Xin; Liu, Shunmei; Cheng, Min

    2014-05-01

    The activation of hepatic stellate cells (HSCs) is involved in the development of hepatic fibrosis. Previous studies have indicated that the acquisition of certain properties by activated HSCs is highly dependent on the reorganization of the actin cytoskeleton. However, direct evidence showing that the reorganization of the actin cytoskeleton is responsible for HSC activation is lacking. The aim of the present study was to investigate the role of cytoskeletal reorganization during HSC activation and to clarify the underlying mechanism. HSC-T6 cells were treated either with the F-actin stabilizer jasplakinolide (Jas) or the depolymerizer cytochalasin D (Cyto D). The actin cytoskeleton was evaluated via assessment of stress fiber formation. Furthermore, the activation properties of HSCs, including proliferation, adhesion, migration and the expression of α-smooth muscle actin (α-SMA) and collagen 1, were investigated in vitro. The results showed that Jas and Cyto D affected the actin distribution in HSC-T6 cells. Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells. In parallel, polymerization of actin microfilaments induced by Jas upregulated the expression of α-SMA and collagen 1, and also enhanced the migration and adhesion properties of HSC-T6 cells. Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway. In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process. The inhibition of F-actin reorganization may thus be a potential key factor or molecular target for the control of liver fibrosis or cirrhosis.

  17. Regulation of myotube formation by the actin-binding factor drebrin

    Directory of Open Access Journals (Sweden)

    Mancini Annalisa

    2011-12-01

    Full Text Available Abstract Background Myogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified. Methods RNA interference (RNAi, chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells. Results In a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated. Conclusions Our findings reveal that Dbn1 expression is

  18. Cellulose Depolymerization over Heterogeneous Catalysts.

    Science.gov (United States)

    Shrotri, Abhijit; Kobayashi, Hirokazu; Fukuoka, Atsushi

    2018-02-14

    Cellulosic biomass is the largest source of renewable organic carbon on our planet. Cellulose accounts for 40-50 wt % of this lignocellulose, and it is a feedstock for industrially important chemicals and fuels. The first step in cellulose conversion involves its depolymerization to glucose or to its hydrogenated product sorbitol. The hydrolysis of cellulose to glucose by homogeneous mineral acids was the subject of research for almost a century. However, homogeneous acids have significant drawbacks and are neither economical nor environmentally friendly. In 2006, our group reported for the first time the ability of heterogeneous catalysts to depolymerize cellulose through hydrolytic hydrogenation to produce sorbitol. Later, we reported the hydrolysis of cellulose to glucose using carbon catalyst containing weakly acidic functional groups. Understanding the reaction between cellulose and heterogeneous catalyst is a challenge as the reaction occurs between a solid substrate and a solid catalyst. In this Account, we describe our efforts for the conversion of cellulose to sorbitol and glucose using heterogeneous catalysts. Sorbitol is produced by sequential hydrolysis and hydrogenation of cellulose in one pot. We reported sorbitol synthesis from cellulose in the presence of supported metal catalysts and H 2 gas. The reducing environment of the reaction prevents byproduct formation, and harsh reaction conditions can be used to achieve sorbitol yield of up to 90%. Glucose is produced by acid catalyzed hydrolysis of cellulose, a more challenging reaction owing to the tendency of glucose to rapidly decompose in hot water. Sulfonated carbons were first reported as active catalysts for cellulose hydrolysis, but they were hydrothermally unstable under the reaction conditions. We found that carbon catalysts bearing weakly acidic functional groups such as hydroxyl and carboxylic acids are also active. Weakly acidic functional groups are hydrothermally stable, and a soluble

  19. Redox modification of nuclear actin by MICAL-2 regulates SRF signaling.

    Science.gov (United States)

    Lundquist, Mark R; Storaska, Andrew J; Liu, Ting-Chun; Larsen, Scott D; Evans, Todd; Neubig, Richard R; Jaffrey, Samie R

    2014-01-30

    The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. However, pathways that regulate nuclear actin levels are poorly understood. Here, we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    Science.gov (United States)

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Depolymerization-driven flow and the crawling of nematode sperm

    Science.gov (United States)

    Wolgemuth, Charles

    2008-03-01

    Cell crawling motility is integral in many biological and biomedical processes, such as wound healing, cancer metastasis, and morphogenesis. A complete understanding of the mechanisms by which cells crawl is still lacking, but it is known to entail at least three separate physical processes: (i) cytoskeletal extension at the front of the cell; (ii) adhesion to the substrate at the cell front and release at the rear; and (iii) advance of the cell body. In most cells, the cytoskeletal network is composed of actin. The mechanism by which force is generated to drive translocation of the cell body is still debated. Originally, this force was attributed to an actomyosin system similar to muscle. However, nematode sperm utilize a cytoskeleton composed of a network of Major Sperm Protein (MSP) that forms non-polar filaments for which molecular motors have not been identified. The motility of these cells still exhibits all three fundamental processes required for standard crawling motility. Experiments suggest that depolymerization of the cytoskeletal network is the force-producing mechanism for pulling up the rear. In this talk I will present a mechanical model that describes how depolymerization of the cytoskeleton can drive motility. This model accounts for both cytoskeletal displacements and cytsolic (the fluid component of the cell) flow. The model accurately fits in vitro data using nematode sperm extracts where depolymerization induces contraction of MSP polymer bundles. Application of this model to cell crawling produces testable predictions about how the size and shape of a cell affect crawling speed. Experiments using Caenorhabditis elegans sperm show good agreement with the model predictions. Interestingly, the model requires that cells are anisotropically elastic, being more stiff in the direction of motion than perpendicular to it. A simple physical picture can account for this anisotropy. The model also predicts that cell speed increases with anisotropy and

  2. Non-Straub type actin from molluscan catch muscle

    Energy Technology Data Exchange (ETDEWEB)

    Shelud' ko, Nikolay S., E-mail: sheludko@stl.ru; Girich, Ulyana V.; Lazarev, Stanislav S.; Vyatchin, Ilya G.

    2016-05-27

    We have developed a method of obtaining natural actin from smooth muscles of the bivalves on the example of the Crenomytilus grayanus catch muscle. The muscles were previously rigorized to prevent a loss of thin filaments during homogenization and washings. Thin filaments were isolated with a low ionic strength solution in the presence of ATP and sodium pyrophosphate. Surface proteins of thin filaments-tropomyosin, troponin, calponin and some minor actin-binding proteins-were dissociated from actin filaments by increasing the ionic strength to 0.6 M KCL. Natural fibrillar actin obtained in that way depolymerizes easily in low ionic strength solutions commonly used for the extraction of Straub-type actin from acetone powder. Purification of natural actin was carried out by the polymerization–depolymerization cycle. The content of inactivated actin remaining in the supernatant is much less than at a similar purification of Straub-type actin. A comparative investigation was performed between the natural mussel actin and the Straub-type rabbit skeletal actin in terms of the key properties of actin: polymerization, activation of Mg-ATPase activity of myosin, and the electron-microscopic structure of actin polymers. -- Highlights: •We developed method of repolymerizable invertebrate smooth muscle actin obtaining. •Our method does not involve use of denaturating agents, which could modify proteins. •Viscosity and polymerization rate of actin, gained that way, is similar to Straub one. •Electron microscopy showed that repolymerized mussel actin is similar to Straub one. •Repolymerized mussel actin has greater ATPase activating capacity, than Straub actin.

  3. Prediction and dissection of widely-varying association rate constants of actin-binding proteins.

    Science.gov (United States)

    Pang, Xiaodong; Zhou, Kenneth H; Qin, Sanbo; Zhou, Huan-Xiang

    2012-01-01

    Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as k(a) = k(a0) e(-ΔG(el*)/k(B)T), where k(a0) is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold k(a) variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions.

  4. Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor.

    Science.gov (United States)

    Radha, Vegesna; Mitra, Aninda; Dayma, Kunal; Sasikumar, Kotagiri

    2011-08-01

    C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.

  5. Fibroblast growth factor (Fgf) 23 gene transcription depends on actin cytoskeleton reorganization.

    Science.gov (United States)

    Fajol, Abul; Honisch, Sabina; Zhang, Bingbing; Schmidt, Sebastian; Alkahtani, Saad; Alarifi, Saud; Lang, Florian; Stournaras, Christos; Föller, Michael

    2016-03-01

    FGF23 regulates renal phosphate and vitamin D metabolism. Loss of FGF23 results in massive calcification and rapid aging. FGF23 production is stimulated by 1,25(OH)2D3 and NFκB signaling. Here, we report that treatment of UMR106 osteoblast-like cells with 1,25(OH)2D3, inducing Fgf23 transcription, resulted in actin polymerization which was blocked by NFκB inhibitor wogonin. Interestingly, 1,25(OH)2D3-induced Fgf23 gene transcription was abolished by the actin microfilament-disrupting agent cytochalasin B, as well as by the inhibition of actin-regulating Rac1/PAK1 signaling. Our results provide strong evidence that actin redistribution regulated by the Rac1/PAK1 pathway participates in 1,25(OH)2D3-induced Fgf23 gene transcription. © 2016 Federation of European Biochemical Societies.

  6. Inhibitory effects of pectenotoxins from marine algae on the polymerization of various actin isoforms.

    Science.gov (United States)

    Butler, Suzanne C; Miles, Christopher O; Karim, Amna; Twiner, Michael J

    2012-04-01

    Pectenotoxins (PTXs) are marine toxins produced by dinoflagellates and which accumulate in shellfish. There are at least 14 different analogs of PTX with slight variations in structure leading to different chemical properties and consequently different toxicities. Since preliminary studies have shown that the parent compound PTX1 targets actin, we investigated the effects of two analogs, PTX2 and PTX2 seco acid, on the polymerization and depolymerization of skeletal muscle actin, smooth muscle actin, cardiac muscle actin, and non-muscle actin. Optimized actin assays using fluorescently labeled skeletal muscle actin and SDS-PAGE were jointly used to determine the relative amounts of filamentous and globular actin formed during polymerization and depolymerization experiments. Our findings suggest that PTX2 causes a dose-dependent decrease in both the rate and yield of skeletal muscle actin polymerization (IC50 values of 44 and 177 nM; respectively), with no significant effects on depolymerization. Moreover, the inhibitory effects of PTX2 are conserved towards other actin isoforms (i.e., smooth muscle, cardiac muscle, and non-muscle), as the inhibitory effects on actin polymerization were also observed with similar IC50 values (range: 19-94 nM). No inhibitory effects on polymerization were observed for PTX2 seco acid, suggesting an intact lactone ring is necessary for bioactivity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Self-assembly of actin monomers into long filaments: Brownian dynamics simulations

    Science.gov (United States)

    Guo, Kunkun; Shillcock, Julian; Lipowsky, Reinhard

    2009-07-01

    Brownian dynamics simulations are used to study the dynamical process of self-assembly of actin monomers into long filaments containing up to 1000 actin protomers. In order to overcome the large separation of time scales between the diffusive motion of the free monomers and the relatively slow attachment and detachment processes at the two ends of the filaments, we introduce a novel rescaling procedure by which we speed all dynamical processes related to actin polymerization and depolymerization up by the same factor. In general, the actin protomers within a filament can attain three different states corresponding to a bound adenosine triphosphate (ATP), adenosine diphosphate with inorganic phosphate (ADP/P), and ADP molecule. The simplest situation that has been studied experimentally is provided by the polymerization of ADP-actin, for which all protomers are identical. This case is used to unravel certain relations between the filament's physical properties and the model parameters such as the attachment rate constant and the size of the capture zone, the detachment rate and the probability of the detached event, as well as the growth rate and waiting times between two successive attachment/detachment events. When a single filament is allowed to grow in a bath of constant concentration of free ADP-actin monomers, its growth rate increases linearly with the free monomer concentration in quantitative agreement with in vitro experiments. The results also show that the waiting time is governed by exponential distributions and that the two ends of a filament undergo biased random walks. The filament length fluctuations are described by a length diffusion constant that is found to attain a constant value at low ADP-actin concentration and to increase linearly with this concentration. It is straightforward to apply our simulation code to more complex processes such as polymerization of ATP-actin coupled to ATP hydrolysis, force generation by filaments, formation of

  8. Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Aguado, Carmen; Mato, Eugenia; Sánchez-Ruíz, Yován; Esteban, Inmaculada; Alberch, Jordi; Knecht, Erwin; Egea, Gustavo

    2008-05-01

    In this study, we report the formation of several cytoplasmic inclusion bodies composed of filamentous actin (F-actin) and generated by experimental treatments using depolymerizing or stabilizing actin toxins in neuronal and non-neuronal mammalian cell lines. The actin-stabilizing toxin jasplakinolide (Jpk) induced, in a microtubule-dependent manner, a single, large F-actin aggregate, which contained beta- and gamma-actin, ADF/cofilin, cortactin, and the actin nucleator Arp2/3. This aggregate was tightly associated with the Golgi complex and mitochondria, and was surrounded by vimentin intermediate filaments, microtubules and MAP4. Therefore, the Jpk-induced single, large F-actin aggregate fits the established criteria for being considered an aggresome. Lysosomes and/or autophagic vacuoles, proteasomes and microtubules were found to directly participate in the dissolution of this F-actin aggresome. Finally, the model reported here is simple, highly reproducible and reversible, and it provides an opportunity to test pharmacological agents that interfere with the formation, maintenance and/or disappearance of F-actin-enriched pathological inclusion bodies.

  9. JMY functions as actin nucleation-promoting factor and mediator for p53-mediated DNA damage in porcine oocytes.

    Directory of Open Access Journals (Sweden)

    Zili Lin

    Full Text Available Junction-mediating and regulatory protein(JMY is a multifunctional protein with roles in the transcriptional co-activation of p53 and the regulation of actin nucleation promoting factors and, hence, cell migration; however, its role in the maturation of porcine oocytes is unclear. In the current study, we investigated functional roles of JMY in porcine oocytes. Porcine oocytes expressed JMY mRNA and protein, and the mRNA expression level decreased during oocyte maturation. Knockdown of JMY by RNA interference decreased the rate of polar body extrusion, validating its role in the asymmetric division of porcine oocytes. JMY knockdown also down-regulated the mRNA and protein levels of actin and Arp2/3. Furthermore, JMY accumulated in the nucleus in response to DNA damage, and JMY knockdown suppressed DNA damage-mediated p53 activation. In conclusion, our results show that JMY has important roles in oocyte maturation as a regulator of actin nucleation-promoting factors and an activator of p53 during DNA damage during DNA damages in porcine oocytes.

  10. Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

    International Nuclear Information System (INIS)

    Saito, Shin-ya; Feng Jue; Kira, Atsushi; Kobayashi, Jun'ichi; Ohizumi, Yasushi

    2004-01-01

    The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function

  11. Enzymatic depolymerization of lignin by laccases

    OpenAIRE

    Hamidi, Nor Hanimah

    2013-01-01

    More than half of platform petrochemicals are aromatic, whereas the only large-scale, naturally-occurring, renewable source of aromatics is lignin. Chemical depolymerization of lignin requires extreme conditions, and results in extensive destruction of the aromatic rings and/or char formation. By contrast, enzymatic lignin depolymerization occurs under mild conditions with retention of the aromatic nuclei. Therefore, laccase from Agaricus bisporus (LAB) and from Trametes versicolor (LTV) with...

  12. Alkaline Depolymerization of Polyethylene Terephthalate Plastic Waste

    OpenAIRE

    Ammar F. Abbas

    2016-01-01

    Depolymerization reaction is considered one of the most significant ways of converting waste polyethylene terephthalate in to terephthalic acid. The water polyethylene terephthalate bottle waste was collected from different places in Baghdad. The collection step shows that there is plenty amount of polyethylene terephthalate suitable to be an important source of terephthalic acid production.PET plastic waste conversion to terephthalic acid by depolymerization process was examined. The effect ...

  13. Distinct actin oligomers modulate differently the activity of actin nucleators.

    Science.gov (United States)

    Qu, Zheng; Silvan, Unai; Jockusch, Brigitte M; Aebi, Ueli; Schoenenberger, Cora-Ann; Mannherz, Hans Georg

    2015-10-01

    Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits; however, their instability has hindered their detailed study. Therefore we used chemically crosslinked actin oligomers to analyse their effect on actin polymerization. Actin dimer (upper dimer, UD), trimer and tetramer intermolecularly crosslinked by phenylene-bismaleimide along the genetic helix (between Lys199 and Cys374) were isolated by gel filtration and found to increasingly stimulate actin polymerization as shown by the pyrene assay and total internal reflection fluorescence microscopy. In contrast, the so-called lower actin dimer (LD) characterized by a Cys374-Cys374 crosslink stimulated actin polymerization only at low but inhibited it at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin β4 (Tβ4) or profilin, whereas the LD stimulated repolymerization only from the profilin : actin but not the actin : Tβ4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin-nucleating activity of gelsolin, mouse diaphanous related (mDia) formin and the actin-related protein 2/3 (Arp2/3) complex were analysed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1-3, to the FH2 domains of the formins mDia1 and mDia3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1-3, but not of the mDia-FH2 domain nor of the Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia-FH2 domains, gelsolin and G1-3, demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin subunits. © 2015 FEBS.

  14. Autism-like Deficits in Shank3-Deficient Mice Are Rescued by Targeting Actin Regulators

    Directory of Open Access Journals (Sweden)

    Lara J. Duffney

    2015-06-01

    Full Text Available Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synapses, is a highly prevalent and penetrant risk factor for autism. Using combined behavioral, electrophysiological, biochemical, imaging, and molecular approaches, we find that Shank3-deficient mice exhibit autism-like social deficits and repetitive behaviors, as well as the significantly diminished NMDA receptor (NMDAR synaptic function and synaptic distribution in prefrontal cortex. Concomitantly, Shank3-deficient mice have a marked loss of cortical actin filaments, which is associated with the reduced Rac1/PAK activity and increased activity of cofilin, the major actin depolymerizing factor. The social deficits and NMDAR hypofunction are rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice and are induced by inhibiting PAK or Rac1 in wild-type mice. These results indicate that the aberrant regulation of synaptic actin filaments and loss of synaptic NMDARs contribute to the manifestation of autism-like phenotypes. Thus, targeting actin regulators provides a strategy for autism treatment.

  15. Microtubule-actin crosslinking factor 1 (Macf1 domain function in Balbiani body dissociation and nuclear positioning.

    Directory of Open Access Journals (Sweden)

    Matias Escobar-Aguirre

    2017-09-01

    Full Text Available Animal-vegetal (AV polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb. The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a. Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs, actin filaments (AF, and intermediate filaments (IF. In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK, a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD and plectin repeat domain (PRD to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the

  16. Crowding of molecular motors determines microtubule depolymerization.

    Science.gov (United States)

    Reese, Louis; Melbinger, Anna; Frey, Erwin

    2011-11-02

    The assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT-associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Using an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis reveals two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise that cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity affects only the end-residence time of depolymerases and not the depolymerization speed. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Actin purification from a gel of rat brain extracts.

    Science.gov (United States)

    Levilliers, N; Peron-Renner, M; Coffe, G; Pudles, J

    1984-01-01

    Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles.

  18. Actin cytoskeleton regulation of epithelial mesenchymal transition in metastatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Jay Shankar

    Full Text Available Epithelial-mesenchymal transition (EMT is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells.

  19. Amination of biorefinery technical lignins using Mannich reaction synergy with subcritical ethanol depolymerization.

    Science.gov (United States)

    Wang, Bing; Chen, Tian-Ying; Wang, Han-Min; Li, Han-Yin; Liu, Chuan-Fu; Wen, Jia-Long

    2018-02-01

    The alcoholic depolymerization and Mannich reaction were conducted to improve the chemical activity of biorefinery technical lignins and introduce amino groups into lignins, respectively. To understand the chemical structural transformations and examine the reaction mechanism, GPC and solution-state NMR techniques were performed. Element analysis was also used to quantify the amount of amine groups. The NMR characterization the depolymerized lignins indicated of the depolymerization, demethoxylation, and bond cleavage of linkages occurred during the depolymerization process. Results showed that the depolymerization temperature instead of the addition of capping reagents was the main factor for improving the reactivity of lignin under the given conditions. The Mannich reaction was very selective, primarily occurred at H 3,5 and G 5 positions, and the H units present a higher chemical reactivity. It is believed that the understanding of the fundamental chemistry of lignin during depolymerization and Mannich reaction process will contribute to the extension of high value-added applications of biorefinery lignin. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF)-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells.

    Science.gov (United States)

    Li, Li; Song, Haijing; Mu, Peipei; Xu, Ming; Liu, Chaoxia; Wang, Ying; Qin, Yingsong; Sun, Shen; Gao, Jin; Wang, Ting; Gao, Dianshuai

    2017-09-07

    Glial cell line-derived neurotrophic factor (GDNF), a potential therapeutic factor for Parkinson's disease (PD), exerts its biological effects through the Ret receptor tyrosine kinase. The redistribution of Ret into lipid rafts substantially influences Ret signaling, but the mechanisms underlying Ret translocation remain unclear. The purpose of our study was to further explore the signaling mechanisms of GDNF and to determine whether the actin cytoskeleton is involved in the GDNF-induced Ret translocation into lipid rafts. In MN9D dopaminergic neuronal cells, we used density gradient centrifugation and immunofluorescence confocal microscopy to separate and visualize lipid rafts, co-immunoprecipitation to analyze protein-protein interactions, and latrunculin B (Lat B) and jasplakinolide (Jas) to disrupt and enhance the polymerization of the actin cytoskeleton, respectively. The results showed that Ret translocated into lipid rafts and coimmunoprecipitated with actin in response to GDNF treatment. After Lat B or Jas treatment, the Ret-F-actin association induced by GDNF was impaired or enhanced respectively and then the levels of Ret translocated into lipid rafts were correspondingly inhibited or promoted. These data indicate that actin polymerization and cytoskeletal remodeling are integral to GDNF-induced cell signaling in dopaminergic cells and define a new role of the actin cytoskeleton in promoting Ret redistribution into lipid rafts.

  1. The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells

    Directory of Open Access Journals (Sweden)

    Li Li

    2017-09-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF, a potential therapeutic factor for Parkinson’s disease (PD, exerts its biological effects through the Ret receptor tyrosine kinase. The redistribution of Ret into lipid rafts substantially influences Ret signaling, but the mechanisms underlying Ret translocation remain unclear. The purpose of our study was to further explore the signaling mechanisms of GDNF and to determine whether the actin cytoskeleton is involved in the GDNF-induced Ret translocation into lipid rafts. In MN9D dopaminergic neuronal cells, we used density gradient centrifugation and immunofluorescence confocal microscopy to separate and visualize lipid rafts, co-immunoprecipitation to analyze protein-protein interactions, and latrunculin B (Lat B and jasplakinolide (Jas to disrupt and enhance the polymerization of the actin cytoskeleton, respectively. The results showed that Ret translocated into lipid rafts and coimmunoprecipitated with actin in response to GDNF treatment. After Lat B or Jas treatment, the Ret–F-actin association induced by GDNF was impaired or enhanced respectively and then the levels of Ret translocated into lipid rafts were correspondingly inhibited or promoted. These data indicate that actin polymerization and cytoskeletal remodeling are integral to GDNF-induced cell signaling in dopaminergic cells and define a new role of the actin cytoskeleton in promoting Ret redistribution into lipid rafts.

  2. Alkaline Depolymerization of Polyethylene Terephthalate Plastic Waste

    Directory of Open Access Journals (Sweden)

    Ammar F. Abbas

    2016-02-01

    Full Text Available Depolymerization reaction is considered one of the most significant ways of converting waste polyethylene terephthalate in to terephthalic acid. The water polyethylene terephthalate bottle waste was collected from different places in Baghdad. The collection step shows that there is plenty amount of polyethylene terephthalate suitable to be an important source of terephthalic acid production.PET plastic waste conversion to terephthalic acid by depolymerization process was examined. The effect of ethylene glycol amount, reaction time (up to 90 minutes and reaction temperature (from 70 to 170° C on the polyethylene terephthalate conversion was obtained.The kinetic study shows that the ordination of the depolymerization reaction of PET is first order irreversible reaction with 31103.5 J/mole activation energy.A 97.9 % terephthalic acid purity has been obtained by purification with N, N-dimethylformamide.

  3. Alkaline Depolymerization of Polyethylene Terephthalate Plastic Waste

    Directory of Open Access Journals (Sweden)

    Ammar S. Abbas

    2016-02-01

    Full Text Available Depolymerization reaction is considered one of the most significant ways of converting waste polyethylene terephthalate in to terephthalic acid. The water polyethylene terephthalate bottle waste was collected from different places in Baghdad. The collection step shows that there is plenty amount of polyethylene terephthalate suitable to be an important source of terephthalic acid production. PET plastic waste converting to terephthalic acid by depolymerization process was examined. The effect of ethylene glycol amount, reaction time (up to 90 minutes and reaction temperature (from 70 to 170° C on the polyethylene terephthalate conversion was obtained. The kinetic study shows that the ordination of the depolymerization reaction of PET is first order irreversible reaction with 31103.5 J/mole activation energy. A 97.9 % terephthalic acid purity has been obtained by purification with N, N-dimethylformamide. Normal 0 false false false EN-US X-NONE AR-SA

  4. Radiation depolymerization of chitosan to prepare oligomers

    International Nuclear Information System (INIS)

    Hai, Le; Bang Diep, Tran; Nagasawa, Naotsugu; Yoshii, Fumio; Kume, Tamikazu

    2003-01-01

    Radiation depolymerization of chitosan was carried out by gamma irradiation in the solid state. The radiation-chemical depolymerization yield of chitosan in the solid state, Gd, determined by gel permeation chromatography, is 0.9 for chitosan 10B and 1.8 for chitosan 8B. Low molecular weight chitosan/or oligochitosans were separated from a chitosan depolymerized by gamma radiation, using mixtures of methanol-water and acetone as the solvents. Due to the differences in solubility revealed upon radiolysis, extracts became subdivided into precipitates and soluble fractions. The biological effect of oligochitosan in each fraction was evaluated; the preliminary results indicated that the oligochitosan with M w -bar=2x10 4 inhibited the growth of fungi at 100 ppm and that with M w -bar=800 only enhanced the growth of the same typical fungi

  5. Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin

    International Nuclear Information System (INIS)

    Lee, Y.-J.; Sheu, T.-J.; Keng, Peter C.

    2005-01-01

    Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity

  6. Self-assembly of actin monomers into long filaments: Brownian Dynamics simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2009-01-01

    states corresponding to a bound adenosine triphosphate (ATP), adenosine diphosphate with inorganic phosphate (ADP/P), and ADP molecule. The simplest situation that has been studied experimentally is provided by the polymerization of ADP-actin, for which all protomers are identical. This case is used...... attachment and detachment processes at the two ends of the filaments, we introduce a novel rescaling procedure by which we speed all dynamical processes related to actin polymerization and depolymerization up by the same factor. In general, the actin protomers within a filament can attain three different....../detachment events. When a single filament is allowed to grow in a bath of constant concentration of free ADP-actin monomers, its growth rate increases linearly with the free monomer concentration in quantitative agreement with in vitro experiments. Theresults also show that the waiting time is governed by...

  7. The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri.

    Science.gov (United States)

    Sohn, Hae-Jin; Kim, Jong-Hyun; Shin, Myeong-Heon; Song, Kyoung-Ju; Shin, Ho-Joon

    2010-03-01

    Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

  8. The preliminary observation of the changes of β-actin,coagulant and inflammatory factors in mice serum induced by γ rays irradiation

    International Nuclear Information System (INIS)

    Zhang Qingzhi; Wang Jia; Cheng Ying; Li Mingjuan; Min Rui

    2010-01-01

    In order to learn the effect of β-actin in acute radiation injury, the changeable pattern with time of plasma β-actin, PT, APTT, FIB and IL-8 in mice spleen tissue exposed to 6 Gy γ-rays radiation was investigated.Blood and spleen were collected at immediate, 1, 2, 3, 4, 7 and 14 d after irradiation, respectively. The contents of blood β-actin were detected by magnetic bead separation enzyme-linked immunosorbent. An STAGO blood coagulation instrument was used to determine PT, APTT and FIB. DNA expression of IL-8 was detected by real time-PCR analyzer. The results show that the level of β-actin in serum of irradiated mice is higher than that of normal control group at all different post-irradiation time points although the change of β-actin in serum of irradiated mice with time schedule shows a pattern which increases within 1d and declines beyond 1d. The trend of the changes in plasma PT, APTT, FIB and in spleen IL-8 and time pattern of these changes are similar to that in plasma β-actin in irradiated mice. The difference in values and the time phase between plasma β-actin and other indexes is the reaching time of peak values and the declining levels of the values. These results are valuable for studying the role of β-actin in acute radiation sickness pathology process and can be used to explore new factors influencing and regulating pathology process. (authors)

  9. Phosphoric Acid-Mediated Depolymerization and Decrystallization ...

    African Journals Online (AJOL)

    Methods: α- cellulose (AC-CC) obtained from corn cob by a delignification process, was depolymerized with 85% phosphoric acid to yield LCC-CC product, whose physical properties were compared with those of AC-CC. Results: The yield of AC-CC and LCC-CC were 14 and 11 %, respectively. The physical properties ...

  10. Actin polymerization contributes to neutrophil chemotactic dysfunction following thermal injury.

    Science.gov (United States)

    Hasslen, S R; Ahrenholz, D H; Solem, L D; Nelson, R D

    1992-11-01

    The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients' cells may contribute to their generalized chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury.

  11. Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

    International Nuclear Information System (INIS)

    Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O.

    2010-01-01

    The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties

  12. depolymerizing factor gene in Hevea brasiliensis

    African Journals Online (AJOL)

    Yomi

    2010-11-08

    Nov 8, 2010 ... 1Key Laboratory of Rubber Biology, Ministry of Agriculture, Rubber Research Institute, Chinese Academy of Tropical. Agricultural Sciences ... 3Institute of Biological Science and Technology, College of Agriculture, Hainan University, Haikou, 570228, China. ... rice ADF proteins were divided into four ancient.

  13. Smooth muscle actin-expressing stromal fibroblasts in head and neck squamous cell carcinoma: Increased expression of galectin-1 and induction of poor prognosis factors

    Czech Academy of Sciences Publication Activity Database

    Valach, J.; Fík, Z.; Strnad, Hynek; Chovanec, M.; Plzák, J.; Čada, Z.; Szabo, Pavol; Šáchová, Jana; Hroudová, Miluše; Urbanová, Markéta; Steffl, M.; Pačes, Jan; Mazánek, J.; Vlček, Čestmír; Betka, J.; Kaltner, H.; André, S.; Gabius, H. J.; Kodet, R.; Smetana, K.; Gál, P.; Kolář, Michal

    2012-01-01

    Roč. 131, č. 11 (2012), s. 2499-2508 ISSN 0020-7136 R&D Projects: GA MŠk 2B06106 Institutional research plan: CEZ:AV0Z50520514 Keywords : carcinoma * stroma * actin * lectin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.198, year: 2012

  14. Characterization of actin filament severing by actophorin from Acanthamoeba castellanii

    Science.gov (United States)

    1991-01-01

    Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate. PMID:1757465

  15. Actin in Mung Bean Mitochondria and Implications for Its Function[W][OA

    Science.gov (United States)

    Lo, Yih-Shan; Cheng, Ning; Hsiao, Lin-June; Annamalai, Arunachalam; Jauh, Guang-Yuh; Wen, Tuan-Nan; Dai, Hwa; Chiang, Kwen-Sheng

    2011-01-01

    Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed. PMID:21984697

  16. Kinetics and Thermodynamics of Ultrasound-Assisted Depolymerization of κ-Carrageenan

    Directory of Open Access Journals (Sweden)

    Ratnawati Ratnawati

    2016-03-01

    Full Text Available The ultrasound-assisted depolymerization of κ-carrageenan has been studied at various temperatures and times. The κ-carrageenan with initial molecular weight of 545 kDa was dispersed in water to form a 5 g/L solution, which was then depolymerized in an ultrasound device at various temperatures and times. The viscosity of the solution was measured using Brookfield viscometer, which was then used to find the number-average molecular weight by Mark-Houwink equation. To obtain the kinetics of κ-carrageenan depolymerization, the number-average molecular weight data was treated using midpoint-chain scission kinetics model. The pre-exponential factor and activation energies for the reaction are 2.683×10-7 mol g-1 min-1 and 6.43 kJ mol-1, respectively. The limiting molecular weight varies from 160 kDa to 240 kDa, and it is linearly correlated to temperature. The results are compared to the result of thermal depolymerization by calculating the half life. It is revealed that ultrasound assisted depolymerization of κ-carrageenan is faster than thermal depolymerization at temperatures below 72.2°C. Compared to thermal depolymerization, the ultrasound-assisted process has lower values of Ea, ΔG‡, ΔH‡, and ΔS‡, which can be attributed to the ultrasonically induced breakage of non-covalent bonds in κ-carrageenan molecules. Copyright © 2016 BCREC GROUP. All rights reserved Received: 10th November 2015; Revised: 18th January 2016; Accepted: 19th January 2016 How to Cite: Ratnawati, R., Prasetyaningrum, A., Wardhani, D.H. (2016. Kinetics and Thermodynamics of Ultrasound-Assisted Depolymerization of κ-Carrageenan. Bulletin of Chemical Reaction Engineering & Catalysis, 11(1: 48-58. (doi:10.9767/bcrec.11.1.415.48-58 Permalink/DOI: http://dx.doi.org/10.9767/bcrec.11.1.415.48-58

  17. Actin binding proteins and spermiogenesis

    Science.gov (United States)

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  18. Memory effect on ion beam-induced depolymerization of PMMA

    Energy Technology Data Exchange (ETDEWEB)

    Compagnini, G.; Angilella, G.G.N. E-mail: giuseppe.angilella@ct.infn.it; Raudino, A.; Puglisi, O. E-mail: opuglisi@dipchi.unict.it

    2001-04-01

    Recently a new ion beam-induced phenomenology - depolymerization of macromolecular target - has been found and studied in our laboratory. This has been observed in poly(methylmethacrylate) (PMMA) using He{sup +} 300 keV ion beam, in a temperature range much lower than that required for thermal depolymerization. Here we report an in situ monitoring of PMMA depolymerization performed by mass spectrometry. We have found that at a given temperature the monomer flux due to depolymerization of the bombarded polymer is much more intense when the temperature is quenched, with respect to the same temperature during heating. The results have been rationalized by using an accurate model that involves initiation and propagation kinetics followed by the diffusion process. The use of the model allowed us to exclude any influence of transport properties of PMMA on this effect but indicates that the hysteresis phenomenon detected at macroscopic level has a microscopic counterpart linked to the chemical yields of the depolymerization kinetics.

  19. Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β-catenin signaling and osteoblast proliferation.

    Science.gov (United States)

    Yin, Chong; Zhang, Yan; Hu, Lifang; Tian, Ye; Chen, Zhihao; Li, Dijie; Zhao, Fan; Su, Peihong; Ma, Xiaoli; Zhang, Ge; Miao, Zhiping; Wang, Liping; Qian, Airong; Xian, Cory J

    2017-12-08

    Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation. © 2017 Wiley Periodicals, Inc.

  20. Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte.

    Directory of Open Access Journals (Sweden)

    Tripti Gupta

    2010-08-01

    Full Text Available Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1 gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.

  1. Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte.

    Science.gov (United States)

    Gupta, Tripti; Marlow, Florence L; Ferriola, Deborah; Mackiewicz, Katarzyna; Dapprich, Johannes; Monos, Dimitri; Mullins, Mary C

    2010-08-19

    Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.

  2. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    Science.gov (United States)

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity. © 2013 Elsevier Inc. All rights reserved.

  3. Dynamics of actin cables in polarized growth of the filamentous fungus Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Anna eBergs

    2016-05-01

    Full Text Available Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although specific marker proteins to visualize actin cables have been developed in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here we visualized actin cables using tropomyosin (TpmA and Lifeact fused to fluorescent proteins in Aspergillus nidulans and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules.

  4. Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells.

    Science.gov (United States)

    Schudt, Gordian; Dolnik, Olga; Kolesnikova, Larissa; Biedenkopf, Nadine; Herwig, Astrid; Becker, Stephan

    2015-10-01

    Transport of ebolavirus (EBOV) nucleocapsids from perinuclear viral inclusions, where they are formed, to the site of budding at the plasma membrane represents an obligatory step of virus assembly. Until now, no live-cell studies on EBOV nucleocapsid transport have been performed, and participation of host cellular factors in this process, as well as the trajectories and speed of nucleocapsid transport, remain unknown. Live-cell imaging of EBOV-infected cells treated with different inhibitors of cellular cytoskeleton was used for the identification of cellular proteins involved in the nucleocapsid transport. EBOV nucleocapsids were visualized by expression of green fluorescent protein (GFP)-labeled nucleocapsid viral protein 30 (VP30) in EBOV-infected cells. Incorporation of the fusion protein VP30-GFP into EBOV nucleocapsids was confirmed by Western blot and indirect immunofluorescence analyses. Importantly, VP30-GFP fluorescence was readily detectable in the densely packed nucleocapsids inside perinuclear viral inclusions and in the dispersed rod-like nucleocapsids located outside of viral inclusions. Live-cell imaging of EBOV-infected cells revealed exit of single nucleocapsids from the viral inclusions and their intricate transport within the cytoplasm before budding at the plasma membrane. Nucleocapsid transport was arrested upon depolymerization of actin filaments (F-actin) and inhibition of the actin-nucleating Arp2/3 complex, and it was not altered upon depolymerization of microtubules or inhibition of N-WASP. Actin comet tails were often detected at the rear end of nucleocapsids. Marginally located nucleocapsids entered filopodia, moved inside, and budded from the tip of these thin cellular protrusions. Live-cell imaging of EBOV-infected cells revealed actin-dependent long-distance transport of EBOV nucleocapsids before budding at the cell surface. These findings provide useful insights into EBOV assembly and have potential application in the development

  5. Actin dynamics in mouse fibroblasts in microgravity

    Science.gov (United States)

    Moes, Maarten J. A.; Bijvelt, Jose J.; Boonstra, Johannes

    2007-09-01

    After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.

  6. Co-transcriptional nuclear actin dynamics.

    Science.gov (United States)

    Percipalle, Piergiorgio

    2013-01-01

    Actin is a key player for nuclear structure and function regulating both chromosome organization and gene activity. In the cell nucleus actin interacts with many different proteins. Among these proteins several studies have identified classical nuclear factors involved in chromatin structure and function, transcription and RNA processing as well as proteins that are normally involved in controlling the actin cytoskeleton. These discoveries have raised the possibility that nuclear actin performs its multi task activities through tight interactions with different sets of proteins. This high degree of promiscuity in the spectrum of protein-to-protein interactions correlates well with the conformational plasticity of actin and the ability to undergo regulated changes in its polymerization states. Several of the factors involved in controlling head-to-tail actin polymerization have been shown to be in the nucleus where they seem to regulate gene activity. By focusing on the multiple tasks performed by actin and actin-binding proteins, possible models of how actin dynamics controls the different phases of the RNA polymerase II transcription cycle are being identified.

  7. Lignin depolymerization by fungal secretomes and a microbial sink

    Energy Technology Data Exchange (ETDEWEB)

    Salvachúa, Davinia; Katahira, Rui; Cleveland, Nicholas S.; Khanna, Payal; Resch, Michael G.; Black, Brenna A.; Purvine, Samuel O.; Zink, Erika M.; Prieto, Alicia; Martínez, María J.; Martínez, Angel T.; Simmons, Blake A.; Gladden, John M.; Beckham, Gregg T.

    2016-08-25

    In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining, and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/ methanol/choline (GMC) oxidoreductases and laccases. Overall, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high

  8. Latrunculin B-induced plant dwarfism: Plant cell elongation is F-actin-dependent.

    Science.gov (United States)

    Baluska, F; Jasik, J; Edelmann, H G; Salajová, T; Volkmann, D

    2001-03-01

    Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

  9. Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

    DEFF Research Database (Denmark)

    Jorgensen, Nanna K; Pedersen, Stine F; Rasmussen, Hanne B

    2003-01-01

    -induced activation of hIK channels was strongly inhibited by cytochalasin D (CD), in concentrations that caused depolymerization of F-actin filaments, indicating a role for the F-actin cytoskeleton in modulation of hIK by changes in cell volume. In conclusion, hIK and rSK3 channels are activated by cell swelling...... and inhibited by shrinkage. A role for the F-actin cytoskeleton in the swelling-induced activation of hIK channels is suggested....

  10. Directional Transport of a Bead Bound to Lamellipodial Surface Is Driven by Actin Polymerization

    Directory of Open Access Journals (Sweden)

    Daisuke Nobezawa

    2017-01-01

    Full Text Available The force driving the retrograde flow of actin cytoskeleton is important in the cellular activities involving cell movement (e.g., growth cone motility in axon guidance, wound healing, or cancer metastasis. However, relative importance of the forces generated by actin polymerization and myosin II in this process remains elusive. We have investigated the retrograde movement of the poly-D-lysine-coated bead attached with the optical trap to the edge of lamellipodium of Swiss 3T3 fibroblasts. The velocity of the attached bead drastically decreased by submicromolar concentration of cytochalasin D, latrunculin A, or jasplakinolide, indicating the involvement of actin turnover. On the other hand, the velocity decreased only slightly in the presence of 50 μM (−-blebbistatin and Y-27632. Comparative fluorescence microscopy of the distribution of actin filaments and that of myosin II revealed that the inhibition of actin turnover by cytochalasin D, latrunculin A, or jasplakinolide greatly diminished the actin filament network. On the other hand, inhibition of myosin II activity by (−-blebbistatin or Y-27632 little affected the actin network but diminished stress fibers. Based on these results, we conclude that the actin polymerization/depolymerization plays the major role in the retrograde movement, while the myosin II activity is involved in the maintenance of the dynamic turnover of actin in lamellipodium.

  11. Expression of Tau Produces Aberrant Plasma Membrane Blebbing in Glial Cells Through RhoA-ROCK-Dependent F-Actin Remodeling.

    Science.gov (United States)

    Torres-Cruz, Francisco M; Rodríguez-Cruz, Fanny; Escobar-Herrera, Jaime; Barragán-Andrade, Norma; Basurto-Islas, Gustavo; Ripova, Daniela; Ávila, Jesús; Garcia-Sierra, Francisco

    2016-03-21

    Abnormal aggregation of Tau in glial cells has been reported in Alzheimer's disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

  12. Treatment of Actinic Purpura

    Science.gov (United States)

    2017-01-01

    Mature skin is prone to bruising, resulting in a condition known as actinic purpura, characterized by unsightly ecchymosis and purple patches. Similar to other skin conditions, the incidence of actinic purpura increases with advancing age and occurs with equal frequency among men and women. The unsightly appearance of actinic purpura may be a source of emotional distress among the elderly. A new product has been formulated specifically for the treatment of actinic purpura. This product contains retinol, α-hydroxy acids, arnica oil, ceramides, niacinamide, and phytonadione, which effectively treat actinic purpura by improving local circulation, thickening the skin, and repairing the skin barrier. The objective of this paper is to review the beneficial properties of these ingredients and their respective roles in the treatment of actinic purpura. PMID:28979656

  13. Mechanocatalytic Depolymerization of Cellulose With Perfluorinated Sulfonic Acid Ionomers

    Directory of Open Access Journals (Sweden)

    Ayman Karam

    2018-03-01

    Full Text Available Here, we investigated that the mechanocatalytic depolymerization of cellulose in the presence of Aquivion, a sulfonated perfluorinated ionomer. Under optimized conditions, yields of water soluble sugars of 90–97% were obtained using Aquivion PW98 and PW66, respectively, as a solid acid catalyst. The detailed characterization of the water soluble fraction revealed (i the selective formation of oligosaccharides with a DP up to 11 and (ii that depolymerization and reversion reactions concomitantly occurred during the mechanocatalytic process, although the first largely predominated. More importantly, we discussed on the critical role of water contained in Aquivion and cellulose on the efficiency of the mechanocatalytic process.

  14. Actin-dependence of the chloroplast cold positioning response in the liverwort Marchantia polymorpha L.

    Directory of Open Access Journals (Sweden)

    Shun Kimura

    2016-09-01

    Full Text Available The subcellular positioning of chloroplasts can be changed by alterations in the environment such as light and temperature. For example, in leaf mesophyll cells, chloroplasts localize along anticlinal cell walls under high-intensity light, and along periclinal cell walls under low-intensity light. These types of positioning responses are involved in photosynthetic optimization. In light-mediated chloroplast positioning responses, chloroplasts move to the appropriate positions in an actin-dependent manner, although some exceptions also depend on microtubule. Even under low-intensity light, at low temperature (e.g., 5°C, chloroplasts localize along anticlinal cell walls; this phenomenon is termed chloroplast cold positioning. In this study, we analyzed whether chloroplast cold positioning is dependent on actin filaments and/or microtubules in the liverwort Marchantia polymorpha L. When liverwort cells were treated with drugs for the de-polymerization of actin filaments, chloroplast cold positioning was completely inhibited. In contrast, chloroplast cold positioning was not affected by treatment with a drug for the de-polymerization of microtubules. These observations indicate the actin-dependence of chloroplast cold positioning in M. polymorpha. Actin filaments during the chloroplast cold positioning response were visualized by using fluorescent probes based on fluorescent proteins in living liverwort cells, and thus, their behavior during the chloroplast cold positioning response was documented.

  15. EhCoactosin stabilizes actin filaments in the protist parasite Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Nitesh Kumar

    2014-09-01

    Full Text Available Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics.

  16. The Carboxy-Terminal Third Of Dystrophin Enhances Actin Binding Activity

    Science.gov (United States)

    Henderson, Davin M.; Lin, Ava Yun; Thomas, David D.; Ervasti, James M.

    2012-01-01

    Dystrophin is an actin-binding protein thought to stabilize cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule (from N-terminal ABD1 through ABD2) bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays compared to full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction in terms of restricting actin’s rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin. PMID:22226838

  17. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

    Science.gov (United States)

    Spracklen, Andrew J.; Kelpsch, Daniel J.; Chen, Xiang; Spracklen, Cassandra N.; Tootle, Tina L.

    2014-01-01

    Prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton—temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling. PMID:24284900

  18. CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.

    Science.gov (United States)

    Xu, L; Daly, T; Gao, C; Flotte, T R; Song, S; Byrne, B J; Sands, M S; Parker Ponder, K

    2001-03-20

    Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken beta-actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1 alpha promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-beta-actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice. Neonatal intramuscular injection resulted in expression of hFX at 248 ng/ml (3.1% of normal), which derived from both liver and muscle. We conclude that neonatal gene therapy with an AAV vector with the CMV-beta-actin promoter might correct hemophilia due to hFX deficiency.

  19. Actinic Granuloma with Focal Segmental Glomerulosclerosis

    Directory of Open Access Journals (Sweden)

    Ruedee Phasukthaworn

    2016-02-01

    Full Text Available Actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun-damaged skin. The pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. We report a case of a 52-year-old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. The clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. During that period of time, she also developed focal segmental glomerulosclerosis. The association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies.

  20. Characterization of anaerobic consortia coupled lignin depolymerization with biomethane generation.

    Science.gov (United States)

    Wu, Yi-Rui; He, Jianzhong

    2013-07-01

    Two sediment-free microbial consortia (LI3 and LP3) were established to depolymerize lignin under anaerobic conditions. During depolymerizing high molecular weight lignin to low molecular weight molecules, the two cultures produced biomethane up to 151.7 and 113.0 mL g(-1) total lignin. Furthermore, LI3 and LP3 could also utilize the biomass - oil palm empty fruit bunch fiber (OPEFB) to produce 190.6 and 195.6 mL methaneg(-1) total lignin in OPEFB, and at the same time improve the bioavailability of lignocellulosic matters for further enzymatic hydrolysis. The microbial community analysis by denature gradient gel electrophoresis (DGGE) and the high-density 16S rDNA gene microarray (PhyloChip) exhibited that Methanomethylovorans sp. (LI3) and Methanoculleus sp. (LP3) were the main methanogens present, and phylum Firmicutes and Bacteroidetes were mainly involved in the lignin depolymerization. The established microbial consortia with both lignin depolymerization and biomethane production provide profound application on the environmental friendly pretreatment of lignocellulosic materials. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Microtubule depolymerization induces traction force increase through two distinct pathways

    Science.gov (United States)

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-li

    2011-01-01

    Traction forces increase after microtubule depolymerization; however, the signaling mechanisms underlying this, in particular the dependence upon myosin II, remain unclear. We investigated the mechanism of traction force increase after nocodazole-induced microtubule depolymerization by applying traction force microscopy to cells cultured on micropatterned polyacrylamide hydrogels to obtain samples of homogeneous shape and size. Control cells and cells treated with a focal adhesion kinase (FAK) inhibitor showed similar increases in traction forces, indicating that the response is independent of FAK. Surprisingly, pharmacological inhibition of myosin II did not prevent the increase of residual traction forces upon nocodazole treatment. This increase was abolished upon pharmacological inhibition of FAK. These results suggest two distinct pathways for the regulation of traction forces. First, microtubule depolymerization activates a myosin-II-dependent mechanism through a FAK-independent pathway. Second, microtubule depolymerization also enhances traction forces through a myosin-II-independent, FAK-regulated pathway. Traction forces are therefore regulated by a complex network of complementary signals and force-generating mechanisms. PMID:22193960

  2. A polymerization–depolymerization model for generation of ...

    Indian Academy of Sciences (India)

    A polymerization–depolymerization model for generation of contractile force during bacterial cell division. BIPLAB GHOSH and ANIRBAN SAIN∗. Physics Department, Indian Institute of Technology Bombay, Powai, Mumbai 400 076,. India. *Corresponding author. E-mail: asain@phy.iitb.ac.in. Abstract. During the last phase ...

  3. Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

    Science.gov (United States)

    Shirai, Yuki M.; Tsunoyama, Taka A.; Hiramoto-Yamaki, Nao; Hirosawa, Koichiro M.; Shibata, Akihiro C. E.; Kondo, Kenichi; Tsurumune, Atsushi; Ishidate, Fumiyoshi; Kusumi, Akihiro

    2017-01-01

    Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”. PMID:29190677

  4. Unveiling interactions among mitochondria, caspase-like proteases, and the actin cytoskeleton during plant programmed cell death (PCD.

    Directory of Open Access Journals (Sweden)

    Christina E N Lord

    Full Text Available Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD. PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B, a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1 activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs. The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.

  5. Catalytic Oxidation and Depolymerization of Lignin in Aqueous Ionic Liquid

    International Nuclear Information System (INIS)

    Das, Lalitendu; Xu, Siquan; Shi, Jian

    2017-01-01

    Lignin is an integral part of the plant cell wall, which provides rigidity to plants, also contributes to the recalcitrance of the lignocellulosic biomass to biochemical and biological deconstruction. Lignin is a promising renewable feedstock for aromatic chemicals; however, an efficient and economic lignin depolymerization method needs to be developed to enable the conversion. In this study, we investigated the depolymerization of alkaline lignin in aqueous 1-ethyl-3-methylimidazolium acetate [C 2 C 1 Im][OAc] under oxidizing conditions. Seven different transition metal catalysts were screened in presence of H 2 O 2 as oxidizing agent in a batch reactor. CoCl 2 and Nb 2 O 5 proved to be the most effective catalysts in degrading lignin to aromatic compounds. A central composite design was used to optimize the catalyst loading, H 2 O 2 concentration, and temperature for product formation. Results show that lignin was depolymerized, and the major degradation products found in the extracted oil were guaiacol, syringol, vanillin, acetovanillone, and homovanillic acid. Lignin streams were characterized by Fourier transform infrared spectroscopy and gel permeation chromatography to determine effects of the experimental parameters on lignin depolymerization. The weight-average molecular weight (M w ) of liquid stream lignin after oxidation, for CoCl 2 and Nb 2 O 5 catalysts were 1,202 and 1,520 g mol −1 , respectively, lower than that of Kraft lignin. Polydispersity index of the liquid stream lignin increased as compared with Kraft lignin, indicating wide span of the molecular weight distribution as a result of lignin depolymerization. Results from this study provide insights into the role of oxidant and transition metal catalysts and the oxidative degradation reaction sequence of lignin toward product formation in presence of aqueous ionic liquid.

  6. Catalytic Oxidation and Depolymerization of Lignin in Aqueous Ionic Liquid

    Energy Technology Data Exchange (ETDEWEB)

    Das, Lalitendu [Biosystems and Agricultural Engineering, University of Kentucky, Lexington, KY (United States); Xu, Siquan [Biosystems and Agricultural Engineering, University of Kentucky, Lexington, KY (United States); College of Chemical Engineering, Nanjing Forestry University, Nanjing (China); Shi, Jian, E-mail: j.shi@uky.edu [Biosystems and Agricultural Engineering, University of Kentucky, Lexington, KY (United States)

    2017-08-10

    Lignin is an integral part of the plant cell wall, which provides rigidity to plants, also contributes to the recalcitrance of the lignocellulosic biomass to biochemical and biological deconstruction. Lignin is a promising renewable feedstock for aromatic chemicals; however, an efficient and economic lignin depolymerization method needs to be developed to enable the conversion. In this study, we investigated the depolymerization of alkaline lignin in aqueous 1-ethyl-3-methylimidazolium acetate [C{sub 2}C{sub 1}Im][OAc] under oxidizing conditions. Seven different transition metal catalysts were screened in presence of H{sub 2}O{sub 2} as oxidizing agent in a batch reactor. CoCl{sub 2} and Nb{sub 2}O{sub 5} proved to be the most effective catalysts in degrading lignin to aromatic compounds. A central composite design was used to optimize the catalyst loading, H{sub 2}O{sub 2} concentration, and temperature for product formation. Results show that lignin was depolymerized, and the major degradation products found in the extracted oil were guaiacol, syringol, vanillin, acetovanillone, and homovanillic acid. Lignin streams were characterized by Fourier transform infrared spectroscopy and gel permeation chromatography to determine effects of the experimental parameters on lignin depolymerization. The weight-average molecular weight (M{sub w}) of liquid stream lignin after oxidation, for CoCl{sub 2} and Nb{sub 2}O{sub 5} catalysts were 1,202 and 1,520 g mol{sup −1}, respectively, lower than that of Kraft lignin. Polydispersity index of the liquid stream lignin increased as compared with Kraft lignin, indicating wide span of the molecular weight distribution as a result of lignin depolymerization. Results from this study provide insights into the role of oxidant and transition metal catalysts and the oxidative degradation reaction sequence of lignin toward product formation in presence of aqueous ionic liquid.

  7. Catalytic Oxidation and Depolymerization of Lignin in Aqueous Ionic Liquid

    Directory of Open Access Journals (Sweden)

    Lalitendu Das

    2017-08-01

    Full Text Available Lignin is an integral part of the plant cell wall, which provides rigidity to plants, also contributes to the recalcitrance of the lignocellulosic biomass to biochemical and biological deconstruction. Lignin is a promising renewable feedstock for aromatic chemicals; however, an efficient and economic lignin depolymerization method needs to be developed to enable the conversion. In this study, we investigated the depolymerization of alkaline lignin in aqueous 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc] under oxidizing conditions. Seven different transition metal catalysts were screened in presence of H2O2 as oxidizing agent in a batch reactor. CoCl2 and Nb2O5 proved to be the most effective catalysts in degrading lignin to aromatic compounds. A central composite design was used to optimize the catalyst loading, H2O2 concentration, and temperature for product formation. Results show that lignin was depolymerized, and the major degradation products found in the extracted oil were guaiacol, syringol, vanillin, acetovanillone, and homovanillic acid. Lignin streams were characterized by Fourier transform infrared spectroscopy and gel permeation chromatography to determine effects of the experimental parameters on lignin depolymerization. The weight-average molecular weight (Mw of liquid stream lignin after oxidation, for CoCl2 and Nb2O5 catalysts were 1,202 and 1,520 g mol−1, respectively, lower than that of Kraft lignin. Polydispersity index of the liquid stream lignin increased as compared with Kraft lignin, indicating wide span of the molecular weight distribution as a result of lignin depolymerization. Results from this study provide insights into the role of oxidant and transition metal catalysts and the oxidative degradation reaction sequence of lignin toward product formation in presence of aqueous ionic liquid.

  8. Nuclear Actin and Myosins in Adenovirus Infection

    Science.gov (United States)

    Fuchsova, Beata; Serebryannyy, Leonid A.; de Lanerolle, Primal

    2015-01-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host’s transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. PMID:26226218

  9. The role of mechanics in actin stress fiber kinetics.

    Science.gov (United States)

    Elson, E L; Genin, G M

    2013-10-01

    The dynamic responses of actin stress fibers within a cell's cytoskeleton are central to the development and maintenance of healthy tissues and organs. Disturbances to these underlie a broad range of pathologies. Because of the importance of these responses, extensive experiments have been conducted in vitro to characterize actin cytoskeleton dynamics of cells cultured upon two-dimensional substrata, and the first experiments have been conducted for cells within three-dimensional tissue models. Three mathematical models exist for predicting the dynamic behaviors observed. Surprisingly, despite differing viewpoints on how actin stress fibers are stabilized or destabilized, all of these models are predictive of a broad range of available experimental data. Coarsely, the models of Kaunas and co-workers adopt a strategy whereby mechanical stretch can hasten the depolymerization actin stress fibers that turn over constantly, while the models of Desphande and co-workers adopt a strategy whereby mechanical stress is required to activate the formation of stress fibers and subsequently stabilize them. In three-dimensional culture, elements of both approaches appear necessary to predict observed phenomena, as embodied by the model of Lee et al. After providing a critical review of existing models, we propose lines of experimentation that might be able to test the different principles underlying their kinetic laws. © 2013 Elsevier Inc. All rights reserved.

  10. Actin filaments regulate the adhesion between the plasma membrane and the cell wall of tobacco guard cells.

    Science.gov (United States)

    Yu, Qin; Ren, Jing-Jing; Kong, Lan-Jing; Wang, Xiu-Ling

    2018-01-01

    During the opening and closing of stomata, guard cells undergo rapid and reversible changes in their volume and shape, which affects the adhesion of the plasma membrane (PM) to the cell wall (CW). The dynamics of actin filaments in guard cells are involved in stomatal movement by regulating structural changes and intracellular signaling. However, it is unclear whether actin dynamics regulate the adhesion of the PM to the CW. In this study, we investigated the relationship between actin dynamics and PM-CW adhesion by the hyperosmotic-induced plasmolysis of tobacco guard cells. We found that actin filaments in guard cells were depolymerized during mannitol-induced plasmolysis. The inhibition of actin dynamics by treatment with latrunculin B or jasplakinolide and the disruption of the adhesion between the PM and the CW by treatment with RGDS peptide (Arg-Gly-Asp-Ser) enhanced guard cell plasmolysis. However, treatment with latrunculin B alleviated the RGDS peptide-induced plasmolysis and endocytosis. Our results reveal that the actin depolymerization is involved in the regulation of the PW-CW adhesion during hyperosmotic-induced plasmolysis in tobacco guard cells.

  11. Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

    Energy Technology Data Exchange (ETDEWEB)

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto (IBS); (BBRI); (UPENN-MED)

    2013-11-20

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  12. Integration of linear and dendritic actin nucleation in Nck-induced actin comets.

    Science.gov (United States)

    Borinskaya, Sofya; Velle, Katrina B; Campellone, Kenneth G; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I; Loew, Leslie M; Mayer, Bruce J

    2016-01-15

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. © 2016 Borinskaya et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

    Science.gov (United States)

    McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

    2016-01-01

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

  14. Piracy of decay-accelerating factor (CD55) signal transduction by the diffusely adhering strain Escherichia coli C1845 promotes cytoskeletal F-actin rearrangements in cultured human intestinal INT407 cells.

    Science.gov (United States)

    Peiffer, I; Servin, A L; Bernet-Camard, M F

    1998-09-01

    Diffusely adhering Escherichia coli (DAEC) C1845 (clinical isolate) harboring the fimbrial adhesin F1845 can infect cultured human differentiated intestinal epithelial cells; this process is followed by the disassembly of the actin network in the apical domain. The aim of this study was to examine the mechanism by which DAEC C1845 promotes F-actin rearrangements. For this purpose, we used a human embryonic intestinal cell line (INT407) expressing the membrane-associated glycosylphosphatidylinositol (GPI) protein-anchored decay-accelerating factor (DAF), the receptor of the F1845 adhesin. We show here that infection of INT407 cells by DAEC C1845 can provoke dramatic F-actin rearrangements without cell entry. Clustering of phosphotyrosines was observed, revealing that the DAEC C1845-DAF interaction involves the recruitment of signal transduction molecules. A pharmacological approach with a subset of inhibitors of signal transduction molecules was used to identify the cascade of signal transduction molecules that are coupled to the DAF, that are activated upon infection, and that promote the F-actin rearrangements. DAEC C1845-induced F-actin rearrangements can be blocked dose dependently by protein tyrosine kinase, phospholipase Cgamma, phosphatidylinositol 3-kinase, protein kinase C, and Ca2+ inhibitors. F-actin rearrangements and blocking by inhibitors were observed after infection of the cells with two E. coli recombinants carrying the plasmids containing the fimbrial adhesin F1845 or the fimbrial hemagglutinin Dr, belonging to the same family of adhesins. These findings show that the DAEC Dr family of pathogens promotes alterations in the intestinal cell cytoskeleton by piracy of the DAF-GPI signal cascade without bacterial cell entry.

  15. Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton.

    Science.gov (United States)

    Marat, Andrea L; Ioannou, Maria S; McPherson, Peter S

    2012-01-01

    The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)-domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1-3/DENND1A-C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

  16. Kinetics of Oxidative Depolymerization of κ-carrageenan by Ozone

    Directory of Open Access Journals (Sweden)

    Aji Prasetyaningrum

    2017-05-01

    Full Text Available Depolymerization kinetics of κ-carrageenan by ozone treatment has been studied at various pHs and times. The purified κ-carrageenan with the initial molecular weight of 271 kDa was dispersed in water to form (1 % w/v solution. Ozone with 80±2 ppm concentration and constant flow rate of 3 L.min-1 was bubbled into the κ-carrageenan solution. The experiments were conducted at pH of 3, 7, and 10 at     different times (5, 10, 15, and 20 minutes of ozonation. The viscosity of the solution was measured   using Ubbelohde capillary viscometer, which was then used to find the number-average molecular weight by Mark-Houwink equation. The number-average molecular weight data was treated using zero, first, and the second-order reaction kinetics model, to obtain the kinetics of κ-carrageenan depolymerization. The depolymerization is assumed to occur by random scission. The results show that the kinetics rate constant of κ-carrageenan depolymerization is higher at lower pHs. The second-order model is more suitable for describing the kinetics of depolymerization of κ-carrageenan by ozonation process. The rate constants for the second-order kinetics model are 5.45×10-4 min-1, 1.27×10-4 min-1, and 7.21×10-5 min-1 for pH 3, 7, and 10, respectively. The actual values of reaction order under acid and    alkali conditions are ranging from 1.88 to 1.90. Copyright © 2017 BCREC Group. All rights reserved. Received: 21st November 2016; Revised: 27th January 2017; Accepted: 18th February 2017 How to Cite: Prasetyaningrum, A., Ratnawati, R., Jos, B. (2017. Kinetics of Oxidative Depolymerization of κ-carrageenan by Ozone. Bulletin of Chemical Reaction Engineering & Catalysis, 12 (2: 235-242 (doi:10.9767/bcrec.12.2.805.235-242 Permalink/DOI: http://dx.doi.org/10.9767/bcrec.12.2.805.235-242

  17. The effect of Cytochalasin D on F-Actin behavior of single-cell electroendocytosis using multi-chamber micro cell chip

    KAUST Repository

    Lin, Ran

    2012-03-01

    Electroendocytosis (EED) is a pulsed-electric-field (PEF) induced endocytosis, facilitating cells uptake molecules through nanometer-sized EED vesicles. We herein investigate the effect of a chemical inhibitor, Cytochalasin D (CD) on the actin-filaments (F-Actin) behavior of single-cell EED. The CD concentration (C CD) can control the depolymerization of F-actin. A multi-chamber micro cell chip was fabricated to study the EED under different conditions. Large-scale single-cell data demonstrated EED highly depends on both electric field and C CD. © 2012 IEEE.

  18. Study of ion beam induced depolymerization using positron annihilation techniques

    Energy Technology Data Exchange (ETDEWEB)

    Puglisi, O. E-mail: opuglisi@dipchi.unict.it; Fragala, M.E.; Lynn, K.G.; Petkov, M.; Weber, M.; Somoza, A.; Dupasquier, A.; Quasso, F

    2001-04-01

    Ion beam induced depolymerization of polymers is a special class of ion beam induced chemical reaction which gives rise to catastrophic 'unzipping' of macromolecules with production of large amounts of the monomer, of the order of many hundreds monomer molecules per each macromolecule. The possible modification of the density at microscopic level prompted us to undertake a study of this effect utilizing positron annihilation techniques in Poly(methylmethacrylate) (PMMA) before and after bombardment with He{sup +} 300 keV ions at 200 deg. C. Preliminary results shown here indicate that before bombardment there is a reproducible dependence of nano-hole distribution on the sample history. Moreover at 200 deg. C we do not detect formation of new cavities as a consequence of the strong depolymerization that occurs under the ion beam. The possible correlation of these findings with transport properties of PMMA at temperature higher than the glass transition temperature will be discussed.

  19. Formic-acid-induced depolymerization of oxidized lignin to aromatics

    Science.gov (United States)

    Rahimi, Alireza; Ulbrich, Arne; Coon, Joshua J.; Stahl, Shannon S.

    2014-11-01

    Lignin is a heterogeneous aromatic biopolymer that accounts for nearly 30% of the organic carbon on Earth and is one of the few renewable sources of aromatic chemicals. As the most recalcitrant of the three components of lignocellulosic biomass (cellulose, hemicellulose and lignin), lignin has been treated as a waste product in the pulp and paper industry, where it is burned to supply energy and recover pulping chemicals in the operation of paper mills. Extraction of higher value from lignin is increasingly recognized as being crucial to the economic viability of integrated biorefineries. Depolymerization is an important starting point for many lignin valorization strategies, because it could generate valuable aromatic chemicals and/or provide a source of low-molecular-mass feedstocks suitable for downstream processing. Commercial precedents show that certain types of lignin (lignosulphonates) may be converted into vanillin and other marketable products, but new technologies are needed to enhance the lignin value chain. The complex, irregular structure of lignin complicates chemical conversion efforts, and known depolymerization methods typically afford ill-defined products in low yields (that is, less than 10-20wt%). Here we describe a method for the depolymerization of oxidized lignin under mild conditions in aqueous formic acid that results in more than 60wt% yield of low-molecular-mass aromatics. We present the discovery of this facile C-O cleavage method, its application to aspen lignin depolymerization, and mechanistic insights into the reaction. The broader implications of these results for lignin conversion and biomass refining are also considered.

  20. Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis

    Directory of Open Access Journals (Sweden)

    Lip Nam Loh

    2017-01-01

    Full Text Available The Gram-positive bacterial cell wall (CW peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2 ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl amiloride (EIPA and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling.

  1. RhoA and RhoC are involved in stromal cell-derived factor-1-induced cell migration by regulating F-actin redistribution and assembly.

    Science.gov (United States)

    Luo, Jixian; Li, Dingyun; Wei, Dan; Wang, Xiaoguang; Wang, Lan; Zeng, Xianlu

    2017-12-01

    Stromal cell-derived factor-1 (SDF-1) signaling is important to the maintenance and progression of T-cell acute lymphoblastic leukemia by inducing chemotaxis migration. To identify the mechanism of SDF-1 signaling in the migration of T-ALL, Jurkat acute lymphoblastic leukemia cells were used. Results showed that SDF-1 induces Jurkat cell migration by F-actin redistribution and assembly, which is dependent on Rho activity. SDF-1 induced RhoA and RhoC activation, as well as reactive oxygen species (ROS) production, which was inhibited by Rho inhibitor. The Rho-dependent ROS production led to subsequent cytoskeleton redistribution and assembly in the process of migration. Additionally, RhoA and RhoC were involved in SDF-1-induced Jurkat cell migration. Taken together, we found a SDF-1/CXCR4-RhoA and RhoC-ROS-cytoskeleton pathway that regulates Jurkat cell migration in response to SDF-1. This work will contribute to a clearer insight into the migration mechanism of acute lymphoblastic leukemia.

  2. Reorganization of the actin cytoskeleton via transcriptional regulation of cytoskeletal/focal adhesion genes by myocardin-related transcription factors (MRTFs/MAL/MKLs)

    International Nuclear Information System (INIS)

    Morita, Tsuyoshi; Mayanagi, Taira; Sobue, Kenji

    2007-01-01

    RhoA is a crucial regulator of stress fiber and focal adhesion formation through the activation of actin nucleation and polymerization. It also regulates the nuclear translocation of myocardin-related transcription factor-A and -B (MRTF-A/B, MAL or MKL 1/2), which are co-activators of serum response factor (SRF). In dominant-negative MRTF-A (DN-MRTF-A)-expressing NIH 3T3 cell lines, the expressions of several cytoskeletal/focal adhesion genes were down-regulated, and the formation of stress fiber and focal adhesion was severely diminished. MRTF-A/B-knockdown cells also exhibited such cytoskeletal defects. In reporter assays, both RhoA and MRTF-A enhanced promoter activities of these genes in a CArG-box-dependent manner, and DN-MRTF-A inhibited the RhoA-mediated activation of these promoters. In dominant-negative RhoA (RhoA-N19)-expressing NIH 3T3 cell lines, the nuclear translocation of MRTF-A/B was predominantly prevented, resulting in the reduced expression of cytoskeletal/focal adhesion proteins. Further, constitutive-active MRTF-A/B increased the expression of endogenous cytoskeletal/focal adhesion proteins, and thereby rescued the defective phenotype of stress fibers and focal adhesions in RhoA-N19 expressing cells. These results indicate that MRTF-A/B act as pivotal mediators of stress fiber and focal adhesion formation via the transcriptional regulation of a subset of cytoskeletal/focal adhesion genes

  3. Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

    Science.gov (United States)

    Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

    2017-01-03

    The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017

  4. ACTIN-DIRECTED TOXIN. ACD toxin-produced actin oligomers poison formin-controlled actin polymerization.

    Science.gov (United States)

    Heisler, David B; Kudryashova, Elena; Grinevich, Dmitry O; Suarez, Cristian; Winkelman, Jonathan D; Birukov, Konstantin G; Kotha, Sainath R; Parinandi, Narasimham L; Vavylonis, Dimitrios; Kovar, David R; Kudryashov, Dmitri S

    2015-07-31

    The actin cross-linking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin cross-linking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here, we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently "poisoned" the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by using actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses. Copyright © 2015, American Association for the Advancement of Science.

  5. Non-enzymatic depolymerization of cotton cellulose by fungal mimicking metabolites

    DEFF Research Database (Denmark)

    Hastrup, Anne Christine Steenkjær; Howell, Caitlin; Jensen, Bo

    2011-01-01

    significant depolymerization effect on cotton cellulose. An oxalic acid/sodium oxalate buffered pH gradient had an inhibitory effect on the reduction of cellulose polymers at increased pH values. The organic iron chelator, EDTA, was found to promote depolymerization of cellulose in combination with Fenton....... The results confirm that low molecular weight metabolites are capable of depolymerizing cellulose and suggest an importance of these mechanisms during incipient decay by brown rot fungi....

  6. Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

    Science.gov (United States)

    Yamamoto, Kazuyoshi; Kiss, John Z.

    2002-01-01

    The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.

  7. Yeast Translation Elongation Factor-1A Binds Vacuole-localized Rho1p to Facilitate Membrane Integrity through F-actin Remodeling*

    Science.gov (United States)

    Bodman, James A. R.; Yang, Yang; Logan, Michael R.; Eitzen, Gary

    2015-01-01

    Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin. PMID:25561732

  8. Axonal Actin Transport Driven By Metastable Actin Filaments

    Science.gov (United States)

    Chakrabarty, Nilaj; Ganguly, Archan; Roy, Subhojit; Jung, Peter

    Actin is one of the key constituents of the neuronal cytoskeleton and is responsible for driving important cellular processes like axon elongation. Axonal actin is synthesized in the cell body and transported at rates of 0.25 - 3 mm/day, as shown by in-vivo pulse-chase radiolabelling studies. However, the underlying transport mechanisms are unknown. Recent experiments in cultured neurons have revealed a dynamic network of metastable actin filaments (actin trails). Actin trails seem to originate from focal actin hotspots which colocalize with stationary endosomes. Interestingly, the number of actin trails extending anterogradely is higher than the ones extending retrogradely. We hypothesize that the bulk axonal transport of actin originates from this directional asymmetry of the number of actin trails. To test this, we constructed a computational model of actin trail growth and simulated the pulse-chase experiment. In our model, local, metastable trails, which grow with their barbed ends anchored to the hotspots, drive the bulk anterograde transport. Our results indicate that the observed bias of the nucleation probabilities and the elongation rate of actin trails are sufficient to drive the bulk transport of actin at rates that agree with in-vivo pulse chase experiments.

  9. Partial depolymerization of enzymolysis lignin via mild hydrogenolysis over Raney Nickel.

    Science.gov (United States)

    Xin, Junna; Zhang, Pei; Wolcott, Michael P; Zhang, Xiao; Zhang, Jinwen

    2014-03-01

    In this work, partial depolymerization of enzymolysis lignin collected from a woody biomass-to-ethanol process was studied via mild hydrogenolysis under the catalysis of Raney Ni. The depolymerized lignin products were low molecular weight oligomers with increased hydroxyl values. The solvent selected, use of base and various reaction parameters were all found to influence yield of depolymerization, the molecular weight and hydroxyl value of the hydrogenated product. The depolymerized lignins displayed greatly enhanced solubility in organic solvents, and therefore would have great potential to be used as feedstock for many valuable thermosetting polymer applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Prokaryotic DNA segregation by an actin-like filament

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Löwe, Jan

    2002-01-01

    The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments...... was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation...... point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus....

  11. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    Science.gov (United States)

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Base-catalyzed depolymerization of lignin : separation of monomers

    Energy Technology Data Exchange (ETDEWEB)

    Vigneault, A. [Sherbrooke Univ., PQ (Canada). Dept. of Chemical Engineering; Johnson, D.K. [National Renewable Energy Laboratory, Golden, CO (United States); Chornet, E. [Sherbrooke Univ., PQ (Canada). Dept. of Chemical Engineering; National Renewable Energy Laboratory, Golden, CO (United States)

    2007-12-15

    Biofuels produced from residual lignocellulosic biomass range from ethanol to biodiesel. The use of lignin for the production of alternate biofuels and green chemicals has been studied with particular emphasis on the structure of lignin and its oxyaromatic nature. In an effort to fractionate lignocellulosic biomass and valorize specific constitutive fractions, the authors developed a strategy for the separation of 12 added value monomers produced during the hydrolytic base catalyzed depolymerization (BCD) of a Steam Exploded Aspen Lignin. The separation strategy was similar to vanillin purification to obtain pure monomers, but combining more steps after the lignin depolymerization such as acidification, batch liquid-liquid-extraction (LLE), followed by vacuum distillation, liquid chromatography (LC) and crystallization. The purpose was to develop basic data for an industrial size process flow diagram, and to evaluate both the monomer losses during the separation and the energy requirements. Experimentally testing of LLE, vacuum distillation and flash LC in the laboratory showed that batch vacuum distillation produced up to 4 fractions. Process simulation revealed that a series of 4 vacuum distillation columns could produce 5 distinct monomer streams, of which 3 require further chromatography and crystallization operations for purification. 22 refs., 4 tabs., 8 figs.

  13. Plastic waste depolymerization as a source of energetic heating oils

    Directory of Open Access Journals (Sweden)

    Wołosiewicz-Głąb Marta

    2017-01-01

    Full Text Available In the past years there has been an increase in production and consumption of plastics, which are widely used in many areas of life. Waste generated from this material are a challenge for the whole of society, regardless of awareness of sustainable development and its technological progress. Still the method of disposal of plastic waste are focused mainly on their storage and incineration, not using energy contained there. In this paper technology for plastic waste depolymerization with characteristics of fuel oil resulting in the process, as an alternative to traditional energy carriers such as: coal, fine coal or coke used in households will be presented. Oil has a high calorific value and no doubt could replace traditional solutions which use conventional energy sources. Furthermore, the fuel resulting from this process is sulfur-free and chemically pure. The paper presents the installation for plastics waste depolymerization used in selected Polish Institute of Plastics Processing, along with the ability to use the main thermocatalytic transformation product.

  14. The Depolymerization of Poly(Ethylene Terephthalate) (PET) Using N-Heterocyclic Carbenes from Ionic Liquids

    Science.gov (United States)

    Kamber, Nahrain E.; Tsujii, Yasuhito; Keets, Kate; Waymouth, Robert M.; Pratt, Russell C.; Nyce, Gregory W.; Hedrick, James L.

    2010-01-01

    The depolymerization of the plastic polyethylene terephthalate (PET or PETE) is described in this laboratory procedure. The transesterification reaction used to depolymerize PET employs a highly efficient N-heterocyclic carbene catalyst derived from a commercially available imidazolium ionic liquid. N-heterocyclic carbenes are potent nucleophilic…

  15. Actin cytoskeleton mediates BMP2-Smad signaling via calponin 1 in preosteoblast under simulated microgravity.

    Science.gov (United States)

    Xu, Hongjie; Wu, Feng; Zhang, Hongyu; Yang, Chao; Li, Kai; Wang, Hailong; Yang, Honghui; Liu, Yue; Ding, Bai; Tan, Yingjun; Yuan, Ming; Li, Yinghui; Dai, Zhongquan

    2017-07-01

    Microgravity influences the activity of osteoblast, induces actin microfilament disruption and leads to bone loss during spaceflight. Mechanical stress such as gravity, regulates cell function, response and differentiation through dynamic cytoskeleton changes, but the mechanotransduction mechanism remains to be fully elucidated. Previous, we demonstrated actin microfilament mediated osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity (SMG). Here, we explored a potential molecular and its detailed mechanism of actin cytoskeleton functioning on BMP2-Smad signaling in MC3T3-E1 under SMG. Results showed that the actin microfilament-disrupting agent, cytochalasin B (CB), reduced BMP2-induced activation, translocation of Smad1/5/8 and Runx2 expression. SMG also inhibited BMP2-Smad signaling, which was rescued by actin cytoskeleton stabilizing agent, Jasplakinolide (JAS). Furthermore, we found that siRNA mediated knockdown of calponin 1 (CNN1), an actin binding protein, markedly promoted BMP2-Smad signaling and abolished both inhibition of CB, SMG on BMP2-Smad signaling and the rescue action of JAS. Overexpression of CNN1 inhibited the p-Smad induced by BMP2. Bidirectional Co-IP experiments demonstrated CNN1 could interacted with Smad or p-Smad protein. Furthermore, CB or SMG decreased the phosphorylated CNN1 and increased its interaction with Smad or p-Smad. Combined with the phosphorylation of CNN1 inhibites its actin binding activity, these results indicate that actin cytoskeleton depolymerization inhibites BMP2 signaling via blocking of Smad by dephosphorylated CNN1 in osteoblast cells. Thus, we provide new important insights into the mechanism of mechanotransduction under SMG condition, which probably contribute to bone formation decrease induced by SMG. Copyright © 2017. Published by Elsevier B.V.

  16. The apical actin fringe contributes to localized cell wall deposition and polarized growth in the lily pollen tube.

    Science.gov (United States)

    Rounds, Caleb M; Hepler, Peter K; Winship, Lawrence J

    2014-09-01

    In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube. © 2014 American Society of Plant Biologists. All Rights Reserved.

  17. Actinic reticuloid. Diagnostics

    Directory of Open Access Journals (Sweden)

    E. V. Sokolovskiy

    2016-01-01

    Full Text Available This article is about the case of actinic reticuloid - the rare dermatosis which clinical presentation is similar to atopic dermatitis, T-cell lymphoma. Good treatment effect was obtained by long cycles (2 cycles for 3 months of hydroxychloroquine and sun protective therapy included sunscreens SPF 50, nicotinic acid, sun-safe clothes which blocked ultraviolet radiation without any glucocorticosteroid drugs and cytostatic treatment.

  18. Preparation of hollow shell ICF targets using a depolymerizing model

    International Nuclear Information System (INIS)

    Letts, S.A.; Fearon, E.M.; Buckley, S.R.

    1994-11-01

    A new technique for producing hollow shell laser fusion capsules was developed that starts with a depolymerizable mandrel. In this technique we use poly(alpha-methylstyrene) (PAMS) beads or shells as mandrels which are overcoated with plasma polymer. The PAMS mandrel is thermally depolymerized to gas phase monomer, which diffuses through the permeable and thermally more stable plasma polymer coating, leaving a hollow shell. We have developed methods for controlling the size of the PAMS mandrel by either grinding to make smaller sizes or melt sintering to form larger mandrels. Sphericity and surface finish are improved by heating the PAMS mandrels in hot water using a surfactant to prevent aggregation. Using this technique we have made shells from 200 μm to 5 mm diameter with 15 to 100 μm wall thickness having sphericity better than 2 μm and surface finish better than 10 nm RMS

  19. Actin acting at the Golgi.

    Science.gov (United States)

    Egea, Gustavo; Serra-Peinado, Carla; Salcedo-Sicilia, Laia; Gutiérrez-Martínez, Enric

    2013-09-01

    The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in trafficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.

  20. Actin-myosin–based contraction is responsible for apoptotic nuclear disintegration

    Science.gov (United States)

    Croft, Daniel R.; Coleman, Mathew L.; Li, Shuixing; Robertson, David; Sullivan, Teresa; Stewart, Colin L.; Olson, Michael F.

    2005-01-01

    Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization. PMID:15657395

  1. Actin Polymerization and ATP Hydrolysis

    Science.gov (United States)

    Korn, Edward D.; Carlier, Marie-France; Pantaloni, Dominique

    1987-10-01

    F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP \\cdot Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP \\cdot Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.

  2. Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP.

    Science.gov (United States)

    Carlier, M F; Criquet, P; Pantaloni, D; Korn, E D

    1986-02-15

    Cytochalasin D strongly inhibits the faster components in the reactions of actin filament depolymerization and elongation in the presence of 10 mM Tris-Cl-, pH 7.8, 0.2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2, and 0.2 mM ATP or ADP. Assuming an exclusive and total capping of the barbed end by the drug, the kinetic parameters derived at saturation by cytochalasin D refer to the pointed end and are 10-15-fold lower than at the barbed end. In ATP, the critical concentration increases with cytochalasin D up to 12-fold its value when both ends are free; as a result of the lowering of the free energy of nucleation by cytochalasin D, short oligomers of F-actin exist just above and below the critical concentration. Cytochalasin D interacts strongly with the barbed ends independently of the ADP-G-actin concentration (K = 0.5 nM-1). In contrast, the affinity of cytochalasin D decreases cooperatively with increasing ATP-G-actin concentration. These data are equally well accounted for by two different models: either cytochalasin D binds very poorly to ATP-capped filament ends whose proportion increases with actin concentration, or cytochalasin D binds equally well to ATP-ends and ADP-ends and also binds to actin dimers in ATP but not in ADP. A linear actin concentration dependence of the rate of growth was found at the pointed end, consistent with the virtual absence of an ATP cap at that end.

  3. Chemoselective Methylation of Phenolic Hydroxyl Group Prevents Quinone Methide Formation and Repolymerization During Lignin Depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kwang Ho; Dutta, Tanmoy; Walter, Eric D.; Isern, Nancy G.; Cort, John R.; Simmons, Blake A.; Singh, Seema

    2017-03-30

    Chemoselective blocking of the phenolic hydroxyl (Ar-OH) group by methylation was found to suppress secondary repolymerization and charring during lignin depolymerization. Methylation of Ar-OH prevents formation of reactive quinone methide intermediates, which are partly responsible for undesirable secondary repolymerization reactions. Instead, this structurally modified lignin produces more relatively low molecular weight products from lignin depolymerization compared to unmodified lignin. This result demonstrates that structural modification of lignin is desirable for production of low molecular weight phenolic products. This approach could be directed toward alteration of natural lignification processes to produce biomass more amenable to chemical depolymerization.

  4. Impact of the fouling mechanism on enzymatic depolymerization of xylan in different configurations of membrane reactors

    DEFF Research Database (Denmark)

    Mohd Sueb, Mohd Shafiq Bin; Luo, Jianquan; Meyer, Anne S.

    2017-01-01

    In order to maximize enzymatic xylan depolymerization while simultaneously purifying the resulting monosaccharide (xylose), different ultrafiltration (UF) membrane reactor configurations were evaluated. Initial results showed that the two hydrolytic enzymes required for complete depolymerization......) and the simultaneous reaction-filtration with both enzymes, respectively. This study thus confirmed that the reactor configuration has a crucial impact on the performance of both the reaction and the separation process of xylose during enzymatic xylan degradation, and that the type of fouling mechanism varies...

  5. Thermal depolymerization of plastics - PDU testing. Task 15. Topical report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-01-01

    The process development unit (PDU) test program is part of an ongoing effort at the Energy & Environmental Research Center (EERC) to expand the base of knowledge for the thermal depolymerization of plastics process. This phase of the development effort, initiated after successful completion of a bench-scale program, has concentrated on maximizing liquid yield. The purposes of the PDU program were (1) to demonstrate the process on a commercially scalable unit, (2) to produce quantities of product that could be used to initiate discussions with potential end users, and (3) to gather engineering and yield data. Experimentation consisted of eleven test points on the PDU and seven on the continuous fluid-bed reactor (CFBR) bench-scale unit. Initial PDU tests (PO35-PO39) were carried out using a base blend, which consists of 60% high-density polyethylene (HDPE), 20% polypropylene (PP), and 20% polystyrene (PS) virgin resin pellets. Test PO39 used base blend with 5% polyvinyl chloride (PVC). The base blend decomposed to produce a flowable liquid, with liquid yields ranging from 33% to 45%. The next series of tests, PO40-PO44, used a postconsumer plastics feed. This material did not decompose as readily as the base blend and formed a very waxy, heavy liquid, with {open_quotes}liquid{close_quotes} yields ranging from 18% to 63% (low liquid yields are the result of using excess air in the natural gas burner in some tests in an attempt to increase gas residence time).

  6. Profilin connects actin assembly with microtubule dynamics

    Czech Academy of Sciences Publication Activity Database

    Nejedla, M.; Sadi, S.; Sulimenko, Vadym; de Almeida, F.N.; Blom, H.; Dráber, Pavel; Aspenstrom, P.; Karlsson, R.

    2016-01-01

    Roč. 27, č. 15 (2016), s. 2381-2393 ISSN 1059-1524 R&D Projects: GA ČR GA16-25159S Institutional support: RVO:68378050 Keywords : cross-linked profilin * arp2/3 complex * f-actin * microfilament system * migrating cell s * focal adhesions * cultured- cell s * messenger-rna * living cell s * protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.685, year: 2016

  7. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  8. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation.

    Science.gov (United States)

    Carruthers, Nicholas J; Stemmer, Paul M; Chen, Ben; Valeriote, Frederick; Gao, Xiaohua; Guatam, Subhash C; Shaw, Jiajiu

    2017-09-15

    UTL-5g is a novel small-molecule TNF-alpha modulator. It reduces cisplatin-induced side effects by protecting kidney, liver, and platelets, thereby increasing tolerance for cisplatin. UTL-5g also reduces radiation-induced acute liver toxicity. The mechanism of action for UTL-5g is not clear at the present time. A phosphoproteomic analysis to a depth of 4943 phosphopeptides and a luminescence-based transcription factor activity assay were used to provide complementary analyses of signaling events that were disrupted by UTL-5g in RAW 264.7 cells. Transcriptional activity downstream of the interferon gamma, IL-6, type 1 Interferon, TGF-β, PKC/Ca 2+ and the glucocorticoid receptor pathways were disrupted by UTL-5g. Phosphoproteomic analysis indicated that hyperphosphorylation of proteins involved in actin remodeling was suppressed by UTL-5g (gene set analysis, FDR 5g. This global characterization of UTL-5g activity in a macrophage cell line discovered that it disrupts selected aspects of LPS signaling including Stat3 activation and actin remodeling providing new insight on how UTL-5g acts to reduce cisplatin-induced side effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. The role of antihistamines in chronic actinic dermatitis treatment

    Directory of Open Access Journals (Sweden)

    E. V. Orlov

    2016-01-01

    Full Text Available Inveterate actinic dermatitis is an immunologically mediated photodermatosis characterized by itchy eczematous dermhelminthiasis exposed to sunlight. The disease proceeds in the same way as the atopic eczema or atopic dermatitis. The treatment of patients with inveterate actinic dermatitis is similar to the treatment of patients with atopic dermatitis and eczema. Administration of the modern antihistaminic preparation desloratadine (Aerius in the treatment has a positive effect on the skin process relief and on some cellular and humoral immunity factors.

  11. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bingyu [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Luo, Qing, E-mail: qing.luo@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Mao, Xinjian [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Xu, Baiyao [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Ju, Yang [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Song, Guanbin, E-mail: song@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-03-10

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the

  12. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

    International Nuclear Information System (INIS)

    Zhang, Bingyu; Luo, Qing; Mao, Xinjian; Xu, Baiyao; Yang, Li; Ju, Yang; Song, Guanbin

    2014-01-01

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the

  13. EZH2-mediated α-actin methylation needs lncRNA TUG1, and promotes the cortex cytoskeleton formation in VSMCs.

    Science.gov (United States)

    Chen, Rong; Kong, Peng; Zhang, Fan; Shu, Ya-Nan; Nie, Xi; Dong, Li-Hua; Lin, Yan-Ling; Xie, Xiao-Li; Zhao, Li-Li; Zhang, Xiang-Jian; Han, Mei

    2017-06-15

    Recent studies have revealed that long non-coding RNAs (lncRNAs) participate in vascular homeostasis and pathophysiological conditions development. But still very few literatures elucidate the regulatory mechanism of non-coding RNAs in this biological process. Here we identified lncRNA taurine up-regulated gene 1 (TUG1) in rat vascular smooth muscle cells (VSMCs), and got 4612bp nucleotide sequence. The expression level of TUG1 RNA was increased in synthetic VSMCs by real-time PCR analysis. Meanwhile, the expression of enhancer of zeste homolog 2 (EZH2) (TUG1 binding protein) increased in cytoplasm of VSMCs under the same conditions. Immunofluoresce analysis displayed the colocalization of EZH2 with α-actin in cytoplasm and F-actin in cell edge ruffles. This leads us to hypothesize the existence of cytoplasmic TUG1/EZH2/α-actin complex. Using RNA pull down assay, we found that TUG1 interacted with both EZH2 and α-actin. Disruption of TUG1 abolished the interaction of EZH2 with α-actin, and accelerated depolymerization of F-actin in VSMCs. Based on EZH2 methyltransferase activity and the potential methylation sites in α-actin structure, we revealed that α-actin was lysine-methylated. Furthermore, the methylation of α-actin was inhibited by knockdown of TUG1. In conclusion, these findings partly suggested that EZH2-mediated methylation of α-actin may be dependent on TUG1, and thereby promotes cortex F-actin polymerization in synthetic VSMCs. Copyright © 2017. Published by Elsevier B.V.

  14. Nuclear actin filaments recruit cofilin and actin-related protein 3, and their formation is connected with a mitotic block

    Czech Academy of Sciences Publication Activity Database

    Kalendová, Alžběta; Kalasová, Ilona; Yamazaki, S.; Uličná, Lívia; Harata, M.; Hozák, Pavel

    2014-01-01

    Roč. 142, č. 2 (2014), s. 139-152 ISSN 0948-6143 R&D Projects: GA ČR GAP305/11/2232; GA MŠk LD12063; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : nuclear actin * transcription * mitosis * actin-related protein 3 * cofilin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.927, year: 2013

  15. Mechanical Properties of Re-constituted Actin Networks at an Oil/Water Interface Determined by Microrheology

    NARCIS (Netherlands)

    Ershov, D.S.; Cohen Stuart, M.A.; Gucht, van der J.

    2012-01-01

    There have been various attempts to investigate the mechanical properties of the actin cortex in cells, but the factors that control them remain poorly understood. To make progress, we develop a reconstituted model of the actin cortex that mimics its structure. We attach actin filaments to lipids

  16. The Apical Actin Fringe Contributes to Localized Cell Wall Deposition and Polarized Growth in the Lily Pollen Tube1[W][OPEN

    Science.gov (United States)

    Rounds, Caleb M.; Hepler, Peter K.; Winship, Lawrence J.

    2014-01-01

    In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube. PMID:25037212

  17. Depolymerization and de-N-acetylation of chitin oligomers in hydrochloric acid.

    Science.gov (United States)

    Einbu, Aslak; Vårum, Kjell M

    2007-01-01

    The monosaccharide 2-amino-2-deoxy-D-glucose (glucosamine, GlcN) has recently drawn much attention in relation to its use to treat or prevent osteoarthritis in humans. Glucosamine is prepared from chitin, a process that is performed in concentrated acid, such as hydrochloric acid. This process involves two acid-catalyzed processes, that is, the hydrolysis of the glycosidic linkages (depolymerization) and of the N-acetyl linkages (de-N-acetylation). The depolymerization reaction has previously been found to be much faster compared to the deacetylation, with the consequence that the chitin chain will first be hydrolyzed to the monomer 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine, GlcNAc) which is subsequently deacetylated. We have found that the chitin disaccharide GlcNAc(1-->4)GlcNAc could be completely hydrolyzed to the monosaccharide GlcNAc with negligible concomitant de-N-acetylation, and the chitin disaccharide and monosaccharide were further used to study the depolymerization reaction and the de-N-acetylation reaction, respectively. The reactions were performed in hydrochloric acid as a function of acid concentration (3-12 M) and temperature (20-35 degrees C), and 1H-NMR spectroscopy was used to monitor the reaction rates. The 1H NMR spectrum of GlcNAc in concentrated (12 M) and deuterated hydrochloric acid at 25 degrees C was assigned. The glucofuranosyl oxazolinium (3) ion was found to exist in equilibrium with the alpha- and beta-anomers of the pyranose form of GlcNAc, where 3 was present in half the total molar concentrations of the two anomeric forms of GlcNAc. At lower acid concentration (3-6 M), only trace concentrations of 3 could be detected. The rate of de-N-acetylation of GlcNAc was determined as a function of hydrochloric acid concentration, showing a maximum at 6 M and decreasing by a factor of 2 upon decreasing or increasing the acid concentration to 3 or 12 M. The activation energy for hydrolysis of the N-acetyl linkage of GlcNAc was

  18. Actin polymerization drives polar growth in Arabidopsis root hair cells.

    Science.gov (United States)

    Vazquez, Luis Alfredo Bañuelos; Sanchez, Rosana; Hernandez-Barrera, Alejandra; Zepeda-Jazo, Isaac; Sánchez, Federico; Quinto, Carmen; Torres, Luis Cárdenas

    2014-01-01

    In plants, the actin cytoskeleton is a prime regulator of cell polarity, growth, and cytoplasmic streaming. Tip growth, as observed in root hairs, caulonema, and pollen tubes, is governed by many factors, including calcium gradients, exocytosis and endocytosis, reactive oxygen species, and the cytoskeleton. Several studies indicate that the polymerization of G-actin into F-actin also contributes to tip growth. The structure and function of F-actin within the apical dome is variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in the elongating apices of tip-growing cells suggests that this cytoskeletal array is tightly regulated. We recently reported that sublethal concentrations of fluorescently labeled cytochalasin could be used to visualize the distribution of microfilament plus ends using fluorescence microscopy, and found that the tip region of the growing root hair cells of a legume plant exhibits a clear response to the nodulation factors secreted by Rhizobium. (1) In this current work, we expanded our analysis using confocal microscopy and demonstrated the existence of highly dynamic fluorescent foci along Arabidopsis root hair cells. Furthermore, we show that the strongest fluorescence signal accumulates in the tip dome of the growing root hair and seems to be in close proximity to the apical plasma membrane. Based on these findings, we propose that actin polymerization within the dome of growing root hair cells regulates polar growth.

  19. Boolean gates on actin filaments

    Energy Technology Data Exchange (ETDEWEB)

    Siccardi, Stefano, E-mail: ssiccardi@2ssas.it [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom); Tuszynski, Jack A., E-mail: jackt@ualberta.ca [Department of Oncology, University of Alberta, Edmonton, Alberta (Canada); Adamatzky, Andrew, E-mail: andrew.adamatzky@uwe.ac.uk [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom)

    2016-01-08

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications. - Highlights: • We simulate interaction between voltage pulses using on actin filaments. • We use a coupled nonlinear transmission line model. • We design Boolean logical gates via interactions between the voltage pulses. • We construct one-bit half-adder with interacting voltage pulses.

  20. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    Science.gov (United States)

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  1. Iron-catalyzed depolymerizations of end-of-life silicones with fatty alcohols

    Directory of Open Access Journals (Sweden)

    Maik Weidauer

    2015-12-01

    Full Text Available During the last decades, polymers became one of the major materials in our society and a future without polymers is hardly imaginable. However, as negative issue of this success enormous amount of end-of-life materials are accumulated, which are mainly treated by landfill storage, thermal recycling or down-cycling. On the other hand, feedstock recycling can be an interesting option to convert end-of-life polymers to high quality polymers, via depolymerization reactions to low-molecular weight building blocks and subsequent transformation via polymerization reactions. In this regard, we present herein the depolymerization of polysiloxanes (silicones applying fatty alcohols as depolymerization reagents. In more detail, in the presence of catalytic amounts of simple iron salts, low-molecular weight products with the motif R(OSiMe2mOR (R = alkyl, m = 1–2 were attained. Remarkably, the reaction of R(OSiMe2mOR with water showed the formation of new cyclic siloxanes, which are useful starting materials for long-chain silicones, and the corresponding fatty alcohol as side product, which can be directly reused in subsequent depolymerization reactions. Importantly, a recycling of the silicones and a straightforward recycling of the depolymerization reagent are feasible.

  2. RhoA/Rho kinase signaling regulates transforming growth factor-β1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway.

    Science.gov (United States)

    Xu, Ting; Wu, Mengjie; Feng, Jianying; Lin, Xinping; Gu, Zhiyuan

    2012-11-01

    Recent studies have suggested that synovium-derived mesenchymal stem cells (SMSCs) may be promising candidates for tissue engineering and play an important role in cartilage regeneration. However, the mechanisms of SMSC chondrogenesis remain to be identified and characterized. The aim of this study was to evaluate the activation of the RhoA/Rho kinase (ROCK) pathway, as well as the manner by which it may contribute to chondrogenesis and the actin cytoskeletal organization of rat temporomandibular SMSCs in response to transforming growth factor-β1 (TGF-β1). Primary isolated SMSCs were treated with TGF-β1, and their actin organization was examined by fluorescein isothiocyanate-phalloidin staining. The specific biochemical inhibitors, C3 transferase, Y27632 and SB431542, were employed to evaluate the function of RhoA/ROCK and Smads. The effect of C3 transferase and Y27632 on the gene expression of chondrocyte-specific markers was evaluated by quantitative real-time polymerase chain reaction. To examine the effect of Y27632 on Smad2/3 phosphorylation induced by TGF-β1, western blot analysis was also performed. The stimulation of TGF-β1 in SMSCs resulted in the activation of the RhoA/ROCK pathway and concomitantly induced cytoskeletal reorganization, which was specifically blocked by C3 transferase and Y27632. The TGF-β-induced gene expression of Sox9, type I collagen, type II collagen and aggrecan was also inhibited by both C3 transferase and Y27632, at different levels. Y27632 treatment reduced the phosphorylation of Smad2/3 in a concentration-dependent manner. These results demonstrate the RhoA/ROCK activation regulates chondrocyte-specific gene transcription and cytoskeletal organization induced by TGF-β1 by interacting with the Smad pathway. This may have significant implications for the successful utilization of SMSCs as a cell source for articular cartilage tissue engineering.

  3. The effect of membrane-regulated actin polymerization on a two-phase flow model for cell motility

    KAUST Repository

    Kimpton, L. S.

    2014-07-23

    Two-phase flow models have been widely used to model cell motility and we have previously demonstrated that even the simplest, stripped-down, 1D model displays many observed features of cell motility [Kimpton, L.S., Whiteley, J.P., Waters, S.L., King, J.R. & Oliver, J.M. (2013) Multiple travelling-wave solutions in a minimal model for cell motility. Math. Med. Biol. 30, 241 - 272]. In this paper, we address a limitation of the previous model.We show that the two-phase flow framework can exhibit travelling-wave solutions with biologically plausible actin network profiles in two simple models that enforce polymerization or depolymerization of the actin network at the ends of the travelling, 1D strip of cytoplasm. © 2014 The authors 2014. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.

  4. Improved resistance of chemically-modified nanocellulose against thermally-induced depolymerization.

    Science.gov (United States)

    Agustin, Melissa B; Nakatsubo, Fumiaki; Yano, Hiroyuki

    2017-05-15

    The study demonstrated the improvement in the resistance of nanocellulose against thermally-induced depolymerization by esterification with benzoyl (BNZ) and pivaloyl (PIV). The change in the degree of polymerization (DP) and molecular weight distribution (MWD) after thermal treatment in nitrogen and in air was investigated using viscometry and gel permeation chromatography. BNZ and PIV nanocellulose esters without α-hydrogens gave higher DP and narrower MWD than pure bacterial cellulose; and the acetyl and myristoyl esters, which possess α-hydrogens. Results also showed that when depolymerization is suppressed, thermal discoloration is also reduced. Resistance against depolymerization inhibits the formation of reducing ends which can be active sites for thermal discoloration. Finally, the findings suggest that benzoylation and pivaloylation can be an excellent modification technique to improve the thermal stability of nanocellulose. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Disruption of actin filaments induces mitochondrial Ca2+ release to the cytoplasm and [Ca2+]c changes in Arabidopsis root hairs

    Directory of Open Access Journals (Sweden)

    Baluška František

    2010-03-01

    Full Text Available Abstract Background Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca2+ storage. Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca2+ storage, cytoplasmic Ca2+ concentration ([Ca2+]c, and the interaction between mitochondrial Ca2+ and cytoplasmic Ca2+ in Arabidopsis root hairs. Results In this study, we found that treatments with latrunculin B (Lat-B and jasplakinolide (Jas, which depolymerize and polymerize actin filaments respectively, decreased membrane potential and Ca2+ stores in the mitochondria of Arabidopsis root hairs. Simultaneously, these treatments induced an instantaneous increase of cytoplasmic Ca2+, followed by a continuous decrease. All of these effects were inhibited by pretreatment with cyclosporin A (Cs A, a representative blocker of the mitochondrial permeability transition pore (mPTP. Moreover, we found there was a Ca2+ concentration gradient in mitochondria from the tip to the base of the root hair, and this gradient could be disrupted by actin-acting drugs. Conclusions Based on these results, we concluded that the disruption of actin filaments caused by Lat-B or Jas promoted irreversible opening of the mPTP, resulting in mitochondrial Ca2+ release into the cytoplasm, and consequent changes in [Ca2+]c. We suggest that normal polymerization and depolymerization of actin filaments are essential for mitochondrial Ca2+ storage in root hairs.

  6. Boolean gates on actin filaments

    Science.gov (United States)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  7. Kinematic Modeling of Mechanocatalytic Depolymerization of α-Cellulose and Beechwood.

    Science.gov (United States)

    Kessler, Martin; Woodward, Robert T; Wong, Narumi; Rinaldi, Roberto

    2018-02-09

    Mechanocatalytic depolymerization of lignocellulose presents a promising method for the solid-state transformation of acidified raw biomass into water-soluble products (WSPs). However, the mechanisms underlining the utilization of mechanical forces in the depolymerization are poorly understood. A kinematic model of the milling process is applied to assess the energy dose transferred to cellulose during its mechanocatalytic depolymerization under varied conditions (rotational speed, milling time, ball size, and substrate loading). The data set is compared to the apparent energy dose calculated from the kinematic model and reveals key features of the mechanocatalytic process. At low energy doses, a rapid rise in the WSP yield associated with the apparent energy dose is observed. However, at a higher energy dose obtained by extended milling duration or high milling speeds, the formation of a substrate cake layer on the mill vials appear to buffer the mechanical forces, preventing full cellulose conversion into WSPs. By contrast, for beechwood, there exists a good linear dependence between the WSP yield and the energy dose provided to the substrate over the entire range of WSP yields. As the formation of a substrate cake in depolymerization of beechwood is less severe than that for the cellulose experiments, the current results verify the hypothesis regarding the negative effect of a substrate layer formed on the mill vials upon the depolymerization process. Overall, the current findings provide valuable insight into relationships between the energy dose and the extent of cellulose depolymerization effected by the mechanocatalytic process. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cooperation, competition, and coalitions in enzyme-producing microbes: Social evolution and nutrient depolymerization rates

    Directory of Open Access Journals (Sweden)

    Henry Joseph Folse

    2012-09-01

    Full Text Available Extracellular enzymes represent a public good for microbial communities, as they break down complex molecules into simple molecules that microbes can take up. These communities are vulnerable to cheating by microbes that do not produce enzymes, but benefit from those produced by others. However, extracellular enzymes are ubiquitous and play an important role in the depolymerization of nutrients. We developed a multi-genotype, multi-nutrient model of a community of exoenzyme-producing microbes, in order to investigate the relationship between diversity, social interactions, and nutrient depolymerization. We focused on coalitions between complementary types of microbes and their implications for spatial pattern formation and nutrient depolymerization. The model included polymers containing carbon, nitrogen, or phosphorus, and eight genotypes of bacteria, which produced different subsets of the three enzymes responsible for hydrolyzing these polymers. We allowed social dynamics to emerge from a mechanistic model of enzyme production, action, and diffusion. We found that diversity was maximized at high rates of either diffusion or enzyme production (but not both. Conditions favoring cheating also favored the emergence of coalitions. We characterized the spatial patterns formed by different interactions, showing that same-type cooperation leads to aggregation, but between-type cooperation leads to an interwoven, filamentous pattern. Contrary to expectations based on niche complementarity, we found that nutrient depolymerization declined with increasing diversity due to a negative competitive effect of coalitions on generalist producers, leading to less overall enzyme production. This decline in depolymerization was stronger for non-limiting nutrients in the system. This study shows that social interactions among microbes foraging for complementary resources can influence microbial diversity, microbial spatial distributions, and rates of nutrient

  9. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  10. Ultrastructural localization of actin and actin-binding proteins in the nucleus

    Czech Academy of Sciences Publication Activity Database

    Dingová, Hana; Fukalová, Jana; Maninová, Miloslava; Philimonenko, Vlada; Hozák, Pavel

    2009-01-01

    Roč. 131, č. 3 (2009), s. 425-434 ISSN 0948-6143 R&D Projects: GA MŠk LC545 Grant - others:MŠk(CZ) LC06063 Program:LC Institutional research plan: CEZ:AV0Z50520514 Keywords : nuclear actin * ultrastructure * actin–binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.021, year: 2009

  11. Influence of the degree of crosslinking on the depolymerization of disulfide polymer

    International Nuclear Information System (INIS)

    Rekalicj, J.V.; Radosavljevicj, D.S.; Popovicj, E.M.; Stashicj, L.

    1976-01-01

    The action of nucleophilic reagents (hydrogen sulfide ion, dithionite ion and hydrazine) on disulfide polymers prepd. from bis-2-chloroethyl formal and 1,2,3-trichloropropane, taken in various mol rations is studied. The depolymerization efficiency is higher with hydrazine and dithionite than with a mixt. of sodium hydrogen sulfide and sodium sulfite. An interpretation of the results is given, attempting to correlate the content of SH-groups in the obtained product with the same quantity in some defined compds. which can be present after the depolymerization

  12. Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

    Science.gov (United States)

    Borgstahl, G.; Lovelace, J.; Snell, E. H.; Bellamy, H.

    2003-01-01

    microfilament system to be restructured in a controlled manner via polymerization, depolymerization, severing, cross-linking, and anchorage. The structure the semicrystalline state of profilin:actin will challenge and validate current models of muscle contraction and cell motility. The methodology and theory under development will be easily extendable to other systems.

  13. Actin microfilament dynamics in locomoting cells

    Science.gov (United States)

    Theriot, Julie A.; Mitchison, Timothy J.

    1991-07-01

    The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge.

  14. Regulation of α-Smooth Muscle Actin Expression in Granulation Tissue Myofibroblasts Is Dependent on the Intronic CArG Element and the Transforming Growth Factor-β1 Control Element

    Science.gov (United States)

    Tomasek, James J.; McRae, Joel; Owens, Gary K.; Haaksma, Carol J.

    2005-01-01

    Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of α-smooth muscle actin (α-SMA); however, little is known regarding molecular mechanisms that control activation of α-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for α-SMA expression in myofibroblasts in vivo and to determine whether activation of α-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-β1 control element (TCE) that are required for α-SMA expression in smooth muscle cells. A Lac Z transgene construct from −2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous α-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-β1 in cultured transgenic fibroblasts. These same elements were also critical in regulating α-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of α-SMA expression in myofibroblasts in granulation tissue. PMID:15855636

  15. Actin' as a Death Signal

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.

    2012-01-01

    Cell death needs to be detected by immune cells. In this issue of Immunity, Ahrens et al. (2012) and Zhang et al. (2012) show that actin filaments become exposed on necrotic cells and act as ligands for the C-type lectin receptor Clec9a

  16. Depolymerization of organosolv lignin using doped porous metal oxides in supercritical methanol

    NARCIS (Netherlands)

    Warner, Genoa; Hansen, Thomas S; Riisager, Anders; Beach, Evan S; Barta, Katalin; Anastas, Paul T

    An isolated, solvent-extracted lignin from candlenut (Aleurites moluccana) biomass was subjected to catalytic depolymerization in the presence of supercritical methanol, using a range of porous metal oxides derived from hydrotalcite-like precursors. The most effective catalysts in terms of lignin

  17. Mechanism of Catalytic Microtubule Depolymerization via KIF2-Tubulin Transitional Conformation

    Directory of Open Access Journals (Sweden)

    Tadayuki Ogawa

    2017-09-01

    Full Text Available Microtubules (MTs are dynamic structures that are fundamental for cell morphogenesis and motility. MT-associated motors work efficiently to perform their functions. Unlike other motile kinesins, KIF2 catalytically depolymerizes MTs from the peeled protofilament end during ATP hydrolysis. However, the detailed mechanism by which KIF2 drives processive MT depolymerization remains unknown. To elucidate the catalytic mechanism, the transitional KIF2-tubulin complex during MT depolymerization was analyzed through multiple methods, including atomic force microscopy, size-exclusion chromatography, multi-angle light scattering, small-angle X-ray scattering, analytical ultracentrifugation, and mass spectrometry. The analyses outlined the conformation in which one KIF2core domain binds tightly to two tubulin dimers in the middle pre-hydrolysis state during ATP hydrolysis, a process critical for catalytic MT depolymerization. The X-ray crystallographic structure of the KIF2core domain displays the activated conformation that sustains the large KIF2-tubulin 1:2 complex.

  18. Lewis-acid catalyzed depolymerization of Protobind lignin in supercritical water and ethanol

    NARCIS (Netherlands)

    Guvenatam, Burcu; Heeres, Erik H.J.; Pidko, Evgeny A.; Hensen, Ernie J. M.

    2016-01-01

    The use of metal acetates, metal chlorides and metal triflates as Lewis acid catalysts for the depolymerization of soda lignin under supercritical conditions was investigated. The reactions were carried out at 400 degrees C in water and ethanol. Lignin conversion in supercritical water led to

  19. Lewis acid-catalyzed depolymerization of soda lignin in supercritical ethanol/water mixtures

    NARCIS (Netherlands)

    Güvenatam, Burcu; Heeres, Erik H.J.; Pidko, Evgeny A.; Hensen, Emiel J M

    2016-01-01

    The depolymerization of lignin model compounds and soda lignin by super Lewis acidic metal triflates has been investigated in a mixture of ethanol and water at 400 °C. The strong Lewis acids convert representative model compounds for the structure-forming linkages in lignin, namely α-O-4, 5-O-4

  20. Depolymerization of lignin via co-pyrolysis with 1,4-butanediol in a microwave reactor

    Science.gov (United States)

    The production of valuable compounds from low cost but abundant residual lignin has proven to be challenging. The lack of effective biochemical lignin depolymerization processes has led many to focus on thermochemical conversion methods. Bench scale microwave pyrolysis of lignin has been performed...

  1. Depolymerized glycosaminoglycan and its anticoagulant activities from sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Yang, Jie; Wang, Yuanhong; Jiang, Tingfu; Lv, Lv; Zhang, Boyuan; Lv, Zhihua

    2015-01-01

    A controlled Cu(2+) catalytic free-radical depolymerization process of fucosylated chondroitin sulfate from sea cucumber Apostichopus japonicus was established. The results showed a good linear relationship between 1/Mw and time during the depolymerization. A series of fractions with different molecular weight were obtained, and the physicochemical properties of them were investigated and compared utilizing the chemical method, IR spectra and NMR spectra. The results showed no significant variations of the backbone and branches structures during the depolymerization. Furthermore, the anticoagulant activities of the depolymerized fractions were evaluated by the activated partial thromboplastin time (APTT). The APTT decreases in proportion to the molecular weight following a linear relationship and the prolongation of APTT activity requires at least oligosaccharide of 4 trisaccharide units (about 4000 Da). Their anticoagulant activity of low molecular weight fraction (Mw = 24,755 Da) is similar to LMWH with significantly less bleeding risk. The results suggest that the low molecular weight fraction could be used as a novel anticoagulant with less undesired side effects. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Optimization and kinetic analysis on the sulfuric acid - Catalyzed depolymerization of wheat straw.

    Science.gov (United States)

    Wu, Qian-Qian; Ma, Yu-Long; Chang, Xuan; Sun, Yong-Gang

    2015-09-20

    The objectives of this work were to optimize the experimental condition and to study the kinetic behavior of wheat straw depolymerization with sulfuric acid (2 wt%, 3 wt%, and 4 wt%) at different temperatures (120°C, 130°C, and 140°C). The two-fraction kinetic model was obtained for the prediction of the generations of product and by-product during depolymerization. The kinetic parameters of the two-fraction model were analyzed using an Arrhenius-type equation. Applying the kinetic two-fraction model, the optimum condition for wheat straw depolymerization was 3 wt% H2SO4 at 130°C for 75 min, which yielded a high concentration of fermentable sugars (xylose 8.934 g/L, glucose 1.363 g/L, and arabinose 1.203 g/L) and low concentrations of microbial inhibitors (furfural 0.526 g/L and acetic acid 1.192 g/L). These results suggest that the model obtained in this study can satisfactorily describe the formation of degradation products and the depolymerization mechanism of wheat straw. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. When fat is not bad: the regulation of actin dynamics by phospholipid signaling molecules

    Czech Academy of Sciences Publication Activity Database

    Pleskot, Roman; Pejchar, Přemysl; Staiger, Ch. J.; Potocký, Martin

    2014-01-01

    Roč. 5, JAN 2014 (2014) ISSN 1664-462X R&D Projects: GA ČR GA13-19073S Institutional support: RVO:61389030 Keywords : actin * actin-binding proteins * capping protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.948, year: 2014

  4. Sites of actin filament initiation and reorganization in cold-treated tobacco cells

    Czech Academy of Sciences Publication Activity Database

    Pokorná, J.; Schwarzerová, K.; Zelenková, S.; Petrášek, Jan; Janotová, I.; Čapková, Věra; Opatrný, Z.

    2004-01-01

    Roč. 27, č. 5 (2004), s. 641-653 ISSN 0140-7791 R&D Projects: GA AV ČR IAA5038207 Institutional research plan: CEZ:AV0Z5038910 Keywords : Nicotiana tabacum * actin * actin filaments Subject RIV: EF - Botanics Impact factor: 3.634, year: 2004

  5. Oral acetylsalicylic acid and prevalence of actinic keratosis

    Directory of Open Access Journals (Sweden)

    Juliano Schmitt

    2014-01-01

    Full Text Available Objective: To investigate the influence of a regular oral use of acetylsalicylic acid in the prevalence of actinic keratosis. Methods: A case-control study with dermatologic outpatients above 50 years of age assessed between 2009 and 2011. Cases were defined as those who had been under regular use of oral acetylsalicylic acid for more than six consecutive months. The assessment focused on: age, sex, skin-type, tobacco smoking, use of medication, occurrence of individual or family skin cancer, and sunscreen and sun exposure habits. Actinic keratoses were counted in the medial region of the face and upper limbs. Counts were adjusted by co-variables based on a generalized linear model. Results: A total of 74 cases and 216 controls were assessed. The median time of acetylsalicylic acid use was 36 months. Cases differed from controls as to the highest age, highest prevalence of use of angiotensin-converting enzyme inhibitors and fewer keratosis on the face and on the upper limbs (p<0.05. The multivariate model showed that the use of acetylsalicylic acid was associated to lower counts of face actinic keratosis and upper-limb erythematous actinic keratosis (p<0.05, regardless of other risk factors. Conclusion: The regular use of oral acetylsalicylic acid for more than six months was associated to a lower prevalence of actinic keratosis, especially facial and erythematous ones.

  6. Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization.

    Science.gov (United States)

    Domínguez-Iturza, Nuria; Calvo, María; Benoist, Marion; Esteban, José Antonio; Morales, Miguel

    2016-01-01

    Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine.

  7. Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Pedersen, Line Hjortshøj; Byg, Luise

    2010-01-01

    Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin d...... dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton....

  8. Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus

    DEFF Research Database (Denmark)

    Malmström, Johan; Lindberg, Henrik Have; Lindberg, Claes

    2004-01-01

    is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-beta(1) induces not only alpha-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced...

  9. Rpg1p/Tif32p, a subunit of translation initiation factor 3, interacts with actin-associated protein Sla2p

    Czech Academy of Sciences Publication Activity Database

    Paleček, Jan; Hašek, Jiří; Ruis, H.

    2001-01-01

    Roč. 282, č. 5 (2001), s. 1244-1250 ISSN 0006-291X R&D Projects: GA ČR GA204/99/1531 Institutional research plan: CEZ:AV0Z5004920 Keywords : yeast Saccharomyces cerevisiae * two-hybrid system * coimmunoprecipitation Subject RIV: BO - Biophysics Impact factor: 2.946, year: 2001

  10. Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

    Directory of Open Access Journals (Sweden)

    Jan Mueller

    2014-01-01

    Full Text Available Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

  11. Fragmentation of Human Erythrocyte Actin following Exposure to Hypoxia

    Czech Academy of Sciences Publication Activity Database

    Risso, A.; Santamaria, B.; Pistarino, E.; Cosulich, M. E.; Pompach, Petr; Bezouška, Karel; Antonutto, G.

    2010-01-01

    Roč. 123, č. 1 (2010), s. 6-13 ISSN 0001-5792 Institutional research plan: CEZ:AV0Z50200510 Keywords : beta-Actin * Erythrocytes * Hypoxia Subject RIV: EE - Microbiology, Virology Impact factor: 1.316, year: 2010

  12. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  13. The actin multigene family of Paramecium tetraurelia

    Directory of Open Access Journals (Sweden)

    Wagner Erika

    2007-03-01

    Full Text Available Abstract Background A Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium. Results The ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic

  14. Whole animal knockout of smooth muscle alpha-actin does not alter excisional wound healing or the fibroblast-to-myofibroblast transition.

    Science.gov (United States)

    Tomasek, James J; Haaksma, Carol J; Schwartz, Robert J; Howard, Eric W

    2013-01-01

    The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts. © 2012 by the Wound Healing Society.

  15. Rapid room temperature solubilization and depolymerization of polymeric lignin at high loadings

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jian; Dutta, Tanmoy; Parthasarathi, Ramakrishnan; Kim, Kwang Ho; Tolic, Nikola; Chu, Rosalie K.; Isern, Nancy G.; Cort, John R.; Simmons, Blake A.; Singh, Seema

    2016-01-01

    The relatively poor solubility of lignin in most pretreatment solvents remains one of the biggest challegnes in lignin valorization to improve overall biorefinery economics. In this work, rapid room temperature solubilization of lignin at high solid loadings (>30 wt%) can be easily achieved in a single step using ethylene glycol (EG). The solubilized lignin can be rapidly and quantitively recovered with the addtion of ethanol. The computational and nuclear magnetic resonance (NMR) spectroscopic studies confirm that strong hydrogen bond interactions between EG and the free hydroxyl groups present in lignin contribute to the lignin dissolution. In addition, hydrogen peroxide mediated depolymerization of dissolved lignin at low temperature (80 oC) was tested and the effect of EG molecules on depolymerization of ligin was also theoritically studied. The findings of this work provide mechanistic insights of hydrogen bond interactions in high lignin solubilization and valorization.

  16. Selective and recyclable depolymerization of cellulose to levulinic acid catalyzed by acidic ionic liquid.

    Science.gov (United States)

    Ren, Huifang; Girisuta, Buana; Zhou, Yonggui; Liu, Li

    2015-03-06

    Cellulose depolymerization to levulinic acid (LA) was catalyzed by acidic ionic liquids (ILs) selectively and recyclably under hydrothermal conditions. The effects of reaction temperature, time, water amount and cellulose intake were investigated. Dilution effect becomes more pronounced at lower cellulose intake, dramatically improving the yield of LA to 86.1%. A kinetic model has been developed based on experimental data, whereby a good fit was obtained and kinetic parameters were derived. The relationships between IL structure, polymeric structure and depolymerization efficiency were established, shedding light on the in-depth catalytic mechanism of IL, inclusive of acidity and hydrogen bonding ability. The LA product can be readily separated through extraction by methyl isobutyl ketone (MIBK) and IL can be reused over five cycles without loss of activity. This environmentally friendly methodology can be applied to selective production of LA from versatile biomass feedstocks, including cellulose and derivatives, glucose, fructose and HMF. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Zhang, Zhenghong

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-...

  18. Mechanosensitive kinetic preference of actin-binding protein to actin filament.

    Science.gov (United States)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

  19. NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.

    Science.gov (United States)

    Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi

    2016-02-01

    The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.

  20. Actin organization and dynamics in filamentous fungi.

    Science.gov (United States)

    Berepiki, Adokiye; Lichius, Alexander; Read, Nick D

    2011-11-02

    Growth and morphogenesis of filamentous fungi is underpinned by dynamic reorganization and polarization of the actin cytoskeleton. Actin has crucial roles in exocytosis, endocytosis, organelle movement and cytokinesis in fungi, and these processes are coupled to the production of distinct higher-order structures (actin patches, cables and rings) that generate forces or serve as tracks for intracellular transport. New approaches for imaging actin in living cells are revealing important similarities and differences in actin architecture and organization within the fungal kingdom, and have yielded key insights into cell polarity, tip growth and long-distance intracellular transport. In this Review, we discuss the contribution that recent live-cell imaging and mutational studies have made to our understanding of the dynamics and regulation of actin in filamentous fungi.

  1. Selective Depolymerization and Effects of Homolysis of Poly(L-lactic acid) in a Blend with Polypropylene

    OpenAIRE

    Nishida, Haruo; Arazoe, Yoshiro; Tsukegi, Takayuki; Yan, Wang; Shirai, Yoshihito

    2009-01-01

    Blends of poly(L-lactic acid) (PLLA) and polypropylene (PP), which are candidates for the practical use of PLLA, were investigated for selective degradation of PLLA, resulting in quantitative conversion of PLLA components into cyclic monomers, lactides, using magnesium oxide (MgO) as a depolymerization catalyst. Obviously, the catalyst MgO selectively accelerated only the PLLA depolymerization in the blends, dominantly generating L,L-lactide as a volatile product and separating the PP compone...

  2. Fermentation Process of Cocoa Based on Optimum Condition of Pulp PectinDepolymerization by Endogenous Pectolityc Enzymes

    Directory of Open Access Journals (Sweden)

    G.P. Ganda-Putra

    2010-08-01

    Full Text Available Pulp degradation during cocoa fermentation can be carried out by depolymerization process of pulp pectin using endogenous pectolytic enzymes at optimum condition. The objectives of this research were to study the effect of fermentation process based on optimum condition in terms of temperature and pH of pulp pectin depolymerization using endogenous pectolytic enzymes polygalakturonase (PG and pectin metyl esterase (PME and fermentation period in cocoa processing on quality characteristics of cocoa beans produced and to study the role of those fermentation process in reducing fermentation time to produce cocoa beans with standard quality. This research used split plot design, with treatments of process condition of cocoa fermentation as main plot and fermentation period as split plot. Treatment of process condition of cocoa fermentation consisted of optimum condition for pulp pectin depolymerization by PGs (temperature 47.5OC; initial pulp pH 4.6; optimum condition of depolymerization on sequence depolymerization by PGs (temperature 48.5OC; initial pulp pH 8.0 during 1 day; last temperature 47.5OC; initial pulp pH 4.6 during 6 days, and natural fermentation process a control. While treatment of fermentation period consisted of 0, 1, 2, 3, 4, 5, 6 and 7 days. Evaluation of fermentation period was carried out based on pursuant to criteria of unfermented beans content and fermentation index. The results showed that process condition and fermentation time of cocoa affected quality characteristic of cocoa beans produced. Period of cocoa fermentation process based on optimum condition for pulp pectin depolymerization using endogenous pectolytic enzymes was 2 days shorter compared to natural fermentation. Cocoa beans quality of grade I and II were obtained from fermentation time of 4 and 2 days, respectively, using fermentation process based on optimum condition of pulp pectin depolymerization using endogenous pectolytic enzymes, whereas 6 and 4 days

  3. Fermentation Process of Cocoa Based on Optimum Condition of Pulp PectinDepolymerization by Endogenous Pectolityc Enzymes

    OpenAIRE

    Ganda-Putra, G.P; Wrasiati, L.P; Wartini, N.M

    2010-01-01

    Pulp degradation during cocoa fermentation can be carried out by depolymerization process of pulp pectin using endogenous pectolytic enzymes at optimum condition. The objectives of this research were to study the effect of fermentation process based on optimum condition in terms of temperature and pH of pulp pectin depolymerization using endogenous pectolytic enzymes polygalakturonase (PG) and pectin metyl esterase (PME) and fermentation period in cocoa processing on quality characteristics o...

  4. Depolymerization of coal by oxidation and alkylation; Sanka bunkai to alkyl ka ni yoru sekitan kaijugo

    Energy Technology Data Exchange (ETDEWEB)

    Tomita, H.; Isoda, T.; Kusakabe, K.; Morooka, S. [Kyushu University, Fukuoka (Japan). Faculty of Engineering; Hayashi, J. [Hokkaido University, Sapporo (Japan). Center for Advanced Research of Energy Technology

    1996-10-28

    Change in depolymerization degree and coal structure was studied for depolymerization treatment of coal in various alcohol containing aqueous hydrogen peroxide. In experiment, the mixture of Yallourn coal, alcohol and aqueous hydrogen peroxide was agitated in nitrogen atmosphere of normal pressure at 70{degree}C for 12 hours. As the experimental result, the methanol solubility of only 5% of raw coal increased up to 35.2% by hydrogen peroxide treatment, while the yield of insoluble matters also decreased from 94% to 62%. Most of the gas produced during treatment was composed of inorganic gases such as CO and CO2, and its carbon loss was extremely decreased by adding alcohol. From the analytical result of carbon loss in hydrogen peroxide treatment, it was clarified that alkylation advances with introduction of alkyl group derived from alcohol into coal by hydrogen peroxide treatment under a coexistence of alcohol, and depolymerization reaction of coal itself is thus promoted by alcohol. 4 refs., 7 figs., 1 tab.

  5. Improved postharvest quality in patagonian squash (Cucurbita moschata) coated with radiation depolymerized chitosan

    International Nuclear Information System (INIS)

    Pugliese, Maria Alicia; Goitia, Maria Teresa; Yossen, Mariana; Cifone, Norma; Agullo, Enrique; Andreucetti, Noemi

    2011-01-01

    Different molecular weight chitosans were evaluated on the decay of coated Anquito squashes (Cucurbita moschata) as well as the maintenance of the fruit quality along five storage months. The original chitosan (Mw=391 kDa, 83% DD), was depolymerized by gamma radiation. Apart from chain scission, other chemical changes were not detected by FTIR or UV-vis analyses. The molecular weight characterization of chitosans was done by size exclusion chromatography with dual light scattering and concentration detection (SEC-MALLS-RI). The coating effectiveness was evaluated on the following parameters: fungal decay incidence, weight loss, firmness, total reducing sugar, soluble solid, flesh color, carotene content, pH and titratable acidity. No sign of fungal decay was observed in squashes coated with 122 and 56 kDa chitosans, which were also the most effective treatments in reducing the weight loss. The chitosan with Mw=122 kDa was also the best treatment considering firmness, internal aspect, sugar and carotene content. Then, radiation degraded chitosan was better in C. moschata preservation than the original chitosan. - Highlights: → Original Chitosan was radiation depolymerized producing chitosans with lower molecular weights. → Gamma-irradiated chitosans only exhibit chain scission. → SEC-MALLS-RI chromatography is a useful tool in molecular weight analysis. → Depolymerized chitosans were the best in maintaining the quality and the storage life of coated squashes.

  6. Conformational and dynamic differences between actin filaments polymerized from ATP- or ADP-actin monomers.

    Science.gov (United States)

    Nyitrai, M; Hild, G; Hartvig, N; Belágyi, J; Somogyi, B

    2000-12-29

    Conformational and dynamic properties of actin filaments polymerized from ATP- or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys(374) residue of actin was smaller in filaments from ADP-actin than in filaments from ATP-actin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys(374) from the longitudinal filament axis. Temperature-dependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys(374) of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.

  7. GPCRs and actin-cytoskeleton dynamics.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; González-Espinosa, Claudia; Espinosa-Riquer, Zyanya P; Macías-Silva, Marina

    2016-01-01

    A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Bioinformatics study of the mangrove actin genes

    Science.gov (United States)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

    This study describes the bioinformatics methods to analyze eight actin genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, subcellular localization, similarity, and phylogenetic. The physical and chemical properties of eight mangroves showed variation among the genes. The percentage of the secondary structure of eight mangrove actin genes followed the order of a helix > random coil > extended chain structure for BgActl, KcActl, RsActl, and A. corniculatum Act. In contrast to this observation, the remaining actin genes were random coil > extended chain structure > a helix. This study, therefore, shown the prediction of secondary structure was performed for necessary structural information. The values of chloroplast or signal peptide or mitochondrial target were too small, indicated that no chloroplast or mitochondrial transit peptide or signal peptide of secretion pathway in mangrove actin genes. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove actin genes. To clarify the relationship among the mangrove actin gene, a phylogenetic tree was constructed. Three groups of mangrove actin genes were formed, the first group contains B. gymnorrhiza BgAct and R. stylosa RsActl. The second cluster which consists of 5 actin genes the largest group, and the last branch consist of one gene, B. sexagula Act. The present study, therefore, supported the previous results that plant actin genes form distinct clusters in the tree.

  9. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

    OpenAIRE

    Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

    2014-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, an...

  10. Pharmacological treatment of actinic keratosis

    Directory of Open Access Journals (Sweden)

    Ewa Zwierzyńska

    2016-09-01

    Full Text Available Actinic keratosis (AK is a disease characterized by hyperkeratotic lesions on skin damaged by ultraviolet. radiation. These lesions may progress to squamous cell or basal cell carcinoma. Currently pharmacotherapy and different surgical procedures are used in AK therapy. The most common treatment options are 5-fluorouracil, imiquimod, diclofenac, ingenol mebutate, and first and third generation retinoids (retinol, adapalene, tazarotene. Furthermore, research is being carried out in order to test new medications including nicotinamide, resiquimod, piroxicam, potassium dobesilate and oleogel based on a triterpene extract (betulin, betulinic acid. Recently, the preventive effect of acetylsalicylic acid and celecoxib has also been investigated.

  11. Survey of Lignin-Structure Changes and Depolymerization during Ionic Liquid Pretreatment

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, Tanmoy; Isern, Nancy G.; Sun, Jian; Wang, Eileen; Hull, Sarah; Cort, John R.; Simmons, Blake A.; Singh, Seema

    2017-09-26

    A detailed study of chemical changes in lignin structure during the ionic liquid (IL) pretreatment process is not only pivotal for understanding and overcoming biomass recalcitrance during IL pretreatment, but also is necessary for designing new routes for lignin valorization. Chemical changes in lignin were systematically studied as a function of pretreatment temperature, time and type of IL used. Kraft lignin was used as the lignin source and common pretreatment conditions were employed using three different ILs of varying chemical structure in terms of acidic or basic character. The chemical changes in the lignin structure due to IL pretreatment processes were monitored using 1H-13C HSQC NMR, 31P NMR, elemental analysis, GPC, FT-IR, and the depolymerized products were analyzed using GC-MS. Although pretreatment in acidic IL, triethylammonium hydrogensulfate ([TEA][HSO4]) results in maximum decrease in β-aryl ether bond, maximum dehydration and recondensation pathways were also evident, with the net process showing a minimum decrease in the molecular weight of regenerated lignin. However, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) pretreatment yields a smaller decrease in the β-aryl ether content along with minimum evidence of recondensation, resulting in the maximum decrease in the molecular weight. Cholinium lysinate ([Ch][Lys]) pretreatment shows an intermediate result, with moderate depolymerization, dehydration and recondensation observed. The depolymerization products after IL pretreatment are found to be a function of the pretreatment temperature and the specific chemical nature of the IL used. At higher pretreatment temperature, [Ch][Lys] pretreatment yields guaiacol, [TEA][HSO4] yields guaiacylacetone, and [C2C1Im][OAc] yields both guaiacol and guaiacylacetone as major products. These results clearly indicate that the changes in lignin structure as well as the depolymerized product profile depend on the pretreatment conditions and the nature

  12. The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain

    Science.gov (United States)

    González-Jamett, Arlek M.; Guerra, María J.; Olivares, María J.; Haro-Acuña, Valentina; Baéz-Matus, Ximena; Vásquez-Navarrete, Jacqueline; Momboisse, Fanny; Martinez-Quiles, Narcisa; Cárdenas, Ana M.

    2017-01-01

    Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells. PMID:28522963

  13. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

    Science.gov (United States)

    Spracklen, Andrew J; Fagan, Tiffany N; Lovander, Kaylee E; Tootle, Tina L

    2014-09-15

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool

  14. Mechanical hysteresis in actin networks.

    Science.gov (United States)

    Majumdar, Sayantan; Foucard, Louis C; Levine, Alex J; Gardel, Margaret L

    2018-03-14

    Understanding the response of complex materials to external force is central to fields ranging from materials science to biology. Here, we describe a novel type of mechanical adaptation in cross-linked networks of F-actin, a ubiquitous protein found in eukaryotic cells. We show that shear stress changes the network's nonlinear mechanical response even long after that stress is removed. The duration, magnitude and direction of forcing history all change this mechanical response. While the mechanical hysteresis is long-lived, it can be simply erased by force application in the opposite direction. We further show that the observed mechanical adaptation is consistent with stress-dependent changes in the nematic order of the constituent filaments. Thus, this mechanical hysteresis arises from the changes in non-linear response that originates from stress-induced changes to filament orientation. This demonstrates that F-actin networks can exhibit analog read-write mechanical hysteretic properties, which can be used for adaptation to mechanical stimuli.

  15. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    Directory of Open Access Journals (Sweden)

    Fernando Navarro-Garcia

    2013-01-01

    Full Text Available The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.

  16. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    Science.gov (United States)

    Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

    2013-01-01

    The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

  17. Dynamic Regulation of Sarcomeric Actin Filaments in Striated Muscle

    OpenAIRE

    Ono, Shoichiro

    2010-01-01

    In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin sub...

  18. During capacitation in bull spermatozoa, actin and PLC-ζ undergo dynamic interactions.

    Science.gov (United States)

    Mejía-Flores, Itzayana; Chiquete-Félix, Natalia; Palma-Lara, Icela; Uribe-Carvajal, Salvador; de Lourdes Juárez-Mosqueda, María

    2017-10-01

    The migration pattern of sperm-specific phospholipase C-ζ (PLC-ζ) was followed and the role of this migration in actin cytoskeleton dynamics was determined. We investigated whether PLC-ζ exits sperm, opening the possibility that PLC-ζ is the 'spermatozoidal activator factor' (SOAF). As capacitation progresses, the highly dynamic actin cytoskeleton bound different proteins to regulate their location and activity. PLC-ζ participation at the start of fertilization was established. In non-capacitated spermatozoa, PLC-ζ is in the perinuclear theca (PT) and in the flagellum, therefore it was decided to determine whether bovine sperm actin interacts with PLC-ζ to direct its relocation as it progresses from non-capacitated (NC) to capacitated (C) and to acrosome-reacted (AR) spermatozoa. PLC-ζ interacted with actin in NC spermatozoa (100%), PLC-ζ levels decreased in C spermatozoa to 32% and in AR spermatozoa to 57% (P < 0.001). The level of actin/PLC-ζ interaction was twice as high in G-actin (P < 0.001) that reflected an increase in affinity. Upon reaching the AR spermatozoa, PLC-ζ was partially released from the cell. It was concluded that actin cytoskeleton dynamics control the migration of PLC-ζ during capacitation and leads to its partial release at AR spermatozoa. It is suggested that liberated PLC-ζ could reach the egg and favour fertilization.

  19. A Secreted Ankyrin-Repeat Protein from Clinical Stenotrophomonas maltophilia Isolates Disrupts Actin Cytoskeletal Structure.

    Science.gov (United States)

    MacDonald, Logan C; O'Keefe, Sean; Parnes, Mei-Fan; MacDonald, Hanlon; Stretz, Lindsey; Templer, Suzanne J; Wong, Emily L; Berger, Bryan W

    2016-01-08

    Stenotrophomonas maltophilia is an emerging, multidrug-resistant pathogen of increasing importance for the immunocompromised, including cystic fibrosis patients. Despite its significance as an emerging pathogen, relatively little is known regarding the specific factors and mechanisms that contribute to its pathogenicity. We identify and characterize a putative ankyrin-repeat protein (Smlt3054) unique to clinical S. maltophilia isolates that binds F-actin in vitro and co-localizes with actin in transfected HEK293a cells. Smlt3054 is endogenously expressed and secreted from clinical S. maltophilia isolates, but not an environmental isolate (R551-3). The in vitro binding of Smlt3054 to F-actin resulted in a thickening of the filaments as observed by TEM. Ectopic expression of Smlt3054-GFP exhibits strong co-localization with F-actin, with distinct, retrograde F-actin waves specifically associated with Smlt3054 in individual cells as well as formation of dense, internal inclusions at the expense of retrograde F-actin waves. Collectively, our results point to an interaction between Smlt3054 and F-actin. Furthermore, as a potentially secreted protein unique to clinical S. maltophilia isolates, Smlt3054 may serve as a starting point for understanding the mechanisms by which S. maltophilia has become an emergent pathogen.

  20. Lamin A/C and polymeric actin in genome organization

    Czech Academy of Sciences Publication Activity Database

    Ondřej, V.; Lukášová, Emilie; Kroupová, Jana; Matula, P.; Kozubek, Stanislav

    2008-01-01

    Roč. 26, č. 4 (2008), s. 356-361 ISSN 1016-8478 R&D Projects: GA AV ČR(CZ) 1QS500040508; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : lamin A/C * polymeric actin * chromosome territories Subject RIV: BO - Biophysics Impact factor: 2.023, year: 2008

  1. Mutual regulation of plant phospholipase D and the actin cytoskeleton

    Czech Academy of Sciences Publication Activity Database

    Pleskot, Roman; Potocký, Martin; Pejchar, Přemysl; Linek, J.; Bezvoda, R.; Martinec, Jan; Valentová, O.; Novotná, Z.; Žárský, Viktor

    2010-01-01

    Roč. 62, č. 3 (2010), s. 494-507 ISSN 0960-7412 R&D Projects: GA AV ČR IAA601110916; GA MŠk(CZ) LC06034; GA ČR GA522/05/0340 Institutional research plan: CEZ:AV0Z50380511 Keywords : phospholipase D * actin * signaling Subject RIV: ED - Physiology Impact factor: 6.948, year: 2010

  2. LIGNIN, STRUCTURE AND APPLICATIONS: DEPOLYMERIZATION METHODS FOR OBTAINING AROMATIC DERIVATIVES OF INDUSTRIAL INTEREST

    Directory of Open Access Journals (Sweden)

    Marvin Chávez-Sifontes

    2013-12-01

    Full Text Available In this article significant data related to the structural characteristics of lignin, the extraction and isolation processes from biomass, and also the characteristics of different types of commercial lignins are presented. The review focuses on the different depolymerization processes (hydrolysis, hydrogenolysis, hydrodeoxygenation, pyrolysis, among others up to now developed and investigated analyzing the different aromatic derivatives obtained in each case, as well as the interesting reactions some of them may undergo. Application possibilities for lignin and its derivatives in new industrial processes integrated into the bio-refinery of the future are finally assessed

  3. Depolymerization of organosolv lignin using doped porous metal oxides in supercritical methanol

    DEFF Research Database (Denmark)

    Warner, Genoa; Hansen, Thomas Søndergaard; Riisager, Anders

    2014-01-01

    An isolated, solvent-extracted lignin from candlenut (Aleurites moluccana) biomass was subjected to catalytic depolymerization in the presence of supercritical methanol, using a range of porous metal oxides derived from hydrotalcite-like precursors. The most effective catalysts in terms of lignin...... conversion to methanol-soluble products, without char formation, were based on copper in combination with other dopants based on relatively earth-abundant metals. Nearly complete conversion of lignin to bio-oil composed of monomers and low-mass oligomers with high aromatic content was obtained in 6. h at 310...

  4. Effects of solution crowding on actin polymerization reveal the energetic basis for nucleotide-dependent filament stability.

    Science.gov (United States)

    Frederick, Kendra B; Sept, David; De La Cruz, Enrique M

    2008-05-02

    Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (C(c)) for assembly. The structural differences between ATP- and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. Here, we investigate the effect of crowding agents on ATP- and ADP-actin polymerization and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the C(c) of ADP-actin in a concentration-dependent manner. The stabilities of ATP- and ADP-actin filaments are comparable in the presence of physiological amounts (approximately 30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability.

  5. Modulation of microfilament protein composition by transfected cytoskeletal actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Ng, S.Y.; Erba, H.; Latter, G.; Kedes, L.; Leavitt, J.

    1988-04-01

    HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of ..beta..-actin due to coding mutations in one of two ..beta..-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional ..beta..-actin gene but not following transfection of the functional ..gamma..-actin gene. In ..gamma..-actin gene-transfected substrains that have increased rates of ..gamma..-actin synthesis, ..beta..-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both ..beta..- and ..gamma..-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal ..beta..-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.

  6. Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments.

    Science.gov (United States)

    Bubb, M R; Spector, I; Bershadsky, A D; Korn, E D

    1995-02-24

    Swinholide A, isolated from the marien sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

  7. The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation

    Directory of Open Access Journals (Sweden)

    Stephanie Karmann

    2017-06-01

    Full Text Available Poly(hydroxyalkanoates (PHAs are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization.

  8. Depolymerization of insulin amyloid fibrils by albumin-modified magnetic fluid

    Science.gov (United States)

    Siposova, Katarina; Kubovcikova, Martina; Bednarikova, Zuzana; Koneracka, Martina; Zavisova, Vlasta; Antosova, Andrea; Kopcansky, Peter; Daxnerova, Zuzana; Gazova, Zuzana

    2012-02-01

    Pathogenesis of amyloid-related diseases is associated with the presence of protein amyloid deposits. Insulin amyloids have been reported in a patient with diabetes undergoing treatment by injection of insulin and causes problems in the production and storage of this drug and in application of insulin pumps. We have studied the interference of insulin amyloid fibrils with a series of 18 albumin magnetic fluids (MFBSAs) consisting of magnetite nanoparticles modified by different amounts of bovine serum albumin (w/w BSA/Fe3O4 from 0.005 up to 15). We have found that MFBSAs are able to destroy amyloid fibrils in vitro. The extent of fibril depolymerization was affected by nanoparticle physical-chemical properties (hydrodynamic diameter, zeta potential and isoelectric point) determined by the BSA amount present in MFBSAs. The most effective were MFBSAs with lower BSA/Fe3O4 ratios (from 0.005 to 0.1) characteristic of about 90% depolymerizing activity. For the most active magnetic fluids (ratios 0.01 and 0.02) the DC50 values were determined in the range of low concentrations, indicating their ability to interfere with insulin fibrils at stoichiometric concentrations. We assume that the present findings represent a starting point for the application of the active MFBSAs as therapeutic agents targeting insulin amyloidosis.

  9. Kinetics and Thermodynamic Studies of Depolymerization of Nylon Waste by Hydrolysis Reaction

    Directory of Open Access Journals (Sweden)

    D. B. Patil

    2014-01-01

    Full Text Available Depolymerization reaction of nylon waste was carried out by hydrolysis reaction. Yield of depolymerization products was up to 72.20% for a two-hour reaction time. The products obtained were characterized by melting point and FTIR spectra. The values obtained for dibenzoyl derivative of hexamethylenediamine (DBHMD agreed with those of the pure substance. Chemical kinetics of this reaction shows that it is a first-order reaction with respect to hexamethylenediamine (HMD concentration with velocity constant 7.32×10-3 min−1. The energy of activation and Arrhenius constant obtained by Arrhenius plot were 87.22 KJg−1 and 0.129, respectively. The other thermodynamic parameters such as enthalpy of activation (ΔH‡ and entropy of activation (ΔS‡ and free energy of activation were 5975.85 J and −270.86 J·K−1·mol−1 and 101.59 KJ·mol−1, respectively.

  10. Immune-enhancing activities of low molecular weight {beta}-glucan depolymerized by gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Nak-Yun [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Hong [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Laboratory of Food Chemistry, Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 (Japan); Kwon, Sun-Kyu; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Yoo, Young-Choon [Department of Microbiology, College of Medicine, Konyang University, Daejeon 302-718 (Korea, Republic of); Kim, Mee-Ree [Department of Food and Nutrition, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Ju-Woon [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)], E-mail: sjwlee@kaeri.re.kr

    2009-07-15

    {beta}-glucans are structural cell wall polymers of many microorganisms and cereals which possess immunomodulatory properties and have been used in the food, cosmetic and medical industry. In our previous study, {beta}-glucan was depolymerized by gamma irradiation and leads to improve the solubility and viscosity. This study was carried out to evaluate the functional properties, mainly immune-enhancing activities of low molecular weight {beta}-glucan fragmented by gamma irradiation. The results showed that RAW 264.7 macrophage cell stimulation activities of irradiated {beta}-glucan were higher than that of non-irradiated {beta}-glucan. In addition, the oral administration of gamma-irradiated {beta}-glucan significantly increased the proliferation and cytokine (IFN-{gamma} and IL-2) release of spleen and Peyer's patch cells compared with non-irradiated {beta}-glucan. In conclusion, gamma irradiation could be used as an effective method for the production of depolymerized {beta}-glucan improved functional property such as immunomodulatory activity.

  11. Immune-enhancing activities of low molecular weight β-glucan depolymerized by gamma irradiation

    International Nuclear Information System (INIS)

    Sung, Nak-Yun; Byun, Eui-Hong; Kwon, Sun-Kyu; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo; Yoo, Young-Choon; Kim, Mee-Ree; Lee, Ju-Woon

    2009-01-01

    β-glucans are structural cell wall polymers of many microorganisms and cereals which possess immunomodulatory properties and have been used in the food, cosmetic and medical industry. In our previous study, β-glucan was depolymerized by gamma irradiation and leads to improve the solubility and viscosity. This study was carried out to evaluate the functional properties, mainly immune-enhancing activities of low molecular weight β-glucan fragmented by gamma irradiation. The results showed that RAW 264.7 macrophage cell stimulation activities of irradiated β-glucan were higher than that of non-irradiated β-glucan. In addition, the oral administration of gamma-irradiated β-glucan significantly increased the proliferation and cytokine (IFN-γ and IL-2) release of spleen and Peyer's patch cells compared with non-irradiated β-glucan. In conclusion, gamma irradiation could be used as an effective method for the production of depolymerized β-glucan improved functional property such as immunomodulatory activity.

  12. Depolymerization of polysaccharides from Opuntia ficus indica: Antioxidant and antiglycated activities.

    Science.gov (United States)

    Chaouch, Mohamed Aymen; Hafsa, Jawhar; Rihouey, Christophe; Le Cerf, Didier; Majdoub, Hatem

    2015-08-01

    The extraction, purification and degradation of polysaccharides from Opuntia ficus indica cladodes, as well as the evaluation of their antioxidant and antiglycated activities in vitro were investigated. The optimization of the extraction showed that extraction by ultrasound at 40 °C presented the best carbohydrates yield. The degradation of the extracted polysaccharides was achieved by free radical depolymerization with H2O2 in the presence of copper(II) acetate for various reaction times. Sugar contents were determined by colorimetric assays. The macromolecular characteristics of the different isolated and degraded carbohydrates were carried by size exclusion chromatography (SEC/MALS/VD/DRI). These experiments showed that all samples are polysaccharides, which are probably pectins and that molecular weight (Mw) has decreased from 6,800,000 to 14,000 g/mol after 3 h of depolymerization without changing the structure. Preliminary antioxidant and antiglycated tests indicated that degraded polysaccharides for 2 and 3 h showed even better antioxidant and antiglycated activities. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Conformational changes in actin induced by its interaction with gelsolin.

    Science.gov (United States)

    Khaitlina, S; Hinssen, H

    1997-08-01

    Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer.

  14. Structural differences explain diverse functions of Plasmodium actins.

    Directory of Open Access Journals (Sweden)

    Juha Vahokoski

    2014-04-01

    Full Text Available Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.

  15. Selective Depolymerization and Effects of Homolysis of Poly(L-lactic acid in a Blend with Polypropylene

    Directory of Open Access Journals (Sweden)

    Haruo Nishida

    2009-01-01

    Full Text Available Blends of poly(L-lactic acid (PLLA and polypropylene (PP, which are candidates for the practical use of PLLA, were investigated for selective degradation of PLLA, resulting in quantitative conversion of PLLA components into cyclic monomers, lactides, using magnesium oxide (MgO as a depolymerization catalyst. Obviously, the catalyst MgO selectively accelerated only the PLLA depolymerization in the blends, dominantly generating L,L-lactide as a volatile product and separating the PP component. Expected effects of homolysis in the blend system were also determined as slight changes in activation energy of degradation for both the components and through the suppression of degradation by an antioxidant.

  16. Mechanics model for actin-based motility.

    Science.gov (United States)

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  17. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

    Science.gov (United States)

    Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

    2013-01-01

    Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

  18. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea

    Directory of Open Access Journals (Sweden)

    Ligia Cristina Kalb Souza

    2013-08-01

    Full Text Available Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major, insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis and plants (Phytomonas serpens. A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.

  19. Symmetrical retrograde actin flow in the actin fusion structure is involved in osteoclast fusion

    Directory of Open Access Journals (Sweden)

    Jiro Takito

    2017-07-01

    Full Text Available The aim of this study was to elucidate the role of the zipper-like structure (ZLS, a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3. Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

  20. Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation

    DEFF Research Database (Denmark)

    Fritzsche, Marco; Fernandes, Ricardo A.; Chang, Veronica T.

    2017-01-01

    optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse....... This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament...... as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions....

  1. The role of DNA and actin polymers on the polymer structure and rheology of cystic fibrosis sputum and depolymerization by gelsolin or thymosin beta 4

    NARCIS (Netherlands)

    Kater, Arnon; Henke, Markus O.; Rubin, Bruce K.

    2007-01-01

    Mucus clearance is the first line of pulmonary defense against inhaled irritants, microorganisms, and allergens. In health, the gel-forming mucins are the principal polymeric components of airway mucus but in cystic fibrosis (CF), the necrotic death of inflammatory and epithelial cells releases a

  2. In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates produced by Pseudomonas putida Bet001.

    Science.gov (United States)

    Anis, Siti Nor Syairah; Mohamad Annuar, Mohamad Suffian; Simarani, Khanom

    2017-09-14

    In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid was studied. Both processes were studied under optimum conditions for mcl-PHA depolymerization viz. 0.2 M Tris-HCl buffer, pH 9, ionic strength (I) = 0.2 M at 30°C. For in vitro depolymerization studies, cell-free system was obtained from lysing bacterial cells suspension by ultrasonication at optimum conditions (frequency 37 kHz, 30% of power output, <25°C for 120 min). The comparison between in vivo and in vitro depolymerizations of intracellular mcl-PHA was made. In vitro depolymerization showed lower depolymerization rate but higher yield compared to in vivo depolymerization. The monomer liberation rate reflected the mol% distribution of the initial polymer subunit composition, and the resulting direct individual products of depolymerization were identical for both in vivo and in vitro processes. It points to exo-type reaction for both processes, and potential biological route to chiral molecules.

  3. Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

    Science.gov (United States)

    Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

    1990-05-01

    THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

  4. Separation of actin-dependent and actin-independent lipid rafts

    NARCIS (Netherlands)

    Klappe, Karin; Hummel, Ina; Kok, Jan Willem

    2013-01-01

    Lipid rafts have been isolated on the basis of their resistance to various detergents and more recently by using detergent-free procedures. The actin cytoskeleton is now recognized as a dynamic regulator of lipid raft stability. We carefully analyzed the effects of the cortical actin-disrupting

  5. Gestalt-binding of tropomyosin on actin during thin filament activation.

    Science.gov (United States)

    Lehman, William; Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Raunser, Stefan

    2013-08-01

    Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

  6. Coupling of two non-processive myosin 5c dimers enables processive stepping along actin filaments.

    Science.gov (United States)

    Gunther, Laura K; Furuta, Ken'ya; Bao, Jianjun; Urbanowski, Monica K; Kojima, Hiroaki; White, Howard D; Sakamoto, Takeshi

    2014-05-09

    Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30-36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells.

  7. Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire.

    Science.gov (United States)

    Ferreira, Tiago; Ou, Yimiao; Li, Sally; Giniger, Edward; van Meyel, Donald J

    2014-02-01

    The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

  8. Advanced Model Compounds for Understanding Acid-Catalyzed Lignin Depolymerization : Identification of Renewable Aromatics and a Lignin-Derived Solvent

    NARCIS (Netherlands)

    Lahive, Ciaran W; Deuss, Peter J; Lancefield, Christopher S; Sun, Zhuohua; Cordes, David B; Young, Claire; Tran, Fanny; Slawin, Alexandra M Z; de Vries, Johannes G; Kamer, Paul C J; Westwood, Nicholas J; Barta, Katalin

    2016-01-01

    The development of fundamentally new approaches for lignin depolymerization is challenged by the complexity of this aromatic biopolymer. While overly simplified model compounds often lack relevance to the chemistry of lignin, the direct use of lignin streams poses significant analytical challenges

  9. Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation

    Science.gov (United States)

    Kühbacher, Andreas; Emmenlauer, Mario; Rämo, Pauli; Kafai, Natasha; Dehio, Christoph

    2015-01-01

    ABSTRACT Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function. PMID:25991686

  10. Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U synergistically activate actin assembly.

    Directory of Open Access Journals (Sweden)

    Kenneth G Campellone

    2008-10-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC generate F-actin-rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspF(U, a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspF(U repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspF(U are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspF(U fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspF(U. Whereas clustering of a single EspF(U repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspF(U derivatives promote actin assembly more efficiently. Moreover, the EspF(U repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3-mediated signaling pathways.

  11. Depolymerization of post-consumer PET with multifunctional alcohol through melt processing

    International Nuclear Information System (INIS)

    Lessa, Tathiane C.R.F.; Mendes, Luis C.; Dias, Marcos L.

    2009-01-01

    The purpose of this study was to prepare oligomers from post-consumer PET with multifunctional alcohol, through melt processing, aiming to develop a new material, able to play a role as filler or property modifier. Maintaining constants the process conditions, content and kind of catalyst, the influence of the solvolysis agent on the PET depolymerization was investigated. The products were evaluated by wide-angle X-ray diffraction (WAXD) and thermogravimetry (TG/DTG). The changes in the WAXD curves and the shift of the maximum degradation temperature suggested that the ester linkages were broken being the ethylene glycol moieties replaced with hydroxyl-terminal groups of the multifunctional alcohol, as result of a transesterification reaction. The chemical structure of the new ester was named 'star-branching polymer'. (author)

  12. Solid acid-catalyzed depolymerization of barley straw driven by ball milling.

    Science.gov (United States)

    Schneider, Laura; Haverinen, Jasmiina; Jaakkola, Mari; Lassi, Ulla

    2016-04-01

    This study describes a time and energy saving, solvent-free procedure for the conversion of lignocellulosic barley straw into reducing sugars by mechanocatalytical pretreatment. The catalytic conversion efficiency of several solid acids was tested which revealed oxalic acid dihydrate as a potential catalyst with high conversion rate. Samples were mechanically treated by ball milling and subsequently hydrolyzed at different temperatures. The parameters of the mechanical treatment were optimized in order to obtain sufficient amount of total reducing sugar (TRS) which was determined following the DNS assay. Additionally, capillary electrophoresis (CE) and Fourier transform infrared spectrometry (FT-IR) were carried out. Under optimal conditions TRS 42% was released using oxalic acid dihydrate as a catalyst. This study revealed that the acid strength plays an important role in the depolymerization of barley straw and in addition, showed, that the oxalic acid-catalyzed reaction generates low level of the degradation product 5-hydroxymethylfurfural (HMF). Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Temperature effects on protein depolymerization and amino acid immobilization rates in soils.

    Science.gov (United States)

    Noll, Lisa; Hu, Yuntao; Zhang, Shasha; Zheng, Qing; Wanek, Wolfgang

    2017-04-01

    Increasing N deposition, land use change, elevated atmospheric CO2 concentrations and global warming have altered soil nitrogen (N) cycling during the last decades. Those changes affected ecosystem services, such as C and N sequestration in soils, which calls for a better understanding of soil N transformation processes. The cleavage of macromolecular organic N by extracellular enzymes maintains an ongoing flow of new bioavailable organic N into biotic systems and is considered to be the bottle neck of terrestrial N cycling in litter and soils. Recent studies showed that protein depolymerization is susceptible to changes in C and N availabilities. Based on general biological observations the temperature sensitivity of soil organic N processes is expected to depend on whether they are rather enzyme limited (i.e. Q10=2) or diffusion limited (i.e. Q10= 1.0 - 1.3). However, temperature sensitivities of protein depolymerization and amino acid immobilization are still unknown. We therefore here report short-term temperature effects on organic N transformation rates in soils differing in physicochemical parameters but not in climate. Soil samples were collected from two geologically distinct sites close to the LFZ Raumberg-Gumpenstein, Styria, Austria, each from three different management types (arable land, grassland, forest). Four replicates of mineral soil were taken from every site and management type. The area provides a unique opportunity to study geological and management controls in soils without confounding effects of climate and elevation. The soils differ in several soil chemical parameters, such as soil pH, base saturation, soil C: N ratio and SOM content as well as in soil physical parameters, such as soil texture, bulk density and water holding capacity. Soils were pre-incubated at 5, 15 and 25˚ C for one day. Protein depolymerization rates and amino acid immobilization rates were assessed by an isotope pool dilution assay with 15N labeled amino acids at

  14. A novel microtubule depolymerizing colchicine analogue triggers apoptosis and autophagy in HCT-116 colon cancer cells.

    Science.gov (United States)

    Kumar, Ashok; Singh, Baljinder; Sharma, Parduman R; Bharate, Sandip B; Saxena, Ajit K; Mondhe, D M

    2016-03-01

    Colchicine is a tubulin-binding natural product isolated from Colchicum autumnale. Here we report the in vitro anticancer activity of C-ring modified semi-synthetic derivative of colchicine; N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(4-phenyl-piperidin-1-yl)-5,6,7,9 tetrahydrobenzo[a]heptalen-7-yl]acetamide (4h) on colon cancer HCT-116 cell line. The compound 4h was screened for anti-proliferative activity against different human cancer cell lines and was found to exhibit higher cytotoxicity against colon cancer cell lines HCT-116 and Colo-205 with IC50 of 1 and 0.8 μM respectively. Cytotoxicity of the compound to the normal fR2 breast epithelial cells and normal HEK293 human embryonic kidney cells was evaluated in concentration and time-dependent manner to estimate its selectivity for cancer cells which showed much better selectivity than that of colchicine. Compound 4h induced cell death in HCT-116 cells by activating apoptosis and autophagy pathways. Autophagy inhibitor 3-MA blocked the production of LC3-II and reduced the cytotoxicity in response to 4h, but did not affect apoptosis, suggesting thereby that these two were independent events. Reactive oxygen species scavenger ascorbic acid pretreatment not only decreased the reactive oxygen species level but also reversed 4h induced cytotoxicity. Treatment with compound 4h depolymerized microtubules and the majority of cells arrested at the G2/M transition. Together, these data suggest that 4h has better selectivity and is a microtubule depolymerizer, which activates dual cell-death machineries, and thus, it could be a potential novel therapeutic agent in cancer therapy. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

    DEFF Research Database (Denmark)

    Jorgensen, Nanna K; Pedersen, Stine F; Rasmussen, Hanne B

    2003-01-01

    -induced activation of hIK channels was strongly inhibited by cytochalasin D (CD), in concentrations that caused depolymerization of F-actin filaments, indicating a role for the F-actin cytoskeleton in modulation of hIK by changes in cell volume. In conclusion, hIK and rSK3 channels are activated by cell swelling......Cloned Ca(2+)-activated K(+) channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125......%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca(2+)](i), evidenced...

  16. Glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (ACD toxin.

    Directory of Open Access Journals (Sweden)

    Elena Kudryashova

    Full Text Available Actin Crosslinking Domain (ACD is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K(0.5 = 30 µM reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg(2+-GTP to support crosslinking, but the kinetic parameters (K(M = 8 µM and 50 µM for ATP and GTP, respectively suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0-9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.

  17. Glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (ACD) toxin.

    Science.gov (United States)

    Kudryashova, Elena; Kalda, Caitlin; Kudryashov, Dmitri S

    2012-01-01

    Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K(0.5) = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg(2+)-GTP to support crosslinking, but the kinetic parameters (K(M) = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0-9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.

  18. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

    Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

  19. Antibodies to actin in autoimmune haemolytic anaemia

    Directory of Open Access Journals (Sweden)

    Ritzmann Mathias

    2010-03-01

    Full Text Available Abstract Background In autoimmune haemolytic anaemia (AIHA, autoreactive antibodies directed against red blood cells are up-regulated, leading to erythrocyte death. Mycoplasma suis infections in pigs induce AIHA of both the warm and cold types. The aim of this study was to identify the target autoantigens of warm autoreactive IgG antibodies. Sera from experimentally M. suis-infected pigs were screened for autoreactivity. Results Actin-reactive antibodies were found in the sera of 95% of all animals tested. The reactivity was species-specific, i.e. reactivity with porcine actin was significantly higher than with rabbit actin. Sera of animals previously immunised with the M. suis adhesion protein MSG1 showed reactivity with actin prior to infection with M. suis indicating that molecular mimicry is involved in the specific autoreactive mechanism. A potentially cross-reactive epitope was detected. Conclusions This is the first report of autoreactive anti-actin antibodies involved in the pathogenesis of autoimmune haemolytic anaemia.

  20. Marcadores e fatores de risco para queratoses actínicas e carcinomas basocelulares: um estudo de caso-controle Risk markers and risk factors for actinic keratosis and basal cell carcinoma: a case-control study

    Directory of Open Access Journals (Sweden)

    Fernando Passos da Rocha

    2004-08-01

    premalignant lesions. OBJECTIVES: To identify the risk factors and dermatological risk markers for cutaneous malignancies. METHODS: A case-control study nested in a population-based prevalence survey was performed in adults aged 50 living in the urban area of Pelotas. In the prevalence study, a questionnaire was applied to identify possible malignant or premalignant lesions and these subjects were examined by two doctors (gold standard. A sub-sample of the persons who answered No to the questions was also sent to the Ambulatory for the same procedure. The sample was constituted of 288 persons: 74 cases and 214 controls. The number and the type of efflorescence was evaluated at the interview with the doctors. The questionnaire also investigated socioeconomic level, behavioral variables and skin examination. The measure of effect used was the Odds Ratio (OR; and to control confounding factors, logistic regression, with hierarchical pattern, was utilized. RESULTS: In the multivariate analysis, the following variables remained significant: age 80 years (OR = 10.21, white skin (OR = 4.85, blond or red hair (OR = 3.69 and risk markers: solar elastosis (OR = 4.35, cutis rhomboidalis nuchae (OR = 2.88 and more than 10 melanoses on the back of the hands (OR = 6.0. CONCLUSION: Elderly individuals, with fair skin and hair, solar elastosis, cutis rhomboidalis nuchae and a great number of melanoses on the hands, are at high risk for actinic keratosis and basal cell carcinoma.

  1. Baculovirus AC102 is a nucleocapsid protein that is crucial for nuclear actin polymerization and nucleocapsid morphogenesis.

    Science.gov (United States)

    Hepp, Susan E; Borgo, Gina M; Ticau, Simina; Ohkawa, Taro; Welch, Matthew D

    2018-03-14

    The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection, and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex. IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also employed as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through

  2. The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize

    Science.gov (United States)

    Blancaflor, E. B.; Hasenstein, K. H.

    1997-01-01

    To determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays cv Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer-controlled video digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to that of vertical roots. Application of cytochalasin B or cytochalasin D resulted in extensive disruption of MFs in the cortex and epidermis, but only partially affected MFs in the stele. Despite the cytochalasin B-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots.

  3. Nuclear Actin and Lamins in Viral Infections

    Science.gov (United States)

    Cibulka, Jakub; Fraiberk, Martin; Forstova, Jitka

    2012-01-01

    Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections. PMID:22590674

  4. Dorsal stress fibers, transverse actin arcs, and perinuclear actin fibers form an interconnected network that induces nuclear movement in polarizing fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Maninová, Miloslava; Vomastek, Tomáš

    2016-01-01

    Roč. 283, č. 20 (2016), s. 3676-3693 ISSN 1742-464X R&D Projects: GA ČR GA13-06405S Institutional support: RVO:61388971 Keywords : actin dorsal fibers * cell polarity * nuclear reorientation Subject RIV: EE - Microbiology, Virology Impact factor: 3.902, year: 2016

  5. The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tang, H Y; Cai, M

    1996-09-01

    Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.

  6. Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization.

    Science.gov (United States)

    Stanslowsky, Nancy; Reinhardt, Peter; Glass, Hannes; Kalmbach, Norman; Naujock, Maximilian; Hensel, Niko; Lübben, Verena; Pal, Arun; Venneri, Anna; Lupo, Francesca; De Franceschi, Lucia; Claus, Peter; Sterneckert, Jared; Storch, Alexander; Hermann, Andreas; Wegner, Florian

    2016-11-23

    Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function. Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer

  7. HIV infection of T cells: actin-in and actin-out.

    Science.gov (United States)

    Liu, Yin; Belkina, Natalya V; Shaw, Stephen

    2009-04-14

    Three studies shed light on the decade-old observation that the actin cytoskeleton is hijacked to facilitate entry of HIV into its target cells. Polymerization of actin is required to assemble high concentrations of CD4 and CXCR4 at the plasma membrane, which promote viral binding and entry in both the simple model of infection by free virus and the more physiologically relevant route of infection through the virological synapse. Three types of actin-interacting proteins-filamin, ezrin/radixin/moesin (ERM), and cofilin-are now shown to play critical roles in this process. Filamin binds to both CD4 and CXCR4 in a manner promoted by signaling of the HIV gp120 glycoprotein. ERM proteins attach actin filaments to the membrane and may promote polymerization of actin. Early in the process of viral entry, cofilin is inactivated, which is proposed to facilitate the early assembly of actin filaments, but cofilin is reported to be activated soon thereafter to facilitate postentry events. This complex role of cofilin may help to reconcile the paradox that actin polymerization promotes initial binding and fusion steps but inhibits some subsequent early postentry events.

  8. Human muscle LIM protein dimerizes along the actin cytoskeleton and cross-links actin filaments.

    Science.gov (United States)

    Hoffmann, Céline; Moreau, Flora; Moes, Michèle; Luthold, Carole; Dieterle, Monika; Goretti, Emeline; Neumann, Katrin; Steinmetz, André; Thomas, Clément

    2014-08-01

    The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. The role of actin and myosin during spermatogenesis.

    Science.gov (United States)

    Sun, Xiao; Kovacs, Tamas; Hu, Yan-Jun; Yang, Wan-Xi

    2011-08-01

    Spermatogenesis is a transitionary process in which the diploid spermatogonia transform into haploid mature spermatozoa. Actin and myosin have been implicated in various aspects during spermatogenesis. Actin is present in the form of monomer, oligomer and polymer within cells, the latter is called microfilament. There are five actin-containing structures during spermatogenesis, i.e., ectoplasmic specialization, acroplaxome, manchette in mammals, actin cones in Drosophila and acroframosome in Caridean shrimp. They are involved in the shaping and differentiating of spermatids. Along with spermatogenesis, the actin cytoskeletons show active remodeling in this process. Some actin binding or actin regulated proteins have been demonstrated to regulate dynamic changes of the actin-containing structures. Myosin, actin-dependent molecular motor, plays an important role during spermatogenesis, such as involving in acrosome biogenesis, vesicle transport, gene transcription and nuclear shaping. The actin cytoskeleton and actin binding/regulated proteins cooperate to facilitate spermatogenesis. In this review, we summarize the existing knowledge about the cytoskeletal structures consisting of actin, actin binding/regulated proteins and myosin during spermatogenesis.

  10. Nucleotide exchange and rheometric studies with F-actin prepared from ATP- or ADP-monomeric actin

    OpenAIRE

    Newman, J.; Zaner, K.S.; Schick, K.L.; Gershman, L.C.; Selden, L.A.; Kinosian, H.J.; Travis, J.L.; Estes, J.E.

    1993-01-01

    It has recently been reported that polymer actin made from monomer containing ATP (ATP-actin) differed in EM appearance and rheological characteristics from polymer made from ADP-containing monomers (ADP-actin). Further, it was postulated that the ATP-actin polymer was more rigid due to storage of the energy released by ATP hydrolysis during polymerization (Janmey et al. 1990. Nature 347:95-99). Electron micrographs of our preparations of ADP-actin and ATP-actin polymers show no major differe...

  11. STRUCTURE AND FUNCTION OF PALLADIN’S ACTIN BINDING DOMAIN

    Science.gov (United States)

    Beck, Moriah R.; Dixon, Richard D.S.; Goicoechea, Silvia M.; Murphy, Grant S.; Brungardt, Joseph G.; Beam, Matthew T.; Srinath, Pavan; Patel, Julie; Mohiuddin, Jahan; Otey, Carol A.; Campbell, Sharon L.

    2013-01-01

    Here we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct F-actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and crosslinking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin crosslinking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo. PMID:23806659

  12. Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

    Science.gov (United States)

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

  13. ACTIN BINDING PROTEIN 29 from Lilium pollen plays an important role in dynamic actin remodeling.

    Science.gov (United States)

    Xiang, Yun; Huang, Xi; Wang, Ting; Zhang, Yan; Liu, Qinwen; Hussey, Patrick J; Ren, Haiyun

    2007-06-01

    Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.

  14. Actin, actin-related proteins and profilin in diatoms: a comparative genomic analysis.

    Science.gov (United States)

    Aumeier, Charlotte; Polinski, Ellen; Menzel, Diedrik

    2015-10-01

    Diatoms are heterokont unicellular algae with a widespread distribution throughout all aquatic habitats. Research on diatoms has advanced significantly over the last decade due to available genetic transformation methods and publicly available genome databases. Yet up to now, proteins involved in the regulation of the cytoskeleton in diatoms are largely unknown. Consequently, this work focuses on actin and actin-related proteins (ARPs) encoded in the diatom genomes of Thalassiosira pseudonana, Thalassiosira oceanica, Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries. Our comparative genomic study revealed that most diatoms possess only a single conventional actin and a small set of ARPs. Among these are the highly conserved cytoplasmic Arp1 protein and the nuclear Arp4 as well as Arp6. Diatom genomes contain genes coding for two structurally different homologues of Arp4 that might serve specific functions. All diatom species examined here lack ARP2 and ARP3 proteins, suggesting that diatoms are not capable of forming the Arp2/3 complex, which is essential in most eukaryotes for actin filament branching and plus-end dynamics. Interestingly, none of the sequenced representatives of the Bacillariophyta phylum code for profilin. Profilin is an essential actin-binding protein regulating the monomer actin pool and is involved in filament plus-end dynamics. This is the first report of organisms not containing profilin. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

    Directory of Open Access Journals (Sweden)

    Rohini Shrivastava

    Full Text Available Ezrin, a member of the ERM (Ezrin/Radixin/Moesin protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2 or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

  16. Effects of solution crowding on actin polymerization reveal the energetic basis for nucleotide-dependent filament stability

    OpenAIRE

    Frederick, Kendra B.; Sept, David; De La Cruz, Enrique M.

    2008-01-01

    Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (Cc) for assembly. The structural differences between ATP- and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditio...

  17. The origin and evolution of green algal and plant actins.

    Science.gov (United States)

    An, S S; Möpps, B; Weber, K; Bhattacharya, D

    1999-02-01

    The Viridiplantae are subdivided into two groups: the Chlorophyta, which includes the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Prasinophyceae; and the Streptophyta, which includes the Charophyceae and all land plants. Within the Streptophyta, the actin genes of the angiosperms diverge nearly simultaneously from each other before the separation of monocots and dicots. Previous evolutionary analyses have provided limited insights into the gene duplications that have produced these complex gene families. We address the origin and diversification of land plant actin genes by studying the phylogeny of actins within the green algae, ferns, and fern allies. Partial genomic sequences or cDNAs encoding actin were characterized from Cosmarium botrytis (Zygnematales), Selaginella apoda (Selaginellales), Anemia phyllitidis (Polypodiales), and Psilotum triquetrum (Psilotales). Selaginella contains at least two actin genes. One sequence (Ac2) diverges within a group of fern sequences that also includes the Psilotum Ac1 actin gene and one gymnosperm sequence (Cycas revoluta Cyc3). This clade is positioned outside of the angiosperm actin gene radiation. The second Selaginella sequence (Ac1) is the sister to all remaining land plant actin sequences, although the internal branches in this portion of the tree are very short. Use of complete actin-coding regions in phylogenetic analyses provides support for the separation of angiosperm actins into two classes. N-terminal "signature" sequence analyses support these groupings. One class (VEG) includes actin genes that are often expressed in vegetative structures. The second class (REP) includes actin genes that trace their ancestry within the vegetative actins and contains members that are largely expressed in reproductive structures. Analysis of intron positions within actin genes shows that sequences from both Selaginella and Cosmarium contain the conserved 20-3, 152-1, and 356-3 introns found in many members of the

  18. Improved postharvest quality in patagonian squash ( Cucurbita moschata) coated with radiation depolymerized chitosan

    Science.gov (United States)

    Pugliese, Maria Alicia; Goitia, Maria Teresa; Yossen, Mariana; Cifone, Norma; Agulló, Enrique; Andreucetti, Noemi

    2011-12-01

    Different molecular weight chitosans were evaluated on the decay of coated Anquito squashes ( Cucurbita moschata) as well as the maintenance of the fruit quality along five storage months. The original chitosan (Mw=391 kDa, 83% DD), was depolymerized by gamma radiation. Apart from chain scission, other chemical changes were not detected by FTIR or UV-vis analyses. The molecular weight characterization of chitosans was done by size exclusion chromatography with dual light scattering and concentration detection (SEC-MALLS-RI). The coating effectiveness was evaluated on the following parameters: fungal decay incidence, weight loss, firmness, total reducing sugar, soluble solid, flesh color, carotene content, pH and titratable acidity. No sign of fungal decay was observed in squashes coated with 122 and 56 kDa chitosans, which were also the most effective treatments in reducing the weight loss. The chitosan with Mw=122 kDa was also the best treatment considering firmness, internal aspect, sugar and carotene content. Then, radiation degraded chitosan was better in C. moschata preservation than the original chitosan.

  19. Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Ruoshui [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Guo, Mond [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Lin, Kuan-ting [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Hebert, Vincent R. [Food and Environmental Laboratory, Washington State, University-TriCities, 2710 Crimson Way Richland WA 99354 USA; Zhang, Jinwen [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Wolcott, Michael P. [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Quintero, Melissa [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Ramasamy, Karthikeyan K. [Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland WA 99354 USA; Chen, Xiaowen [National Bioenergy Center, National Renewable Energy Lab, 1617 Cole Blvd Golden CO 80127 USA; Zhang, Xiao [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA

    2016-07-04

    Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer, it has been a challenge to effectively depolymerize lignin and produce high-value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) including 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPC yields obtained were 18 and 22 % based on the initial weight of the lignin in SESPL and DACSL, respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47 %. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated.

  20. Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Ruoshui [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Guo, Mond [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Lin, Kuan-ting [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Hebert, Vincent R. [Food and Environmental Laboratory, Washington State, University-TriCities, 2710 Crimson Way Richland WA 99354 USA; Zhang, Jinwen [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Wolcott, Michael P. [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Quintero, Melissa [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Ramasamy, Karthikeyan K. [Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland WA 99354 USA; Chen, Xiaowen [National Bioenergy Center, National Renewable Energy Lab, 1617 Cole Blvd Golden CO 80127 USA; Zhang, Xiao [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA

    2016-07-04

    Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer as well as its complex side chain structures, it has been a challenge to effectively depolymerize lignin and produce high value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) inclduing 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPCs yields obtained were 18% and 22% based on the initial weight of the lignin in SESPL and DACSL respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47%. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated.

  1. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Scaife, R.M. (Fred Hutchinson Cancer Research Center, Seattle, WA (United States)); Wilson, L. (Univ. of California, Santa Barbara (United States)); Purich, D.L. (Univ. of Florida, Gainesville (United States))

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  2. Low-Energy Catalytic Electrolysis for Simultaneous Hydrogen Evolution and Lignin Depolymerization.

    Science.gov (United States)

    Du, Xu; Liu, Wei; Zhang, Zhe; Mulyadi, Arie; Brittain, Alex; Gong, Jian; Deng, Yulin

    2017-03-09

    Here, a new proton-exchange-membrane electrolysis is presented, in which lignin was used as the hydrogen source at the anode for hydrogen production. Either polyoxometalate (POM) or FeCl 3 was used as the catalyst and charge-transfer agent at the anode. Over 90 % Faraday efficiency was achieved. In a thermal-insulation reactor, the heat energy could be maintained at a very low level for continuous operation. Compared to the best alkaline-water electrolysis reported in literature, the electrical-energy consumption could be 40 % lower with lignin electrolysis. At the anode, the Kraft lignin (KL) was oxidized to aromatic chemicals by POM or FeCl 3 , and reduced POM or Fe ions were regenerated during the electrolysis. Structure analysis of the residual KL indicated a reduction of the amount of hydroxyl groups and the cleavage of ether bonds. The results suggest that POM- or FeCl 3 -mediated electrolysis can significantly reduce the electrolysis energy consumption in hydrogen production and, simultaneously, depolymerize lignin to low-molecular-weight value-added aromatic chemicals. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A density functional theory study of a silica-supported zirconium monohydride catalyst for depolymerization of polyethylene

    Energy Technology Data Exchange (ETDEWEB)

    Mortensen, J.J.; Parrinello, M.

    2000-04-06

    A silica-supported zirconium hydride catalyst for depolymerization of polyethylene is studied using density functional theory (DFT) together with a generalized gradient approximation (GGA) for the exchange and correlation energy. The (100) and (111) surfaces of {beta}-cristobalite are used as two possible models of a silica surface. Based on the experimental surface structure determined by J. Corker et al., they propose a detailed atomic model of the zirconium monohydride that is believed to be the active site for depolymerization of polyolefins. The model of the zirconium monohydride on the (100) surface is found to be very stable and the structure is in good agreement with extended X-ray absorption fine structure (EXAFS) measurements. Depolymerization of a small polyolefin chain (C{sub 3}H{sub 8}) was carried out to give CH{sub 4} and C{sub 2}H{sub 6} by addition of H{sub 2}. The rate-limiting step is a {beta}-methyl transfer to the zirconium atom, and the activation energy is 29 kcal/mol on the (100) surface.

  4. Simultaneous imaging and functional studies reveal a tight correlation between calcium and actin networks.

    Science.gov (United States)

    Bascom, Carlisle S; Winship, Lawrence J; Bezanilla, Magdalena

    2018-03-20

    Tip-growing cells elongate in a highly polarized manner via focused secretion of flexible cell-wall material. Calcium has been implicated as a vital factor in regulating the deposition of cell-wall material. However, deciphering the molecular and mechanistic calcium targets in vivo has remained challenging. Here, we investigated intracellular calcium dynamics in the moss Physcomitrella patens , which provides a system with an abundant source of genetically identical tip-growing cells, excellent cytology, and a large molecular genetic tool kit. To visualize calcium we used a genetically encoded cytosolic FRET probe, revealing a fluctuating tipward gradient with a complex oscillatory profile. Wavelet analysis coupled with a signal-sifting algorithm enabled the quantitative comparison of the calcium behavior in cells where growth was inhibited mechanically, pharmacologically, or genetically. We found that cells with suppressed growth have calcium oscillatory profiles with longer frequencies, suggesting that there is a feedback between the calcium gradient and growth. To investigate the mechanistic basis for this feedback we simultaneously imaged cytosolic calcium and actin, which has been shown to be essential for tip growth. We found that high cytosolic calcium promotes disassembly of a tip-focused actin spot, while low calcium promotes assembly. In support of this, abolishing the calcium gradient resulted in dramatic actin accumulation at the tip. Together these data demonstrate that tipward calcium is quantitatively linked to actin accumulation in vivo and that the moss P. patens provides a powerful system to uncover mechanistic links between calcium, actin, and growth.

  5. Phosphatidylinositol 4,5-Bisphosphate Alters the Number of Attachment Sites between Ezrin and Actin Filaments

    Science.gov (United States)

    Braunger, Julia A.; Brückner, Bastian R.; Nehls, Stefan; Pietuch, Anna; Gerke, Volker; Mey, Ingo; Janshoff, Andreas; Steinem, Claudia

    2014-01-01

    Direct linkage between the plasma membrane and the actin cytoskeleton is controlled by the protein ezrin, a member of the ezrin-radixin-moesin protein family. To function as a membrane-cytoskeleton linker, ezrin needs to be activated in a process that involves binding of ezrin to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphorylation of a conserved threonine residue. Here, we used colloidal probe microscopy to quantitatively analyze the interaction between ezrin and F-actin as a function of these activating factors. We show that the measured individual unbinding forces between ezrin and F-actin are independent of the activating parameters, in the range of approximately 50 piconewtons. However, the cumulative adhesion energy greatly increases in the presence of PIP2 demonstrating that a larger number of bonds between ezrin and F-actin has formed. In contrast, the phosphorylation state, represented by phosphor-mimetic mutants of ezrin, only plays a minor role in the activation process. These results are in line with in vivo experiments demonstrating that an increase in PIP2 concentration recruits more ezrin to the apical plasma membrane of polarized cells and significantly increases the membrane tension serving as a measure of the adhesion sites between the plasma membrane and the F-actin network. PMID:24500715

  6. ALKBH4-dependent demethylation of actin regulates actomyosin dynamics

    DEFF Research Database (Denmark)

    Li, M.-M.; Shi, Y.; Niu, Y.

    2013-01-01

    -type but not catalytically inactive ALKBH4. Similar to actin and myosin knock-out mice, homozygous Alkbh4 mutant mice display early embryonic lethality. These findings imply that ALKBH4-dependent actin demethylation regulates actomyosin function by promoting actin-non-muscle myosin II interaction.......-dependent processes such as cytokinesis and cell migration. ALKBH4-deficient cells display elevated K84me1 levels. Non-muscle myosin II only interacts with unmethylated actin and its proper recruitment to and interaction with actin depend on ALKBH4. ALKBH4 co-localizes with the actomyosin-based contractile ring...

  7. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    International Nuclear Information System (INIS)

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-01

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP 2 and PIP 3 to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  8. Actin and microtubule cytoskeleton interactions

    Czech Academy of Sciences Publication Activity Database

    Petrášek, Jan; Schwarzerová, K.

    2009-01-01

    Roč. 12, č. 6 (2009), s. 728-734 ISSN 1369-5266 R&D Projects: GA ČR GA522/06/1030; GA MŠk(CZ) LC06034 Institutional research plan: CEZ:AV0Z50380511 Keywords : KINESIN-RELATED PROTEIN * TOBACCO BY-2 CELLS * HIGHER-PLANT CELLS Subject RIV: ED - Physiology Impact factor: 10.333, year: 2009

  9. AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Rosero, A.; Žárský, Viktor; Cvrčková, F.

    2013-01-01

    Roč. 64, č. 2 (2013), s. 585-597 ISSN 0022-0957 R&D Projects: GA ČR GAP305/10/0433 Institutional research plan: CEZ:AV0Z50380511 Keywords : Actin * Arabidopsis * At5g25500 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.794, year: 2013

  10. Changes in actin dynamics are involved in salicylic acid signaling pathway

    Czech Academy of Sciences Publication Activity Database

    Matoušková, J.; Janda, M.; Fišer, R.; Šašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, J.; Martinec, Jan; Valentová, O.

    2014-01-01

    Roč. 223, JUN 2014 (2014), s. 36-44 ISSN 0168-9452 R&D Projects: GA ČR(CZ) GAP501/11/1654 Institutional support: RVO:61389030 Keywords : Actin dynamics * Salicylic acid * PR genes Subject RIV: CE - Biochemistry Impact factor: 3.607, year: 2014

  11. Actin-related defense mechanism to reject penetration attempt by a non-pathogen is maintained in tobacco BY-2 cells.

    Science.gov (United States)

    Kobayashi, Issei; Hakuno, Humiaki

    2003-06-01

    The actin cytoskeleton is a key player in defense responses during early stages of infection by fungal pathogens. To investigate molecular mechanisms of actin-related defense responses, a cultured tobacco ( Nicotiana tabacum L.) BY-2 cell system was devised. When conidia were directly deposited on BY-2 cells, neither a pathogen, Erysiphe cichoracearum, nor a non-pathogen, Erysiphe pisi, was able to form appressoria or haustoria on BY-2 cells. On the other hand, conidia of the powdery mildews formed appressoria on BY-2 cells if they were covered with a thin hydrophobic membrane of Formvar. Percentages of appressoria formation of the powdery mildews on the Formvar-covered BY-2 cells were mostly the same as those on leaf epidermal cells. The pathogen successfully penetrated through the membrane into BY-2 cells and formed haustoria, whereas penetration attempts of the non-pathogen were completely rejected by the BY-2 cells similar to attempts on leaf epidermal cells. On the other hand, when BY-2 cells were treated with actin cytoskeleton-depolymerizing agents, cytochalasins, the non-pathogen became able to penetrate and form haustoria in BY-2 cells. Simultaneously, cytochalasin inhibited callose deposition at penetration sites of the non-pathogen. These results demonstrated that the actin cytoskeleton plays an important role in defense mechanisms against fungal penetration, even in the dedifferentiated cultured cells. The newly devised Formvar-covered cultured cell system will be a useful tool for molecular dissection of signal perception and defense mechanisms of plant cells during the early stage of fungal attack.

  12. Actinic flux and photolysis in water droplets: Mie calculations and geometrical optics limit

    Directory of Open Access Journals (Sweden)

    B. Mayer

    2004-01-01

    Full Text Available Photolysis of water-soluble components inside cloud droplets by ultraviolet/visible radiation may play an important role in atmospheric chemistry. Two earlier studies have suggested that the actinic flux and hence the photolysis frequency within spherical droplets is enhanced relative to that in the surrounding air, but have given different values for this enhancement. Here, we reconcile these discrepancies by noting slight errors in both studies that, when corrected, lead to consistent results. Madronich (1987 examined the geometric (large droplet limit and concluded that refraction leads to an enhancement factor, averaged over all incident directions, of 1.56. However, the physically relevant quantity is the enhancement of the average actinic flux (rather than the average enhancement factor which we show here to be 1.26 in the geometric limit. Ruggaber et al. (1997 used Mie theory to derive energy density enhancements slightly larger than 2 for typical droplet sizes, and applied these directly to the calculation of photolysis rates. However, the physically relevant quantity is the actinic flux (rather than the energy density which is obtained by dividing the energy density by the refractive index of water, 1.33. Thus, the Mie-predicted enhancement for typical cloud droplet sizes is in the range 1.5, only coincidentally in agreement with the value originally given by Madronich. We also investigated the influence of resonances in the actinic flux enhancement. These narrow spikes which are resolved only by very high resolution calculations are orders of magnitude higher than the intermediate values but contribute only little to the actinic flux enhancement when averaged over droplet size distributions. Finally, a table is provided which may be used to obtain the actinic flux enhancement for the photolysis of any dissolved species.

  13. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  14. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

    Science.gov (United States)

    Hong, Nan Hyung; Qi, Aidong

    2015-01-01

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

  15. Late complications of rxtherapy: actinic sarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Buffat, J.D.

    1975-09-09

    Relation of two cases of benign tumors: a vertebral osteoblastoma and a cerebellar medulloblastoma which, after operation, have had radiotherapy. 20 years later for one case and 14 years for the other one actinic sarcomas will appear, and, in spite of usual therapy, the death is coming rapidly. We are certainly in presence of two exceptional cases, but each physician must be conscious, before to attempt a treatment, that very distant complication can eventually occur.

  16. Colchicine Depolymerizes Microtubules, Increases Junctophilin-2, and Improves Right Ventricular Function in Experimental Pulmonary Arterial Hypertension.

    Science.gov (United States)

    Prins, Kurt W; Tian, Lian; Wu, Danchen; Thenappan, Thenappan; Metzger, Joseph M; Archer, Stephen L

    2017-05-31

    Pulmonary arterial hypertension (PAH) is a lethal disease characterized by obstructive pulmonary vascular remodeling and right ventricular (RV) dysfunction. Although RV function predicts outcomes in PAH, mechanisms of RV dysfunction are poorly understood, and RV-targeted therapies are lacking. We hypothesized that in PAH, abnormal microtubular structure in RV cardiomyocytes impairs RV function by reducing junctophilin-2 (JPH2) expression, resulting in t-tubule derangements. Conversely, we assessed whether colchicine, a microtubule-depolymerizing agent, could increase JPH2 expression and enhance RV function in monocrotaline-induced PAH. Immunoblots, confocal microscopy, echocardiography, cardiac catheterization, and treadmill testing were used to examine colchicine's (0.5 mg/kg 3 times/week) effects on pulmonary hemodynamics, RV function, and functional capacity. Rats were treated with saline (n=28) or colchicine (n=24) for 3 weeks, beginning 1 week after monocrotaline (60 mg/kg, subcutaneous). In the monocrotaline RV, but not the left ventricle, microtubule density is increased, and JPH2 expression is reduced, with loss of t-tubule localization and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves t-tubule morphology in RV cardiomyocytes. Colchicine therapy diminishes RV hypertrophy, improves RV function, and enhances RV-pulmonary artery coupling. Colchicine reduces small pulmonary arteriolar thickness and improves pulmonary hemodynamics. Finally, colchicine increases exercise capacity. Monocrotaline-induced PAH causes RV-specific derangement of microtubules marked by reduction in JPH2 and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves both t-tubule architecture and RV function. Colchicine also reduces adverse pulmonary vascular remodeling. These results provide biological plausibility for a clinical trial to repurpose colchicine as a RV-directed therapy for PAH

  17. Actin cytoskeleton rearrangements in Arabidopsis roots under stress and during gravitropic response

    Science.gov (United States)

    Pozhvanov, Gregory; Medvedev, Sergei; Suslov, Dmitry; Demidchik, Vadim

    Among environmental factors, gravity vector is the only one which is constant in direction and accompanies the whole plant ontogenesis. That said, gravity vector can be considered as an essential factor for correct development of plants. Gravitropism is a plant growth response against changing its position relative to the gravity vector. It is well estableshed that gravitropism is directed by auxin redistribution across the gravistimulated organ. In addition to auxin, actin cytoskeleton was shown to be involved in gravitropism at different stages: gravity perception, signal transduction and gravitropic bending formation. However, the relationship between IAA and actin is still under discussion. In this work we studied rearrangements of actin cytoskeleton during root gravitropic response. Actin microfilaments were visualized in vivo in GFP-fABD2 transgenic Arabidopsis plants, and their angle distribution was acquired from MicroFilament Analyzer software. The curvature of actin microfilaments in root elongation zone was shown to be increased within 30-60 min of gravistimulation, the fraction of axially oriented microfilaments decreased with a concomitant increase in the fraction of oblique and transversally oriented microfilaments. In particular, the fraction of transversally oriented microfilaments (i.e. parallel to the gravity vector) increased 3-5 times. Under 10 min of sub-lethal salt stress impact, actin microfilament orientations widened from an initial axial orientation to a set of peaks at 15(°) , 45(°) and 90(°) . We conclude that the actin cytoskeleton rearrangements observed are associated with the regulation of basic mechanisms of cell extension growth by which the gravitropic bending is formed. Having common stress-related features, gravity-induced actin cytoskeleton rearrangement is slower but results in higher number of g-vector-parallel microfilaments when compared to salt stress-induced rearrangement. Also, differences in gravistimulated root

  18. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    Science.gov (United States)

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. © 2015. Published by The Company of Biologists Ltd.

  19. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    International Nuclear Information System (INIS)

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J.

    1990-01-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation

  20. Stress generation by myosin minifilaments in actin bundles.

    Science.gov (United States)

    Dasanayake, Nilushi L; Carlsson, Anders E

    2013-06-01

    Forces and stresses generated by the action of myosin minifilaments are analyzed in idealized computer-generated actin bundles, and compared to results for isotropic actin networks. The bundles are generated as random collections of actin filaments in two dimensions with constrained orientations, crosslinked and attached to two fixed walls. Myosin minifilaments are placed on actin filament pairs and allowed to move and deform the network so that it exerts forces on the walls. The vast majority of simulation runs end with contractile minifilament stress, because minifilaments rotate into energetically stable contractile configurations. This process is aided by the bending and stretching of actin filaments, which accomodate minifilament rotation. Stresses for bundles are greater than those for isotropic networks, and antiparallel filaments generate more tension than parallel filaments. The forces transmitted by the actin network to the walls of the simulation cell often exceed the tension in the minifilament itself.

  1. Covalent interactions of acetaldehyde with the actin/microfilament system.

    Science.gov (United States)

    Xu, D S; Jennett, R B; Smith, S L; Sorrell, M F; Tuma, D J

    1989-01-01

    The covalent binding of [14C]acetaldehyde to purified rabbit skeletal muscle actin was characterized. As we have found for other cytoskeletal proteins, actin formed stable covalent adducts under reductive and non-reductive conditions. Under non-reductive conditions, individual and competition binding studies versus albumin both showed that the G-form of actin is more reactive toward acetaldehyde than the F-form. When proteins were compared on an 'equi-lysine' basis under non-reducing conditions, G-actin was found to preferentially compete with albumin for binding to acetaldehyde. Time-course dialysis studies indicated that acetaldehyde-actin adducts become more stable with prolonged incubation at 37 degrees C. These data raise the possibility that actin could be a preferential target for adduct formation in cellular systems and will serve as the basis for ongoing studies aimed at defining the role of acetaldehyde-protein adducts in ethanol-induced cell injury.

  2. Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro.

    Directory of Open Access Journals (Sweden)

    Yaron Ideses

    Full Text Available During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]. Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing

  3. Base-Catalyzed Depolymerization of Lignin with Heterogeneous Catalysts: Cooperative Research and Development Final Report, CRADA Number CRD-13-513

    Energy Technology Data Exchange (ETDEWEB)

    Beckham, Gregg T. [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2015-08-04

    We will synthesize and screen solid catalysts for the depolymerization of lignin to monomeric and oligomeric oxygenated species, which could be fractionated and integrated into refinery intermediate streams for selective upgrading, or catalytically upgraded to fuels and chemicals. This work will primarily focus on the synthesis and application of layered double hydroxides (LDHs) as recyclable, heterogeneous catalysts for depolymerization of lignin model compounds and softwood lignin. LDHs have been shown in our group to offer good supports and catalysts to promote base-catalyzed depolymerization of lignin model compounds and in preliminary experiments for the depolymerization of lignin from an Organosolv process. We will also include additional catalyst supports such as silica, alumina, and carbon as identified in ongoing and past efforts at NREL. This work will consist of two tasks. Overall, this work will be synergistic with ongoing efforts at NREL, funded by the DOE Biomass Program, on the development of catalysts for lignin depolymerization in the context of biochemical and thermochemical conversion of corn stover and other biomass feedstocks to advanced fuels and chemicals.

  4. Rapid changes in phospho-MAP/tau epitopes during neuronal stress: cofilin-actin rods primarily recruit microtubule binding domain epitopes.

    Science.gov (United States)

    Whiteman, Ineka T; Minamide, Laurie S; Goh, De Lian; Bamburg, James R; Goldsbury, Claire

    2011-01-01

    Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer's disease (AD), however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP) tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods) also occur in AD. Using a series of antibodies--AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422--raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD.

  5. Rapid changes in phospho-MAP/tau epitopes during neuronal stress: cofilin-actin rods primarily recruit microtubule binding domain epitopes.

    Directory of Open Access Journals (Sweden)

    Ineka T Whiteman

    Full Text Available Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer's disease (AD, however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods also occur in AD. Using a series of antibodies--AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422--raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD.

  6. A Robust Actin Filaments Image Analysis Framework.

    Directory of Open Access Journals (Sweden)

    Mitchel Alioscha-Perez

    2016-08-01

    Full Text Available The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale. Based on this observation, we propose a three-steps actin filaments extraction methodology: (i first the input image is decomposed into a 'cartoon' part corresponding to the filament structures in the image, and a noise/texture part, (ii on the 'cartoon' image, we apply a multi-scale line detector coupled with a (iii quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in

  7. Myosin Vs organize actin cables in fission yeast.

    Science.gov (United States)

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G

    2012-12-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.

  8. Probing actin polymerization by intermolecular cross-linking

    OpenAIRE

    1988-01-01

    We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an app...

  9. Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

    Science.gov (United States)

    Crevenna, Alvaro H.; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L.; Lamb, Don C.; Wedlich-Söldner, Roland

    2013-01-01

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment. PMID:23486468

  10. Electrostatics control actin filament nucleation and elongation kinetics.

    Science.gov (United States)

    Crevenna, Alvaro H; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L; Lamb, Don C; Wedlich-Söldner, Roland

    2013-04-26

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment.

  11. Daylight photodynamic therapy for actinic keratosis

    DEFF Research Database (Denmark)

    Wiegell, Stine; Wulf, H C; Szeimies, R-M

    2011-01-01

    Photodynamic therapy (PDT) is an attractive therapy for non-melanoma skin cancers including actinic keratoses (AKs) because it allows treatment of large areas; it has a high response rate and results in an excellent cosmesis. However, conventional PDT for AKs is associated with inconveniently long...... clinic visits and discomfort during therapy. In this article, we critically review daylight-mediated PDT, which is a simpler and more tolerable treatment procedure for PDT. We review the effective light dose, efficacy and safety, the need for prior application of sunscreen, and potential clinical scope...

  12. Dynamic buckling of actin within filopodia

    DEFF Research Database (Denmark)

    Leijnse, Natascha; Oddershede, Lene B; Bendix, Pól Martin

    2015-01-01

    Filopodia are active tubular structures protruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filopodia has been studied by measuring the traction forces exerted...... on external substrates.(1) These studies have revealed that internal actin flow can transduce a force across the cell surface through transmembrane linkers like integrins. In addition to the elongation-retraction behavior filopodia also exhibit a buckling and rotational behavior. Filopodial buckling...

  13. Plasmin enzymatic activity in the presence of actin

    Directory of Open Access Journals (Sweden)

    Yusova E. I.

    2015-10-01

    Full Text Available Aim. To study the changes in the plasmin activity towards substrates with high and low molecular mass in the presence of actin. Methods. The proteins used for this investigation were obtained by affinity chromatography and gel-filtration. The plasmin enzymatic activity was determined by a turbidimetric assay and a chromogenic substrate-based assay. The enzyme linked immunosorbent assay and biotin-avidin-phosphatase system were used to study the interaction of plasminogen and its fragments with actin. Results. It was shown that G-actin causes 1.5-fold decrease in the rate of polymeric fibrin hydrolysis by plasmin and Glu-plasminogen activated by the tissue plasminogen activator. However, actin did not impede plasmin autolysis and had no influence on its amidase activity. We have studied an interaction of biotinylated Glu-plasminogen and its fragments (kringle 1-3, kringle 4 and mini-plasminogen with immobilized G-actin. Glu-plasminogen and kringle 4 had a high affinity towards actin (C50 is 113 and 117 nM correspondingly. Mini-plasminogen and kringe 4 did not bind to actin. A similar affinity of Glu-plasminogen and kringle 1-3 towards actin proves the involvement of the kringle 1-3 lysine-binding sites of the native plasminogen form in the actin interaction. Conclusions. Actin can modulate plasmin specificity towards high molecular mass substrates through its interaction with lysine-binding sites of the enzyme kringle domains. Actin inhibition of the fibrinolytic activity of plasmin is due to its competition with fibrin for thelysine binding sites of plasminogen/plasmin.

  14. Force Transmission in the Actin Cytoskeleton

    Science.gov (United States)

    Gardel, Margaret

    2012-02-01

    The ability of cells to sense and generate mechanical forces is essential to numerous aspects of their physiology, including adhesion, migration, division and differentiation. To a large degree, cellular tension is regulated by the transmission of myosin II-generated forces through the filamentous actin (F-actin) cytoskeleton. While transmission of myosin-generated stresses from the molecular to cellular length scale is well understood in the context of highly organized sarcomeres found in striated muscle, non-muscle and smooth muscle cells contain a wide variety of bundles and networks lacking sarcomeric organization. I will describe the in vitro and in vivo approaches we use to study force transmission in such disordered actomyosin assemblies. Our in vivo results are showing that highly organized stress fibers contribute surprisingly little to the overall extent of cellular tension as compared to disordered actomyosin meshworks. Our in vitro results are demonstrating the mechanisms of symmetry breaking in disordered actomyosin bundles that facilitate the formation of contractile bundles with well-defined ``contractile elements.'' These results provide insight into the self-organization of actomyosin cytoskeleton in non-muscle cells that regulate and maintain cellular tension.

  15. Actin binding proteins, spermatid transport and spermiation*

    Science.gov (United States)

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  16. Nuclear localisation of the G-actin sequestering peptide thymosin beta4.

    Science.gov (United States)

    Huff, Thomas; Rosorius, Olaf; Otto, Angela M; Müller, Christian S G; Ballweber, Edda; Hannappel, Ewald; Mannherz, Hans Georg

    2004-10-15

    Thymosin beta4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin beta4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin beta4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin beta4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin beta4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin beta4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

  17. A receptor-interacting protein 1 (RIP1)-independent necrotic death under the control of protein phosphatase PP2A that involves the reorganization of actin cytoskeleton and the action of cofilin-1.

    Science.gov (United States)

    Tomasella, Andrea; Blangy, Anne; Brancolini, Claudio

    2014-09-12

    Cell death by necrosis is emerging not merely as a passive phenomenon but as a cell-regulated process. Here, by using different necrotic triggers, we prove the existence of two distinct necrotic pathways. The mitochondrial reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone elicits necrosis characterized by the involvement of RIP1 and Drp1. However, G5, a non-selective isopeptidase inhibitor, triggers a distinct necrotic pathway that depends on the protein phosphatase PP2A and the actin cytoskeleton. PP2A catalytic subunit is stabilized by G5 treatment, and its activity is increased. Furthermore, PP2Ac accumulates into the cytoplasm during necrosis similarly to HMGB1. We have also defined in the actin-binding protein cofilin-1 a link between PP2A, actin cytoskeleton, and necrotic death. Cofilin-1-severing/depolymerization activity is negatively regulated by phosphorylation of serine 3. PP2A contributes to the dephosphorylation of serine 3 elicited by G5. Finally, a cofilin mutant that mimics phosphorylated Ser-3 can partially rescue necrosis in response to G5. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization.

    Science.gov (United States)

    Delphin, Christian; Bouvier, Denis; Seggio, Maxime; Couriol, Emilie; Saoudi, Yasmina; Denarier, Eric; Bosc, Christophe; Valiron, Odile; Bisbal, Mariano; Arnal, Isabelle; Andrieux, Annie

    2012-10-12

    Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

  19. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening.

    Science.gov (United States)

    Santiago-Doménech, Nieves; Jiménez-Bemúdez, Silvia; Matas, Antonio J; Rose, Jocelyn K C; Muñoz-Blanco, Juan; Mercado, José A; Quesada, Miguel A

    2008-01-01

    Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of pectate lyase genes by antisense technology in strawberry (Fragaria x ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in pectate lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell-cell adhesion was substantially reduced in the transgenic fruits. These results indicate that pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly.

  20. Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea

    Science.gov (United States)

    Kawakami-Schulz, Sharolyn V.; Verdoni, Angela M.; Sattler, Shannon G.; Jessen, Erik; Kao, Winston W.-Y.; Ikeda, Akihiro

    2014-01-01

    Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstncorn1 mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstncorn1 mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstncorn1 cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstncorn1 cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstncorn1 mice. Comparison of gene expression values from WT, Dstncorn1 mutant, and Dstncorn1 rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstncorn1 mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstncorn1 mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstncorn1 mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis. PMID:24550211

  1. Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea.

    Science.gov (United States)

    Kawakami-Schulz, Sharolyn V; Verdoni, Angela M; Sattler, Shannon G; Jessen, Erik; Kao, Winston W-Y; Ikeda, Akihiro; Ikeda, Sakae

    2014-04-15

    Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstn(corn1) mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstn(corn1) mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstn(corn1) cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstn(corn1) cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstn(corn1) mice. Comparison of gene expression values from WT, Dstn(corn1) mutant, and Dstn(corn1) rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstn(corn1) mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstn(corn1) mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstn(corn1) mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.

  2. The evolution of compositionally and functionally distinct actin filaments.

    Science.gov (United States)

    Gunning, Peter W; Ghoshdastider, Umesh; Whitaker, Shane; Popp, David; Robinson, Robert C

    2015-06-01

    The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity. © 2015. Published by The Company of Biologists Ltd.

  3. Tropomyosin isoforms bias actin track selection by vertebrate myosin Va

    Science.gov (United States)

    Sckolnick, Maria; Krementsova, Elena B.; Warshaw, David M.; Trybus, Kathleen M.

    2016-01-01

    Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin–Tpm3.1 and actin–Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin–Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an “actin–Tpm code” that allows for spatial and temporal sorting of actomyosin function in the cell. PMID:27535431

  4. Deafness and espin-actin self-organization in stereocilia

    Science.gov (United States)

    Wong, Gerard C. L.

    2009-03-01

    Espins are F-actin-bundling proteins associated with large parallel actin bundles found in hair cell stereocilia in the ear, as well as brush border microvilli and Sertoli cell junctions. We examine actin bundle structures formed by different wild-type espin isoforms, fragments, and naturally-occurring human espin mutants linked to deafness and/or vestibular dysfunction. The espin-actin bundle structure consisted of a hexagonal arrangement of parallel actin filaments in a non-native twist state. We delineate the structural consequences caused by mutations in espin's actin-bundling module. For espin mutation with a severely damaged actin-bundling module, which are implicated in deafness in mice and humans, oriented nematic-like actin filament structures, which strongly impinges on bundle mechanical stiffness. Finally, we examine what makes espin different, via a comparative study of bundles formed by espin and those formed by fascin, a prototypical bundling protein found in functionally different regions of the cell, such as filopodia.

  5. Actin-mediated cytoplasmic organization of plant cells

    NARCIS (Netherlands)

    Honing, van der H.S.

    2011-01-01

    In this thesis, I present results that give insight in the role of the actin cytoskeleton in the production of an organized cytoplasm in plant cells, which is, for instance, required for proper cell morphogenesis.

    Chapter 1 is a review in which we discuss the possible role of actin-based

  6. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    Science.gov (United States)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  7. Actin dynamics, architecture, and mechanics in cell motility.

    Science.gov (United States)

    Blanchoin, Laurent; Boujemaa-Paterski, Rajaa; Sykes, Cécile; Plastino, Julie

    2014-01-01

    Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

  8. Structural modeling and molecular dynamics simulation of the actin filament.

    Science.gov (United States)

    Splettstoesser, Thomas; Holmes, Kenneth C; Noé, Frank; Smith, Jeremy C

    2011-07-01

    Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized. Copyright © 2011 Wiley-Liss, Inc.

  9. Measuring Actin Flow in 3D Cell Protrusions

    Science.gov (United States)

    Chiu, Chi-Li; Digman, Michelle A.; Gratton, Enrico

    2013-01-01

    Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of

  10. Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae

    Directory of Open Access Journals (Sweden)

    Benjamin B. A. Raymond

    2018-02-01

    Full Text Available Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15 using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM, and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

  11. Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Raymond, Benjamin B A; Madhkoor, Ranya; Schleicher, Ina; Uphoff, Cord C; Turnbull, Lynne; Whitchurch, Cynthia B; Rohde, Manfred; Padula, Matthew P; Djordjevic, Steven P

    2018-01-01

    Mycoplasma hyopneumoniae , an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

  12. Ultraviolet actinic flux in clear and cloudy atmospheres: model calculations and aircraft-based measurements

    Directory of Open Access Journals (Sweden)

    G. G. Palancar

    2011-06-01

    Full Text Available Ultraviolet (UV actinic fluxes measured with two Scanning Actinic Flux Spectroradiometers (SAFS aboard the NASA DC-8 aircraft are compared with the Tropospheric Ultraviolet-Visible (TUV model. The observations from 17 days in July-August 2004 (INTEX-NA field campaign span a wide range of latitudes (28° N–53° N, longitudes (45° W–140° W, altitudes (0.1–11.9 km, ozone columns (285–353 DU, and solar zenith angles (2°–85°. Both cloudy and cloud-free conditions were encountered. For cloud-free conditions, the ratio of observed to clear-sky-model actinic flux (integrated from 298 to 422 nm was 1.01±0.04, i.e. in good agreement with observations. The agreement improved to 1.00±0.03 for the down-welling component under clear sky conditions. In the presence of clouds and depending on their position relative to the aircraft, the up-welling component was frequently enhanced (by as much as a factor of 8 relative to cloud-free values while the down-welling component showed both reductions and enhancements of up to a few tens of percent. Including all conditions, the ratio of the observed actinic flux to the cloud-free model value was 1.1±0.3 for the total, or separately 1.0±0.2 for the down-welling and 1.5±0.8 for the up-welling components. The correlations between up-welling and down-welling deviations are well reproduced with sensitivity studies using the TUV model, and are understood qualitatively with a simple conceptual model. This analysis of actinic flux observations illustrates opportunities for future evaluations of photolysis rates in three-dimensional chemistry-transport models.

  13. Lsb1 is a negative regulator of las17 dependent actin polymerization involved in endocytosis.

    Directory of Open Access Journals (Sweden)

    Matthias Spiess

    Full Text Available The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.

  14. Cyclic hardening in bundled actin networks.

    Science.gov (United States)

    Schmoller, K M; Fernández, P; Arevalo, R C; Blair, D L; Bausch, A R

    2010-01-01

    Nonlinear deformations can irreversibly alter the mechanical properties of materials. Most soft materials, such as rubber and living tissues, display pronounced softening when cyclically deformed. Here we show that, in contrast, reconstituted networks of crosslinked, bundled actin filaments harden when subject to cyclical shear. As a consequence, they exhibit a mechano-memory where a significant stress barrier is generated at the maximum of the cyclic shear strain. This unique response is crucially determined by the network architecture: at lower crosslinker concentrations networks do not harden, but soften showing the classic Mullins effect known from rubber-like materials. By simultaneously performing macrorheology and confocal microscopy, we show that cyclic shearing results in structural reorganization of the network constituents such that the maximum applied strain is encoded into the network architecture.

  15. A kinome RNAi screen identified AMPK as promoting poxvirus entry through the control of actin dynamics.

    Directory of Open Access Journals (Sweden)

    Theresa S Moser

    2010-06-01

    Full Text Available Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator.

  16. Titin-Actin Interaction: PEVK-Actin-Based Viscosity in a Large Animal

    Directory of Open Access Journals (Sweden)

    Charles S. Chung

    2011-01-01

    Full Text Available Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5–1.0 than mice (N2BA:N2B ratio ~0.2. To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL to SL of 2.15 μm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1–400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 μm and by 50% at 2.25 μm, suggesting a SL-dependent nature of viscosity that might prevent SL ``overshoot’’ at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.

  17. Molecular investigations into the mechanics of actin in different nucleotide states.

    Science.gov (United States)

    Lee, Ji Y; Iverson, Tyler M; Dima, Ruxandra I

    2011-01-13

    Actin plays crucial roles in the mechanical response of cells to applied forces. For example, during cell adhesion, under the action of forces transmitted through integrins, actin filaments (F-actin) induce intracellular mechanical movements leading to changes in the cell shape. Muscle contraction results from the interaction of F-actin with the molecular motor myosin. Thus, understanding the origin of actin's mechanical flexibility is required to understand the basis of fundamental cellular processes. F-actin results from the polymerization of globular actin (G-actin), which contains one tightly bound nucleotide (ATP or ADP). Experiments revealed that G-actin is more flexible than F-actin, but no molecular-level understanding of this differential behavior exists. To probe the basis of the mechanical behavior of actin, we study the force response of G-actin bound with ATP (G-ATP) or ADP (G-ADP). We investigate the global unfolding of G-actin under forces applied at its ends and its mechanical resistance along the actin-actin and actin-myosin bonds in F-actin. Our study reveals that the nucleotide plays an important role in the global unfolding of actin, leading to multiple unfolding scenarios which emphasize the differences between the G-ATP and G-ADP states. Furthermore, our simulations show that G-ATP is more flexible than G-ADP and that the actin-myosin interaction surface responds faster to force than the actin-actin interaction surface. The deformation of G-actin under tension revealed in our simulations correlates very well with experimental data on G-actin domain flexibility.

  18. Triggering actin comets versus membrane ruffles: distinctive effects of phosphoinositides on actin reorganization.

    Science.gov (United States)

    Ueno, Tasuku; Falkenburger, Björn H; Pohlmeyer, Christopher; Inoue, Takanari

    2011-12-13

    A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. First, using a membrane-recruitable form of PI(4)P 5-kinase, we increased PI(4,5)P(2) synthesis from phosphatidylinositol 4-phosphate [PI(4)P] and found that COS-7, HeLa, and human embryonic kidney 293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P(2) without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P(2) on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions.

  19. Evolutionarily conserved modules in actin nucleation: lessons from Dictyostelium discoideum and plants

    Czech Academy of Sciences Publication Activity Database

    Cvrčková, F.; Rivero, F.; Bavlnka, Břetislav

    2004-01-01

    Roč. 224, 1/2 (2004), s. 15-31 ISSN 0033-183X R&D Projects: GA ČR GA204/02/1461; GA MŠk LN00A081 Institutional research plan: CEZ:AV0Z5038910 Keywords : actin nucleation * Dictyostelium discoideum * Arabidopsis thaliana Subject RIV: EF - Botanics Impact factor: 1.625, year: 2004

  20. The role of actin isoforms in somatic embryogenesis in Norway spruce

    Czech Academy of Sciences Publication Activity Database

    Schwarzerová, K.; Vondráková, Zuzana; Fischer, L.; Boříková, P.; Bellinvia, E.; Eliášová, Kateřina; Havelková, L.; Fišerová, J.; Vágner, Martin; Opatrný, Z.

    2010-01-01

    Roč. 10, č. 89 (2010), s. 1-13 ISSN 1471-2229 R&D Projects: GA MŠk(CZ) LC06034; GA MŠk OC 158; GA MŠk ME 668 Institutional research plan: CEZ:AV0Z50380511 Keywords : PROGRAMMED CELL-DEATH * GENE FAMILY * F-ACTIN Subject RIV: ED - Physiology Impact factor: 4.085, year: 2010

  1. Interplay between Two Bacterial Actin Homologs, MamK and MamK-Like, Is Required for the Alignment of Magnetosome Organelles in Magnetospirillum magneticum AMB-1

    OpenAIRE

    Abreu, Nicole; Mannoubi, Soumaya; Ozyamak, Ertan; Pignol, David; Ginet, Nicolas; Komeili, Arash

    2014-01-01

    Many bacterial species contain multiple actin-like proteins tasked with the execution of crucial cell biological functions. MamK, an actin-like protein found in magnetotactic bacteria, is important in organizing magnetosome organelles into chains that are used for navigation along geomagnetic fields. MamK and numerous other magnetosome formation factors are encoded by a genetic island termed the magnetosome island. Unlike most magnetotactic bacteria, Magnetospirillum magneticum AMB-1 (AMB-1) ...

  2. Crosstalk between Rac1-mediated actin regulation and ROS production.

    Science.gov (United States)

    Acevedo, Alejandro; González-Billault, Christian

    2018-02-20

    The small RhoGTPase Rac1 is implicated in a variety of events related to actin cytoskeleton rearrangement. Remarkably, another event that is completely different from those related to actin regulation has the same relevance; the Rac1-mediated production of reactive oxygen species (ROS) through NADPH oxidases (NOX). Each outcome involves different Rac1 downstream effectors; on one hand, events related to the actin cytoskeleton require Rac1 to bind to WAVEs proteins and PAKs that ultimately promote actin branching and turnover, on the other, NOX-derived ROS production demands active Rac1 to be bound to a cytosolic activator of NOX. How Rac1-mediated signaling ends up promoting actin-related events, NOX-derived ROS, or both is poorly understood. Rac1 regulators, including scaffold proteins, are known to exert tight control over its functions. Hence, evidence of Rac1 regulatory events leading to both actin remodeling and NOX-mediated ROS generation are discussed. Moreover, cellular functions linked to physiological and pathological conditions that exhibit crosstalk between Rac1 outcomes are analyzed, while plausible roles in neuronal functions (and dysfunctions) are highlighted. Together, discussed evidence shed light on cellular mechanisms which requires Rac1 to direct either actin- and/or ROS-related events, helping to understand crucial roles of Rac1 dual functionality. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Catalytic Depolymerization and Upgrading of Lignin for Vanillin Production: Cooperative Research and Development Final Report, CRADA Number CRD-14-545

    Energy Technology Data Exchange (ETDEWEB)

    Beckham, Gregg [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2017-03-31

    Examine catalytic conversion of lignin using multifunctional catalysts that are able to depolymerize and oxidize lignin to a vanillin-rich stream. Examine separation processes for isolation of vanillin from product mixtures. Conduct preliminary experiments to determine if deconstructed lignin streams can be metabolized by Pseudomonas putida.

  4. The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

    Science.gov (United States)

    Douglas, Lois M; Wang, Hong X; Konopka, James B

    2013-11-26

    Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis. The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane

  5. Treadmilling of actin filaments via Brownian dynamics simulations

    DEFF Research Database (Denmark)

    Guo, Kunkun; Shillcock, Julian C.; Lipowsky, Reinhard

    2010-01-01

    Actin polymerization is coupled to the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi). Therefore, each protomer within an actin filament can attain three different nucleotide states corresponding to bound ATP, ADP / Pi, and ADP....... These protomer states form spatial patterns on the growing (or shrinking) filaments. Using Brownian dynamics simulations, the growth behavior of long filaments is studied, together with the associated protomer patterns, as a function of ATP-actin monomer concentration, CT, within the surrounding solution...

  6. Highly Dynamic Host Actin Reorganization around Developing Plasmodium Inside Hepatocytes

    Science.gov (United States)

    Gomes-Santos, Carina S. S.; Itoe, Maurice A.; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Sepúlveda, Nuno; Simões, Pedro D.; Raquel, Helena; Almeida, António Paulo; Moita, Luis F.; Frischknecht, Friedrich; Mota, Maria M.

    2012-01-01

    Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver. PMID:22238609

  7. Role of sodium ions in the vitrification process: glass matrix modification, slag structure depolymerization, and influence of metal immobilization.

    Science.gov (United States)

    Kuo, Yi-Ming

    2014-07-01

    This study investigates the role of Na ions, a common flux, in the vitrification process. Artificial glass systems composed of Al2O3, CaO, and SiO2 with various Na concentrations were melted at 1450 degrees C. The specimens were cooled by air cooling and water quenching and the metal mobility was evaluated using a sequential extraction procedure. The X-ray diffraction analysis and scanning electron microscopy observations showed that Na ions governed the air-cooled slag's structure. Na ions initially depolymerized CaSiO3-linked chains into CaSiO3 chains, and further cut them into shorter and nonuniform ones, making the slag structure amorphous. With even more Na ions, CaSiO3 chains were divided into single SiO4 tetrahedrons and formed Na-related crystals (Na2Ca3Si2O8 and NaAlSiO4). The phase distributions of Al, Cr, Cu Mn, and Ni showed that Na has a positive effect on the immobilization of heavy metals at suitable concentrations, but a negative effect when in excess amounts. Implications: Vitrification has been widely used to treat hazardous materials. The Na-bearing additives were often used as a flux to improve the melting process. This study described the role of Na played in the vitrification process. The Na ions acted as glass modifier and depolymerize the chain structure of slag. With adequate addition amount of Na ions, the immobilization of heavy metals was improved. The results provided much information about the crystalline phase variation, metal mobility, and surface characteristics while Na serves as a flux.

  8. The Cell Surface Structure of Tumor Endothelial Marker 8 (TEM8) is Regulated by the Actin Cytoskeleton

    OpenAIRE

    Yang, Mi Young; Chaudhary, Amit; Seaman, Steven; Dunty, Jill; Stevens, Janine; Elzarrad, Mohammed K.; Frankel, Arthur E.; St. Croix, Brad

    2010-01-01

    Tumor Endothelial Marker 8 (TEM8) is an integrin-like cell surface protein upregulated on tumor blood vessels and a potential vascular target for cancer therapy. Here, we found that the ability of an anti-TEM8 antibody, clone SB5, to recognize the extracellular domain of TEM8 on the cell surface depends on other host-cell factors. By taking advantage of SB5’s ability to distinguish different forms of cell-surface TEM8, we identified alpha-smooth muscle actin and transgelin, an actin binding p...

  9. EGFR/MEK/ERK/CDK5-dependent integrin-independent FAK phosphorylated on serine 732 contributes to microtubule depolymerization and mitosis in tumor cells.

    Science.gov (United States)

    Rea, K; Sensi, M; Anichini, A; Canevari, S; Tomassetti, A

    2013-10-03

    FAK is a non-receptor tyrosine kinase contributing to migration and proliferation downstream of integrin and/or growth factor receptor signaling of normal and malignant cells. In addition to well-characterized tyrosine phosphorylations, FAK is phosphorylated on several serines, whose role is not yet clarified. We observed that phosphorylated FAK on serine 732 (P-FAKSer732) is present at variable levels in vitro, in several melanoma, ovarian and thyroid tumor cell lines and in vivo, in tumor cells present in fresh ovarian cancer ascites. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its levels were inversely related to the levels of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MTs) of the spindle, biochemically associated with MTs and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. We also identified, for the first time, that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MTs. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling but also in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of tumor cells in which the EGFR/MEK/ERK/CDK5 pathway is active.

  10. Electrostatic interactions between the Bni1p Formin FH2 domain and actin influence actin filament nucleation.

    Science.gov (United States)

    Baker, Joseph L; Courtemanche, Naomi; Parton, Daniel L; McCullagh, Martin; Pollard, Thomas D; Voth, Gregory A

    2015-01-06

    Formins catalyze nucleation and growth of actin filaments. Here, we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and interprotein interactions by molecular dynamics simulations. These simulations produced a refined model for the FH2 dimer associated with the barbed end of the filament and showed electrostatic interactions between the formin knob and actin target-binding cleft. Mutations of two formin residues contributing to these interactions (R1423N, K1467L, or both) reduced the interaction energies between the proteins, and in coarse-grained simulations, the formin lost more interprotein contacts with an actin dimer than with an actin 7-mer. Biochemical experiments confirmed a strong influence of these mutations on Bni1p-mediated actin filament nucleation, but not elongation, suggesting that different interactions contribute to these two functions of formins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Increased expression of secretory actin-binding protein (SABP) on human spermatozoa is associated with poor semen quality

    Czech Academy of Sciences Publication Activity Database

    Čapková, Jana; Elzeinová, Fatima; Novák, Petr

    2007-01-01

    Roč. 22, č. 5 (2007), s. 1396-1404 ISSN 0268-1161 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50520701; CEZ:AV0Z50200510 Keywords : monoclonal antibody * secretory actin-binding protein * human spermatozoa Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.543, year: 2007

  12. The anti-actin drugs latrunculin and cytochalasin affect the maturation of spruce somatic embryos in different ways

    Czech Academy of Sciences Publication Activity Database

    Vondráková, Zuzana; Eliášová, Kateřina; Vágner, Martin

    2014-01-01

    Roč. 221, MAY 2014 (2014), s. 90-99 ISSN 0168-9452 R&D Projects: GA MŠk 7AMB12FR017 Institutional support: RVO:61389030 Keywords : Somatic embryogenesis * Cytoskeleton * Actin Subject RIV: GK - Forestry Impact factor: 3.607, year: 2014

  13. Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein.

    Science.gov (United States)

    Westphal, M; Jungbluth, A; Heidecker, M; Mühlbauer, B; Heizer, C; Schwartz, J M; Marriott, G; Gerisch, G

    1997-03-01

    The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a 'leading edge' of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum. Purified GFP-actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10-20% of the total actin. Microfilaments containing GFP-actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP-actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP-actin and phalloidin fluorescence, but there were distinct sites in which GFP-actin was more prominent. Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

  14. Nanosecond electric pulses trigger actin responses in plant cells

    International Nuclear Information System (INIS)

    Berghoefer, Thomas; Eing, Christian; Flickinger, Bianca; Hohenberger, Petra; Wegner, Lars H.; Frey, Wolfgang; Nick, Peter

    2009-01-01

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  15. The role of actin networks in cellular mechanosensing

    Science.gov (United States)

    Azatov, Mikheil

    Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

  16. Disruption of the F-actin cytoskeleton and monolayer barrier integrity induced by PAF and the protective effect of ITF on intestinal epithelium.

    Science.gov (United States)

    Xu, Ling-fen; Xu, Cheng; Mao, Zhi-Qin; Teng, Xu; Ma, Li; Sun, Mei

    2011-02-01

    To explore whether platelet-activating factor (PAF) can disrupt the intestinal epithelial barrier directly and is associated with structural alterations of the F-actin-based cytoskeleton, and to observe the protective effect of intestinal trefoil factor (ITF), we establish an intestinal epithelia barrier model using Caco-2 cells in vitro. Transepithelial electrical resistance and unidirectional flux of lucifer yellow were measured to evaluate barrier permeability; immunofluorescent staining and flow cytometry were applied to observe morphological alterations and to quantify proteins of the F-actin cytoskeleton: the tight junction marker ZO-1 and Claudin-1 were observed using immunofluorescent staining. PAF significantly increased paracellular permeability, at the same time, F-actin and tight junction proteins were disrupted. It was thought that ITF could reverse the high permeability by restoring normal F-actin, ZO-1 and Claudin-1 structures. These results collectively demonstrated that PAF plays an important role in the regulation of mucosal permeability and the effects of PAF are correlated with structural alterations of the F-actin-based cytoskeleton and of tight junctions. ITF can protect intestinal epithelium against PAF-induced disruption by restricting the rearrangement of the F-actin cytoskeleton and of tight junctions.

  17. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  18. Rheology of Membrane-Attached Minimal Actin Cortices.

    Science.gov (United States)

    Noeding, Helen; Schoen, Markus; Kramer, Corinna; Doerrer, Nils; Kuerschner, Aileen; Geil, Burkhard; Mey, Ingo P; Heussinger, Claus; Janshoff, Andreas; Steinem, Claudia

    2018-03-28

    The actin cortex is a thin cross-linked network attached to the plasma membrane, being responsible for the cell's shape during migration, division and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) to which a constitutively active mutant of ezrin, being a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P2/ezrin pinning points revealing an increase in the intersections between actin filaments, i.e., the node density of the MAC. Bead tracking microrheology on the membrane attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G0 to the node density of the MAC.

  19. Liquid droplets of cross-linked actin filaments

    Science.gov (United States)

    Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret

    Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.

  20. Nucleotide effects on the structure and dynamics of actin.

    Science.gov (United States)

    Zheng, Xiange; Diraviyam, Karthikeyan; Sept, David

    2007-08-15

    Adenosine 5'-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5'-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an alpha-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.

  1. Functional characterisation of filamentous actin probe expression in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Shrujna Patel

    Full Text Available Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.

  2. Analysis of the DNA binding proteins interacting with specific upstream sequences of the S. purpuratus CyI actin gene.

    Science.gov (United States)

    Ganster, R; Paul, H; Katula, K S

    1992-12-01

    The CyI actin gene of the sea urchin, Strongylocentrotus purpuratus, is regulated temporally and spatially within the cells of the early embryo. In an effort to understand the molecular basis for the CyI actin pattern of expression, we have begun analyzing the protein-DNA interactions within regions previously shown to be of potential functional importance (Katula et al., 1987). Using DNase I footprinting, 10 protected regions were identified containing both conserved and apparently novel protein binding sites. Gel mobility shift competition assays confirmed the presence of multiple protein factors which specifically recognize CyI actin upstream sequences. Determination of a relative affinity constant value (Kr) indicated that most of the protein factors preferred their respective oligonucleotide sequences vs. a synthetic competitor DNA in a range of 10(4). The highest affinity binding was observed for proteins binding to the oligonucleotide probe containing the octamer element (Kr approximately 10(6)). Heterologous gel shift competition assays were carried out to investigate the interrelatedness of the protein factors. These studies, combined with other data, indicate there are both unique and redundant protein-DNA interactions in the region being examined. Possible alterations in CyI actin DNA binding proteins were investigated during the period of CyI transcriptional activation by gel mobility shift analysis. An increase in binding activity was observed for most of the factors, indicating that early transcriptional activity of CyI actin may involve a general increase in the amount or activity of specific transcription factors. In addition, qualitative changes, as seen by alterations in the shift patterns, were observed for some of the oligonucleotide probes.

  3. An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome.

    Science.gov (United States)

    Gil-Krzewska, Aleksandra; Saeed, Mezida B; Oszmiana, Anna; Fischer, Elizabeth R; Lagrue, Kathryn; Gahl, William A; Introne, Wendy J; Coligan, John E; Davis, Daniel M; Krzewski, Konrad

    2017-12-11

    Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel

  4. ACTIN BINDING PROTEIN29 from Lilium Pollen Plays an Important Role in Dynamic Actin Remodeling[W][OA

    Science.gov (United States)

    Xiang, Yun; Huang, Xi; Wang, Ting; Zhang, Yan; Liu, Qinwen; Hussey, Patrick J.; Ren, Haiyun

    2007-01-01

    Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263–amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca2+- and/or phosphatidylinositol 4,5-bisphosphate–regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth. PMID:17586658

  5. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  6. Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis.

    Science.gov (United States)

    Vehlow, Anne; Soong, Daniel; Vizcay-Barrena, Gema; Bodo, Cristian; Law, Ah-Lai; Perera, Upamali; Krause, Matthias

    2013-10-16

    The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.

  7. RickA expression is not sufficient to promote actin-based motility of Rickettsia raoultii.

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    Premanand Balraj

    Full Text Available BACKGROUND: Rickettsia raoultii is a novel Rickettsia species recently isolated from Dermacentor ticks and classified within the spotted fever group (SFG. The inability of R. raoultii to spread within L929 cells suggests that this bacterium is unable to polymerize host cell actin, a property exhibited by all SFG rickettsiae except R. peacocki. This result led us to investigate if RickA, the protein thought to generate actin nucleation, was expressed within this rickettsia species. METHODOLOGY/PRINCIPAL FINDINGS: Amplification and sequencing of R. raoultii rickA showed that this gene encoded a putative 565 amino acid protein highly homologous to those found in other rickettsiae. Using immunofluorescence assays, we determined that the motility pattern (i.e. microcolonies or cell-to-cell spreading of R. raoultii was different depending on the host cell line in which the bacteria replicated. In contrast, under the same experimental conditions, R. conorii shares the same phenotype both in L929 and in Vero cells. Transmission electron microscopy analysis of infected cells showed that non-motile bacteria were free in the cytosol instead of enclosed in a vacuole. Moreover, western-blot analysis demonstrated that the defect of R. raoultii actin-based motility within L929 cells was not related to lower expression of RickA. CONCLUSION/SIGNIFICANCE: These results, together with previously published data about R. typhi, strongly suggest that another factor, apart from RickA, may be involved with be responsible for actin-based motility in bacteria from the Rickettsia genus.

  8. Participation of actin on Giardia lamblia growth and encystation.

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    Araceli Castillo-Romero

    Full Text Available BACKGROUND: Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. METHODOLOGY AND PRINCIPAL FINDINGS: By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45% and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of fluorescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%. CONCLUSIONS AND SIGNIFICANCE: All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression.

  9. Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

    Science.gov (United States)

    Hou, Guichuan; Mohamalawari, Deepti R.; Blancaflor, Elison B.

    2003-01-01

    The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

  10. The receptor-like MLO protein and the RAC/ROP family G-protein RACB modulate actin reorganization in barley attacked by the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei.

    Science.gov (United States)

    Opalski, Krystina S; Schultheiss, Holger; Kogel, Karl-Heinz; Hückelhoven, Ralph

    2005-01-01

    Cytoskeleton remodelling is a crucial process in determining the polarity of dividing and growing plant cells, as well as during interactions with the environment. Nothing is currently known about the proteins, which regulate actin remodelling during interactions with invading pathogens. The biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) invades susceptible barley (Hordeum vulgare L.) by penetrating epidermal cells, which remain intact during fungal development. In contrast, resistant host plants prevent infection by inhibiting penetration through apoplastic mechanisms, which require focusing defence reactions on the site of attack. We stained actin filaments in a susceptible Mlo-genotype and a near-isogenic race-non-specifically resistant barley mlo5-mutant genotype using fluorescence-labelled phalloidin after chemical fixation. This revealed that the actin cytoskeleton is differentially reorganized in susceptible and resistant hosts challenged by Bgh. Actin filaments were polarized towards the sites of attempted penetration in the resistant host, whereas when susceptible hosts were penetrated, a more subtle reorganization took place around fungal haustoria. Strong actin filament focusing towards sites of fungal attack was closely associated with successful prevention of penetration. Actin focusing was less frequent and seemingly delayed in susceptible wild-type barley expressing the susceptibility factor MLO. Additionally, single cell overexpression of a constitutively activated RAC/ROP G-protein, CA RACB, another potential host susceptibility factor and hypothetical actin cytoskeleton regulator, partly inhibited actin reorganization when under attack from Bgh, whereas knockdown of RACB promoted actin focusing. We conclude that RACB and, potentially, MLO are host proteins involved in the modulation of actin reorganization and cell polarity in the interaction of barley with Bgh.

  11. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

    Energy Technology Data Exchange (ETDEWEB)

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L. (Queensland); (Aust. Synch.)

    2008-08-21

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

  12. Microscale Mechanics of Actin Networks During Dynamic Assembly and Dissociation

    Science.gov (United States)

    Gurmessa, Bekele; Robertson-Anderson, Rae; Ross, Jennifer; Nguyen, Dan; Saleh, Omar

    Actin is one of the key components of the cytoskeleton, enabling cells to move and divide while maintaining shape by dynamic polymerization, dissociation and crosslinking. Actin polymerization and network formation is driven by ATP hydrolysis and varies depending on the concentrations of actin monomers and crosslinking proteins. The viscoelastic properties of steady-state actin networks have been well-characterized, yet the mechanical properties of these non-equilibrium systems during dynamic assembly and disassembly remain to be understood. We use semipermeable microfluidic devices to induce in situ dissolution and re-polymerization of entangled and crosslinked actin networks, by varying ATP concentrations in real-time, while measuring the mechanical properties during disassembly and re-assembly. We use optical tweezers to sinusoidally oscillate embedded microspheres and measure the resulting force at set time-intervals and in different regions of the network during cyclic assembly/disassembly. We determine the time-dependent viscoelastic properties of non-equilibrium network intermediates and the reproducibility and homogeneity of network formation and dissolution. Results inform the role that cytoskeleton reorganization plays in the dynamic multifunctional mechanics of cells. NSF CAREER Award (DMR-1255446) and a Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

  13. All-Round Manipulation of the Actin Cytoskeleton by HIV.

    Science.gov (United States)

    Ospina Stella, Alberto; Turville, Stuart

    2018-02-05

    While significant progress has been made in terms of human immunodeficiency virus (HIV) therapy, treatment does not represent a cure and remains inaccessible to many people living with HIV. Continued mechanistic research into the viral life cycle and its intersection with many aspects of cellular biology are not only fundamental in the continued fight against HIV, but also provide many key observations of the workings of our immune system. Decades of HIV research have testified to the integral role of the actin cytoskeleton in both establishing and spreading the infection. Here, we review how the virus uses different strategies to manipulate cellular actin networks and increase the efficiency of various stages of its life cycle. While some HIV proteins seem able to bind to actin filaments directly, subversion of the cytoskeleton occurs indirectly by exploiting the power of actin regulatory proteins, which are corrupted at multiple levels. Furthermore, this manipulation is not restricted to a discrete class of proteins, but rather extends throughout all layers of the cytoskeleton. We discuss prominent examples of actin regulators that are exploited, neutralized or hijacked by the virus, and address how their coordinated deregulation can lead to changes in cellular behavior that promote viral spreading.

  14. Incorporation of β-actin loading control into zymography.

    Science.gov (United States)

    Govindasamy, Natasha; Yan, MengJie; Jurasz, Paul

    2016-11-01

    Gelatin zymography and immunoblot are widely used gel electrophoresis techniques to study matrix metalloproteinases-2 and -9. Each method has its advantages and disadvantages. Zymography is exquisitely sensitive but offers no loading control to ensure equal sample loading. Immunoblot is a 100-1000-fold less sensitive, but allows for the probing of a sample loading control such as β-actin to ensure accurate protein loading. In this report, we describe two simple protocols that combine gelatin zymography to study MMP-2 and -9 levels with an in-gel β-actin immunoblot loading control, thus combining sensitivity and accuracy in a single assay. The protocols incorporate the loading of molecular weight markers to demarcate MMP-2/-9 from the β-actin. The first protocol utilizes the overlay of a 10% zymography gel over a 5% Tris-Glycine separating gel from which the β-actin is transferred. The second protocol involves the direct transfer of the β-actin from a single 10% zymography gel.

  15. Localization of actin in pollen tubes of Ornithogalum virens L.

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    Małgorzata Stępka

    2014-01-01

    Full Text Available The germinating pollen grain (in vivo on the stigma or in vitro in germination medium forms a pollen tube which transports the vegetative nucleus and generative cell/two sperm cells participating in the process of double fertilization. The growth of the tube and the transport of organelles and the cells occur due to two major motor systems existing in the pollen tubes of higher plants: the tubuline-dynein/kinesin and the actin-myosin system. In pollen tubes of Ornithogalum virens the actin filaments were labelled with TRITC-phalloidin (2 µg/ml in the PIPES buffer and the 10% sucrose, without the fixative and DMSO. Omission of the fixative and permeabilizing agent (DMSO allowed better preservation of the structure, and the "fluorescence" of actin was observed in living pollen tubes. Observations in CLSM (confocal laser scanning microscope showed that actin is distributed in the vicinity of the cell membrane. This could support the view that actin filaments and the plasmalemma form the pollen tube cortex along which the cytoplasmic movement of organelles, and cell transport occurs.

  16. Novel Actin-like Filament Structure from Clostridium tetani*

    Science.gov (United States)

    Popp, David; Narita, Akihiro; Lee, Lin Jie; Ghoshdastider, Umesh; Xue, Bo; Srinivasan, Ramanujam; Balasubramanian, Mohan K.; Tanaka, Toshitsugu; Robinson, Robert C.

    2012-01-01

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines. PMID:22514279

  17. Novel actin-like filament structure from Clostridium tetani.

    Science.gov (United States)

    Popp, David; Narita, Akihiro; Lee, Lin Jie; Ghoshdastider, Umesh; Xue, Bo; Srinivasan, Ramanujam; Balasubramanian, Mohan K; Tanaka, Toshitsugu; Robinson, Robert C

    2012-06-15

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

  18. Addition of electrophilic lipids to actin alters filament structure

    International Nuclear Information System (INIS)

    Gayarre, Javier; Sanchez, David; Sanchez-Gomez, Francisco J.; Terron, Maria C.; Llorca, Oscar; Perez-Sala, Dolores

    2006-01-01

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Δ 12,14 -PGJ 2 (15d-PGJ 2 ) and PGA 1 in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA 1 and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ 2 or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ 2 at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles

  19. Oral acetylsalicylic acid and prevalence of actinic keratosis.

    Science.gov (United States)

    Schmitt, Juliano; Miot, Hélio

    2014-01-01

    To investigate the influence of a regular oral use of acetylsalicylic acid in the prevalence of actinic keratosis. A case-control study with dermatologic outpatients above 50 years of age assessed between 2009 and 2011. Cases were defined as those who had been under regular use of oral acetylsalicylic acid for more than six consecutive months. The assessment focused on: age, sex, skin-type, tobacco smoking, use of medication, occurrence of individual or family skin cancer, and sunscreen and sun exposure habits. Actinic keratoses were counted in the medial region of the face and upper limbs. Counts were adjusted by co-variables based on a generalized linear model. A total of 74 cases and 216 controls were assessed. The median time of acetylsalicylic acid use was 36 months. Cases differed from controls as to the highest age, highest prevalence of use of angiotensin-converting enzyme inhibitors and fewer keratosis on the face and on the upper limbs (pacetylsalicylic acid was associated to lower counts of face actinic keratosis and upper-limb erythematous actinic keratosis (pacetylsalicylic acid for more than six months was associated to a lower prevalence of actinic keratosis, especially facial and erythematous ones.

  20. Multiple roles for the actin cytoskeleton during regulated exocytosis

    Science.gov (United States)

    Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

    2014-01-01

    Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

  1. A Fast and Robust UHPLC-MRM-MS Method to Characterize and Quantify Grape Skin Tannins after Chemical Depolymerization

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    Lucie Pinasseau

    2016-10-01

    Full Text Available A rapid, sensitive, and selective analysis method using ultra high performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ-MS has been developed for the characterization and quantification of grape skin flavan-3-ols after acid-catalysed depolymerization in the presence of phloroglucinol (phloroglucinolysis. The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM mode, this fast gradient robust method allows analysis of constitutive units of grape skin proanthocyanidins, including some present in trace amounts, in a single injection, with a throughput of 6 samples per hour. This method was applied to a set of 214 grape skin samples from 107 different red and white grape cultivars grown under two conditions in the vineyard, irrigated or non-irrigated. The results of triplicate analyses confirmed the robustness of the method, which was thus proven to be suitable for high-throughput and large-scale metabolomics studies. Moreover, these preliminary results suggest that analysis of tannin composition is relevant to investigate the genetic bases of grape response to drought.

  2. Depolymerization of cellulose into high-value chemicals by using synergy of zinc chloride hydrate and sulfate ion promoted titania catalyst.

    Science.gov (United States)

    Wei, Weiqi; Wu, Shubin

    2017-10-01

    Experiments for cellulose depolymerization by synergy of zinc chloride hydrate (ZnCl 2 ·RH 2 O) and sulfated titania catalyst (SO 4 2- /TiO 2 ) were investigated in this study. The results showed the introduction of sulfate into the TiO 2 significantly enhanced the catalyst acid amount, especially for Brønsted acid site, which is beneficial for subsequent cellulose depolymerization. ZnCl 2 ·RH 2 O hydrate, only a narrow composition range of water, specifically 3.0≤R≤4.0, can dissolve cellulose, which finally resulted the cellulose with low crystallinity and weak intrachain and interchain hydrogen bond network. Coupling of ZnCl 2 ·RH 2 O hydrate and SO 4 2- /TiO 2 catalyst as a mixed reaction system promoted cellulose depolymerization, and the products can be adjusted by the control of reaction conditions, the low temperature (80-100°C) seemed beneficial for glucose formation (maximal yield 50.5%), and the high temperature (120-140°C) favored to produce levulinic acid (maximal yield 43.1%). Besides, the addition of organic co-solvent making HMF as the main product (maximal yield 38.3%). Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Spatially restricted actin-regulatory signaling contributes to synapse morphology

    Science.gov (United States)

    Nicholson, Daniel A.; Cahill, Michael E.; Tulisiak, Christopher T.; Geinisman, Yuri; Penzes, Peter

    2012-01-01

    The actin cytoskeleton in dendritic spines is organized into microdomains, but how signaling molecules that regulate actin are spatially governed is incompletely understood. Here we examine how the localization of the RacGEF kalirin-7, a well-characterized regulator of actin in spines, varies as a function of postsynaptic density (PSD) area and spine volume. Using serial section electron microscopy (EM), we find that extrasynaptic, but not synaptic, expression of kalirin-7 varies directly with synapse size and spine volume. Moreover, we find that overall expression levels of kalirin-7 differ in spines bearing perforated and non-perforated synapses, due primarily to extrasynaptic pools of kalirin-7 expression in the former. Overall, our findings indicate that kalirin-7 is differentially compartmentalized in spines as a function of both synapse morphology and spine size. PMID:22458534

  4. Spiral actin-polymerization waves can generate amoeboidal cell crawling

    Energy Technology Data Exchange (ETDEWEB)

    Dreher, A.; Aranson, I. S.; Kruse, K.

    2014-05-01

    Amoeboidal cell crawling on solid substrates is characterized by protrusions that seemingly appear randomly along the cell periphery and drive the cell forward. For many cell types, it is known that the protrusions result from polymerization of the actin cytoskeleton. However, little is known about how the formation of protrusions is triggered and whether the appearance of subsequent protrusions is coordinated. Recently, the spontaneous formation of actin-polymerization waves was observed. These waves have been proposed to orchestrate the cytoskeletal dynamics during cell crawling. Here, we study the impact of cytoskeletal polymerization waves on cell migration using a phase-field approach. In addition to directionally moving cells, we find states reminiscent of amoeboidal cell crawling. In this framework, new protrusions are seen to emerge from a nucleation process, generating spiral actin waves in the cell interior. Nucleation of new spirals does not require noise, but occurs in a state that is apparently displaying spatio-temporal chaos.

  5. Condensation of F-Actin by Dimensional Reduction

    Science.gov (United States)

    Bruinsma, Robijn; Christian, Cyron; Mueller, Kei; Bausch, Andreas; Wall, Wolfgang

    2012-02-01

    We present a Brownian Dynamics simulation of the equilibrium condensation of F-actin in the presence of linker molecules. The filaments are modeled as worm-like chains, using finite element analysis. At low linker concentrations, the systems forms a gel whose physical properties do not depend on the linker molecules. If the linker concentration is increased then for isotropic linkers only a single mode of condensation is encountered: bundle formation. If the linker molecules impose a preferential angle between F-actin filaments, then condensation takes place either into a either a hexatic or squaratic two-dimensional liquid crystal phase or into a heterogeneous cluster. Condensation is driven by competition between linker and filament entropy, which imposes dimensional reduction on the F-actin aggregate.

  6. Formation of actin networks in microfluidic concentration gradients

    Directory of Open Access Journals (Sweden)

    Natalja eStrelnikova

    2016-05-01

    Full Text Available The physical properties of cytoskeletal networks are contributors in a number of mechanical responses of cells including cellular deformation and locomotion, and are crucial for the proper action of living cells. Local chemical gradients modulate cytoskeletal functionality including the interactions of the cytoskeleton with other cellular components. Actin is a major constituent of the cytoskeleton. Introducing a microfluidic-based platform, we explored the impact of concentration gradients on the formation and structural properties of actin networks. Microfluidics-controlled flow-free steady state experimental conditions allow for the generation of chemical gradients of different profiles, such as linear or step-like. We discovered specific features of actin networks emerging in defined gradients. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity.

  7. Estrogen and Resveratrol Regulate Rac and Cdc42 Signaling to the Actin Cytoskeleton of Metastatic Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nicolas G. Azios

    2007-02-01

    Full Text Available Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 µM, an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 µM, resveratrol at 5 µM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 µM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 µM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominantnegative Rac retain filopodia response to 50 µM resveratrol. Lamellipodia response to 5 µM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 µM signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.

  8. The F-actin-binding RapGEF GflB is required for efficient macropinocytosis inDictyostelium.

    Science.gov (United States)

    Inaba, Hironori; Yoda, Koji; Adachi, Hiroyuki

    2017-09-15

    Macropinocytosis involves the uptake of large volumes of fluid, which is regulated by various small GTPases. The Dictyostelium discoideum protein GflB is a guanine nucleotide exchange factor (GEF) of Rap1, and is involved in chemotaxis. Here, we studied the role of GflB in macropinocytosis, phagocytosis and cytokinesis. In plate culture of vegetative cells, compared with the parental strain AX2, gflB -knockout (KO) cells were flatter and more polarized, whereas GflB-overproducing cells were rounder. The gflB -KO cells exhibited impaired crown formation and retraction, particularly retraction, resulting in more crowns (macropinocytic cups) per cell and longer crown lifetimes. Accordingly, gflB -KO cells showed defects in macropinocytosis and also in phagocytosis and cytokinesis. F-actin levels were elevated in gflB -KO cells. GflB localized to the actin cortex most prominently at crowns and phagocytic cups. The villin headpiece domain (VHP)-like N-terminal domain of GflB directly interacted with F-actin in vitro Furthermore, a domain enriched in basic amino acids interacted with specific membrane cortex structures such as the cleavage furrow. In conclusion, GflB acts as a key local regulator of actin-driven membrane protrusion possibly by modulating Rap1 signaling pathways. © 2017. Published by The Company of Biologists Ltd.

  9. IR Laser Ablative Decomposition and Depolymerization/Repolymerization of Poly(ethylene succinate)

    Czech Academy of Sciences Publication Activity Database

    Pokorná, Dana; Galíková, Anna; Šubrt, Jan; Blechta, Vratislav; Pola, Josef

    2006-01-01

    Roč. 91, č. 12 (2006), s. 3383-3389 ISSN 0141-3910 R&D Projects: GA ČR(CZ) GA104/04/2028 Institutional research plan: CEZ:AV0Z40720504; CEZ:AV0Z40320502 Keywords : poly(ethylene succinate) * laser degradation * laser-produced films Subject RIV: CC - Organic Chemistry Impact factor: 2.174, year: 2006

  10. Modelling phagosomal lipid networks that regulate actin assembly

    Directory of Open Access Journals (Sweden)

    Schwarz Roland

    2008-12-01

    Full Text Available Abstract Background When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. Isolated phagosomes are metabolically active organelles that contain enzymes and metabolites necessary for lipid interconversion. Hence, addition of ATP, lipids, and actin to the system alter the steady-state composition of the phagosomal membrane at the same time that the actin nucleation is initiated. Our aim was to model all these processes in parallel. Results We compiled detailed experimental data on the effects of different lipids and ATP on actin nucleation and we investigated experimentally lipid interconversion and ATP metabolism in phagosomes by using suitable radioactive compounds. In a first step, a complex lipid network interconnected by chemical reactions catalyzed by known enzymes was modelled in COPASI (Complex Pathway Simulator. However, several lines of experimental evidence indicated that only the phosphatidylinositol branch of the network was active, an observation that dramatically reduced the number of parameters in the model. The results also indicated that a lipid network-independent ATP-consuming activity should be included in the model. When this activity was introduced, the set of differential equations satisfactorily reproduced the experimental data. On the other hand, a molecular mechanism connecting membrane lipids, ATP, and the actin nucleation process is still missing. We therefore adopted a phenomenological (black-box approach to represent the empirical observations. We proposed that lipids and ATP influence the dynamic interconversion between active and inactive actin nucleation sites. With this simple model, all the experimental data were satisfactorily fitted with a single positive parameter per lipid and ATP. Conclusion By establishing an active 'dialogue' between an

  11. Health related quality of life in patients with actinic keratosis

    DEFF Research Database (Denmark)

    Tennvall, Gunnel Ragnarson; Norlin, J M; Malmberg, I

    2015-01-01

    BACKGROUND: Actinic keratosis (AK) is a common skin condition that may progress to non-melanoma skin cancer (NMSC). The disease may influence Health Related Quality of Life (HRQoL), but studies of HRQoL in patients with AK are limited. The purpose of the study was to analyze HRQoL in patients......-center setting. Dermatologists assessed AK severity and patients completed: Actinic Keratosis Quality of Life Questionnaire (AKQoL), Dermatology Life Quality Index (DLQI), and EQ-5D-5 L including EQ-VAS. Differences between categorical subgroups were tested with Wilcoxon rank-sum test. The relationship between...

  12. Actin and Arp2/3 localize at the centrosome of interphase cells

    Energy Technology Data Exchange (ETDEWEB)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan, E-mail: jan.gettemans@vib-ugent.be

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  13. Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Sundaravadivel Balasubramanian

    2010-07-01

    Full Text Available The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring

  14. Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim.

    Science.gov (United States)

    Shao, Xiaowei; Li, Qingsen; Mogilner, Alex; Bershadsky, Alexander D; Shivashankar, G V

    2015-05-19

    Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼ 2 min. This actin reorganization was triggered by an intracellular Ca(2+) burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca(2+)-dependent perinuclear actin assembly. However, the endoplasmic reticulum- and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca(2+)- and INF2-dependent manner.

  15. Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

    Directory of Open Access Journals (Sweden)

    Eric A. Vitriol

    2015-04-01

    Full Text Available Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin regulated by the ordered assembly from and disassembly into actin monomers (G-actin. Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer-binding protein thymosin β4 (Tβ4 for optimal leading-edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it does not interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions.

  16. Molecular cloning and characterization of an actindepolymerizing ...

    African Journals Online (AJOL)

    The expression profiles and characterizations of HbADF suggested that HbADF might be associated with latex regeneration and flow in H. brasiliensis. Key words: Actin cytoskeleton, actin-depolymerizing factor, expression analysis, Hevea brasiliensis, semiquantitative reverse-transcription polymerase chain reaction.

  17. The role of actin and microtubule networks in plasmid DNA intracellular trafficking

    Czech Academy of Sciences Publication Activity Database

    Ondřej, Vladan; Lukášová, Emilie; Falk, Martin; Kozubek, Stanislav

    2007-01-01

    Roč. 54, č. 3 (2007), s. 657-663 ISSN 0001-527X R&D Projects: GA ČR(CZ) GA202/02/0804; GA ČR(CZ) GA202/04/0907; GA AV ČR(CZ) 1QS500040508; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : plasmid DNA * actin filaments * microtubules Subject RIV: BO - Biophysics Impact factor: 1.261, year: 2007

  18. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    Czech Academy of Sciences Publication Activity Database

    Kitamura, H.; Matsumori, H.; Kalendová, Alžběta; Hozák, Pavel; Goldberg, I.G.; Nakao, M.; Saitoh, N.; Harata, M.

    2015-01-01

    Roč. 464, č. 2 (2015), s. 554-560 ISSN 0006-291X R&D Projects: GA MŠk EE2.3.30.0050; GA MŠk(CZ) ED1.1.00/02.0109 Grant - others: Human Frontier in Science programe(FR) RGP0017/2013 Institutional support: RVO:68378050 Keywords : Actin-related protein * ARP6 * Histone H2A.Z * Nucleolus * Wndchrm Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.371, year: 2015

  19. Latex production with depolymerizing compounds of actin cytoskeleton in rubber trees Produção de látex estimulada pela aplicação de compostos despolimerizantes de citoesqueleto de actina em seringueiras

    Directory of Open Access Journals (Sweden)

    Gao Zheng-Quan

    2008-02-01

    Full Text Available The objective of this work was to assess stimulated latex flow from rubber trees (Hevea brasiliensis with saturated macrolide (latrunculin A, 1, 5, and 10% potassium iodide in 2% methylcellulose compared with 0.3% ethylene in 2% methylcellulose (check and 2% methylcellulose (blank. Latex output and contents of pure rubber, total solid, sucrose, inorganic phosphorus, thiol, and Mg2+ were measured. The treatments containing 1% KI or saturated macrolide increased latex yields compared to the blank with 2% methylcellulose alone. The 1% KI or saturated macrolide treatments were equal to that of 0.3% ethylene check treatment. However, 5 and 10% KI were harmful to bark of rubber trees, even caused prolonged tapping panel dryness.O objetivo deste trabalho foi avaliar a produção de látex em seringueiras (Hevea brasiliensis estimulada pela aplicação de macrolídeo saturado (latrunculina A, iodeto de potássio a 1, 5 e 10%, e etileno a 0,3%, preparados em solução de metilcelulose a 0,2%. O tratamento testemunha consistiu da aplicação de solução pura de metilcelulose a 2%. Foram medidos a produção de látex e o conteúdo de borracha pura, sólidos totais, sacarose, fósforo inorgânico, tiol e Mg2+. A aplicação de KI a 1% ou de macrolídeo saturado aumentou a produção de látex em comparação com o tratamento testemunha. Os tratamentos com KI a 1% e macrolídeo saturado foram iguais ao tratamento com etileno a 0,3%. No entanto, os tratamentos com KI a 5 e 10% foram prejudiciais à casca da seringueira, causando até mesmo o secamento do painel-de-sangria.

  20. The actin cytoskeleton in root hairs: all is fine at the tip

    NARCIS (Netherlands)

    Ketelaar, T.

    2013-01-01

    Filamentous actin forms characteristic bundles in plant cells that facilitate cytoplasmic streaming. In contrast, networks of actin exhibiting fast turnover are found especially near sites of rapid cell expansion. These networks may serve various functions including delivering and retaining vesicles

  1. Sulfated heterorhamnans from the green seaweed Gayralia oxysperma: partial depolymerization, chemical structure and antitumor activity.

    Science.gov (United States)

    Ropellato, Juliana; Carvalho, Mariana M; Ferreira, Luciana G; Noseda, Miguel D; Zuconelli, Cristiane R; Gonçalves, Alan G; Ducatti, Diogo R B; Kenski, Juliana C N; Nasato, Pauline L; Winnischofer, Sheila M B; Duarte, Maria E R

    2015-03-06

    Sulfated heterorhamnans produced by Gayralia oxysperma were utilized for the preparation of two homogeneous and highly sulfated Smith-degraded products (M(w) of 109 and 251 kDa), which were constituted principally by 3-linked α-L-rhamnosyl units 2- or 4-sulfate and 2-linked α-L-rhamnosyl units 4- or 3,4-sulfate, in different percentages. The homogeneous products and the crude extracts containing the sulfated heterorhamnans showed cytotoxic effect against U87MG cells. These sulfated polysaccharides induced an increase in the number of cells in G1 phase with concomitant increase of the mRNA levels of p53 and p21. The presence of 2-linked disulfated rhamnose residues together with the molecular weight could be important factors to be correlated with the inhibitory effect on human glioblastoma cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

    Directory of Open Access Journals (Sweden)

    Almudena García-Ortiz

    2017-04-01

    Full Text Available The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS; however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO generated by endothelial nitric oxide synthase (eNOS controls the coalescence of protein kinase C-θ (PKC-θ at the central supramolecular activation cluster (c-SMAC of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1, as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO. The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S and PFN1 (H119E, respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

  3. Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation

    Science.gov (United States)

    Speldewinde, Shaun H.; Tuite, Mick F.

    2017-01-01

    Mammalian and fungal prions arise de novo; however, the mechanism is poorly understood in molecular terms. One strong possibility is that oxidative damage to the non-prion form of a protein may be an important trigger influencing the formation of its heritable prion conformation. We have examined the oxidative stress-induced formation of the yeast [PSI+] prion, which is the altered conformation of the Sup35 translation termination factor. We used tandem affinity purification (TAP) and mass spectrometry to identify the proteins which associate with Sup35 in a tsa1 tsa2 antioxidant mutant to address the mechanism by which Sup35 forms the [PSI+] prion during oxidative stress conditions. This analysis identified several components of the cortical actin cytoskeleton including the Abp1 actin nucleation promoting factor, and we show that deletion of the ABP1 gene abrogates oxidant-induced [PSI+] prion formation. The frequency of spontaneous [PSI+] prion formation can be increased by overexpression of Sup35 since the excess Sup35 increases the probability of forming prion seeds. In contrast to oxidant-induced [PSI+] prion formation, overexpression-induced [PSI+] prion formation was only modestly affected in an abp1 mutant. Furthermore, treating yeast cells with latrunculin A to disrupt the formation of actin cables and patches abrogated oxidant-induced, but not overexpression-induced [PSI+] prion formation, suggesting a mechanistic difference in prion formation. [PIN+], the prion form of Rnq1, localizes to the IPOD (insoluble protein deposit) and is thought to influence the aggregation of other proteins. We show Sup35 becomes oxidized and aggregates during oxidative stress conditions, but does not co-localize with Rnq1 in an abp1 mutant which may account for the reduced frequency of [PSI+] prion formation. PMID:28369054

  4. Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry*

    Science.gov (United States)

    Stokasimov, Ema; Rubenstein, Peter A.

    2009-01-01

    Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts. PMID:19605362

  5. Non-adherence to topical treatments for actinic keratosis

    Directory of Open Access Journals (Sweden)

    Shergill B

    2013-12-01

    Full Text Available Bav Shergill,1 Simon Zokaie,2 Alison J Carr3 1Department of Dermatology, Brighton and Sussex University Hospitals, Elm Grove, Brighton, UK; 2Leo Pharma, Princes Risborough, 3Hamell, London, UK Background: There is limited information on the patterns of use, adherence rates, and factors that impact adherence with topical treatments for actinic keratosis (AK. Objectives: To establish patterns of use and adherence with topical treatments for AK and to identify treatment-related factors that impact on adherence. Methods: A community-based, cross-sectional study was performed using a standardized questionnaire completed online or via telephone interview. Patients were stratified according to the presence of AK lesions on the scalp and/or other extremities; and presence of scarring resulting from treatment. Results: This study included 305 patients with AK who were currently using a patient-applied topical therapy for AK or had used one within the previous 12 months. In total, 88% (n = 268/305 of patients were either non-adherent, non-persistent or both non-adherent and non-persistent to topical therapy. Duration of treatment was associated with increasing rates of non-adherence (adjusted odds ratio [OR]; for treatment durations greater than 4 weeks, 2.2, P < 0.01: 52% of patients were non-adherent with 3–4 week treatment duration; 69% of patients with 4–8 week treatment duration; and 71% of patients with 6–12 week treatment duration. There were similar increases in non-persistence with increasing treatment duration (adjusted OR; for treatment durations greater than 4 weeks, 2.1, P < 0.05. Conclusion: This study found high rates of non-adherence and non-persistence in patients with AK. Duration of treatment was a significant factor contributing to non-adherence and non-persistence to topical treatments. Patient-applied topical therapies that require less frequent application and have shorter treatment duration may be associated with improved

  6. T cell antigen receptor activation and actin cytoskeleton remodeling

    Science.gov (United States)

    Kumari, Sudha; Curado, Silvia; Mayya, Viveka

    2013-01-01

    T cells constitute a crucial arm of the adaptive immune system and their optimal function is required for a healthy immune response. After the initial step of T cell-receptor (TCR) triggering by antigenic peptide complexes on antigen presenting cell (APC), the T cell exhibits extensive cytoskeletal remodeling. This cytoskeletal remodeling leads to formation of an “immunological synapse” [1] characterized by regulated clustering, segregation and movement of receptors at the interface. Synapse formation regulates T cell activation and response to antigenic peptides and proceeds via feedback between actin cytoskeleton and TCR signaling. Actin polymerization participates in various events during the synapse formation, maturation, and eventually its disassembly. There is increasing knowledge about the actin effectors that couple TCR activation to actin rearrangements [2, 3], and how defects in these effectors translate into impairment of T cell activation. In this review we aim to summarize and integrate parts of what is currently known about this feedback process. In addition, in light of recent advancements in our understanding of TCR triggering and translocation at the synapse, we speculate on the organizational and functional diversity of microfilament architecture in the T cell. PMID:23680625

  7. Interconnection between actin cytoskeleton and plant defense signaling

    Czech Academy of Sciences Publication Activity Database

    Janda, Martin; Matoušková, J.; Burketová, Lenka; Valentová, O.

    2014-01-01

    Roč. 9, č. 11 (2014) ISSN 1559-2316 R&D Projects: GA ČR(CZ) GAP501/11/1654 Institutional support: RVO:61389030 Keywords : Actin * Cytoskeleton * Pathogen Subject RIV: ED - Physiology http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=MEDLINE&DestLinkType=FullRecord&UT=25482795

  8. Control of the actin cytoskeleton in plant cell growth

    NARCIS (Netherlands)

    Hussey, P.J.; Ketelaar, M.J.; Deeks, M.J.

    2006-01-01

    Plant cells grow through increases in volume and cell wall surface area. The mature morphology of a plant cell is a product of the differential rates of expansion between neighboring zones of the cell wall during this process. Filamentous actin arrays are associated with plant cell growth, and the

  9. The actin Cytoskeleton in Root Hairs: a cell elongation device

    NARCIS (Netherlands)

    Ketelaar, T.; Emons, A.M.C.

    2009-01-01

    The actin cytoskeleton plays an important role in root hair development. It is involved in both the delivery of growth materials to the expanding tip of root hairs and the regulation of the area of tip growth. This review starts with a discussion of the techniques that are available to visualize the

  10. Force Exertion and Transmission in Cross-Linked Actin Networks

    Science.gov (United States)

    Stam, Samantha

    Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

  11. Onchocercal DNA amplification using beta actin gene primers ...

    African Journals Online (AJOL)

    Onchocercal DNA amplification using beta actin gene primers compared with first internal transcribed spacer sequences for monitoring onchocerciasis eradication strategy. ... Out of the 12 amplicons in agarose gel, there were 6 sharp and 6 faint bands of 100bp molecular weight as documented. The sharp bands included 3 ...

  12. The roles of the actin cytoskeleton in fear memory formation

    Directory of Open Access Journals (Sweden)

    Raphael eLamprecht

    2011-07-01

    Full Text Available The formation and storage of fear memory is needed to adapt behavior and avoid danger during subsequent fearful events. However, fear memory may also play a significant role in stress and anxiety disorders. When fear becomes disproportionate to that necessary to cope with a given stimulus, or begins to occur in inappropriate situations, a fear or anxiety disorder exists. Thus, the study of cellular and molecular mechanisms underpinning fear memory may shed light on the formation of memory and on anxiety and stress related disorders. Evidence indicates that fear learning leads to changes in neuronal synaptic transmission and morphology in brain areas underlying fear memory formation including the amygdala and hippocampus. The actin cytoskeleton has been shown to participate in these key neuronal processes. Recent findings show that the actin cytoskeleton is needed for fear memory formation and extinction. Moreover, the actin cytoskeleton is involved in synaptic plasticity and in neuronal morphogenesis in brain areas that mediate fear memory. The actin cytoskeleton may therefore mediate between synaptic transmission during fear learning and long-term cellular alterations mandatory for fear memory formation.

  13. Actin and myosin contribute to mammalian mitochondrial DNA maintenance

    Science.gov (United States)

    Reyes, A.; He, J.; Mao, C. C.; Bailey, L. J.; Di Re, M.; Sembongi, H.; Kazak, L.; Dzionek, K.; Holmes, J. B.; Cluett, T. J.; Harbour, M. E.; Fearnley, I. M.; Crouch, R. J.; Conti, M. A.; Adelstein, R. S.; Walker, J. E.; Holt, I. J.

    2011-01-01

    Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance. PMID:21398640

  14. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    NARCIS (Netherlands)

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Mansson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies.

  15. Real-world approach to actinic keratosis management

    DEFF Research Database (Denmark)

    Dirschka, Thomas; Gupta, Girish; Micali, Giuseppe

    2017-01-01

    Actinic keratosis (AK) is a chronic skin disease in which multiple clinical and subclinical lesions co-exist across large areas of sun-exposed skin, resulting in field cancerisation. Lesions require treatment because of their potential to transform into invasive squamous cell carcinoma. This arti...

  16. Decidable and undecidable arithmetic functions in actin filament networks

    Science.gov (United States)

    Schumann, Andrew

    2018-01-01

    The plasmodium of Physarum polycephalum is very sensitive to its environment, and reacts to stimuli with appropriate motions. Both the sensory and motor stages of these reactions are explained by hydrodynamic processes, based on fluid dynamics, with the participation of actin filament networks. This paper is devoted to actin filament networks as a computational medium. The point is that actin filaments, with contributions from many other proteins like myosin, are sensitive to extracellular stimuli (attractants as well as repellents), and appear and disappear at different places in the cell to change aspects of the cell structure—e.g. its shape. By assembling and disassembling actin filaments, some unicellular organisms, like Amoeba proteus, can move in response to various stimuli. As a result, these organisms can be considered a simple reversible logic gate—extracellular signals being its inputs and motions its outputs. In this way, we can implement various logic gates on amoeboid behaviours. These networks can embody arithmetic functions within p-adic valued logic. Furthermore, within these networks we can define the so-called diagonalization for deducing undecidable arithmetic functions.

  17. Roles of cortical actin microfilament patterning in division plane orientation in plants.

    Science.gov (United States)

    Kojo, Kei H; Higaki, Takumi; Kutsuna, Natsumaro; Yoshida, Yuya; Yasuhara, Hiroki; Hasezawa, Seiichiro

    2013-09-01

    In land plant cells, division planes are precisely predicted by the microtubule preprophase band and cortical actin microfilament pattern called the actin-depleted zone or actin microfilament twin peaks. However, the function of cortical actin microfilament patterning is not clear. In this study, we report that treatment with the inhibitor 2,3,5-triiodobenzonic acid (TIBA) or jasplakinolide increased the amount of thick actin microfilaments in tobacco BY-2 cells at interphase. However, during the division of BY-2 cells, these inhibitors did not induce visible alteration of actin microfilament thickness but altered cortical actin microfilament patterning without significant disorganization of the microtubule preprophase band. TIBA treatment induced a single intensity peak of actin microfilament distribution around the cell center, whereas jasplakinolide caused the appearance of triple peaks relative to the distribution of actin microfilament around the cell center, in approximately one-third of the cells at metaphase. Dual observations of microtubules and actin microfilaments revealed that abnormal cortical actin microfilament patterning with single or triple peaks is correlated with oblique mitotic spindles in BY-2 cells. In addition, oblique cell plates were frequently observed in BY-2 cells and Arabidopsis thaliana root cells treated with TIBA or jasplakinolide. These results provide evidence for the critical roles of cortical actin microfilament patterning in spindle and cell plate orientation.

  18. DeActs : genetically encoded tools for perturbing the actin cytoskeleton in single cells

    NARCIS (Netherlands)

    Harterink, Martin; Santos Esteves da Silva, Marta; Will, Lena; Turan, Julia; Ibrahim, Adiljan; Lang, Alexander E; Van Battum, Eljo Y; Pasterkamp, R Jeroen; Kapitein, Lukas C; Kudryashov, Dmitri; Barres, Ben A; Hoogenraad, Casper C; Zuchero, J Bradley

    2017-01-01

    The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively

  19. Multidrug Resistance-Related Protein 1 (MRP1) Function and Localization Depend on Cortical Actin

    NARCIS (Netherlands)

    Hummel, Ina; Klappe, Karin; Ercan, Cigdem; Kok, Jan Willem

    MRP1 (ABCC1) is known to be localized in lipid rafts. Here we show in two different cell lines that localization of Mrp1/MRP1 (Abcc1/ABCC1) in lipid rafts and its function as an efflux pump are dependent on cortical actin. Latrunculin B disrupts both cortical actin and actin stress fibers. This

  20. Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

    NARCIS (Netherlands)

    Ketelaar, T.; Honing, van der H.S.; Emons, A.M.C.

    2010-01-01

    In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be

  1. Analysis of actin FLAP dynamics in the leading lamella.

    Directory of Open Access Journals (Sweden)

    Igor R Kuznetsov

    2010-04-01

    Full Text Available The transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation (see Zicha et al., Science, v. 300, pp. 142-145 [1].We re-examine the Zicha FLAP experiments using a two-phase reactive interpenetrating flow formalism to model the cytoplasm and the transport dynamics of bleached and unbleached actin. By allowing an improved treatment of effects related to the retrograde flow of the cytoskeleton and of the geometry and finite thickness of the lamella, this new analysis reveals a mechanism that can realistically explain the timing and the amplitude of all the FLAP signals observed in [1] without invoking special transport modalities.We conclude that simple diffusion is sufficient to explain the observed transport rates, and that variations in the transport of labeled actin through the lamella are minor and not likely to be the cause of the observed physiological variations among different segments of the leading edge. We find that such variations in labeling can easily arise from differences and changes in the microscopic actin dynamics inside the edge compartment, and that the key dynamical parameter in this regard is the so-called "dilatation rate" (the velocity of cytoskeletal retrograde flow divided by a characteristic dimension of the edge compartment where rapid polymerization occurs. If our dilatation hypothesis is correct, the transient kinetics of bleached actin relocalization constitute a novel and very sensitive method for probing the cytoskeletal dynamics in leading edge micro-environments which are otherwise very difficult to directly interrogate.

  2. Analysis of actin FLAP dynamics in the leading lamella.

    Science.gov (United States)

    Kuznetsov, Igor R; Herant, Marc; Dembo, Micah

    2010-04-15

    The transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation (see Zicha et al., Science, v. 300, pp. 142-145 [1]). We re-examine the Zicha FLAP experiments using a two-phase reactive interpenetrating flow formalism to model the cytoplasm and the transport dynamics of bleached and unbleached actin. By allowing an improved treatment of effects related to the retrograde flow of the cytoskeleton and of the geometry and finite thickness of the lamella, this new analysis reveals a mechanism that can realistically explain the timing and the amplitude of all the FLAP signals observed in [1] without invoking special transport modalities. We conclude that simple diffusion is sufficient to explain the observed transport rates, and that variations in the transport of labeled actin through the lamella are minor and not likely to be the cause of the observed physiological variations among different segments of the leading edge. We find that such variations in labeling can easily arise from differences and changes in the microscopic actin dynamics inside the edge compartment, and that the key dynamical parameter in this regard is the so-called "dilatation rate" (the velocity of cytoskeletal retrograde flow divided by a characteristic dimension of the edge compartment where rapid polymerization occurs). If our dilatation hypothesis is correct, the transient kinetics of bleached actin relocalization constitute a novel and very sensitive method for probing the cytoskeletal dynamics in leading edge micro-environments which are otherwise very difficult to directly interrogate.

  3. Differential sensitivity to detergents of actin cytoskeleton from nerve endings.

    Science.gov (United States)

    Cubí, Roger; Matas, Lluís A; Pou, Marta; Aguilera, José; Gil, Carles

    2013-11-01

    Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied. © 2013.

  4. Roles of the actin cytoskeleton and an actin-binding protein in wheat resistance against Puccinia striiformis f. sp. tritici.

    Science.gov (United States)

    Song, Xiaohe; Ma, Qing; Hao, Xinyuan; Li, Hongli

    2012-01-01

    Elucidating resistance mechanisms of plant cells against pathogens is essential to develop novel strategies of disease control. The actin cytoskeleton was found intimately involved in plant defense. In order to reveal how actin would be involved in the interaction between wheat and the stripe rust Puccinia striiformis f. sp. tritici, prior to fungal inoculation, wheat leaves were treated with cytochalasin A, an inhibitor of actin polymerization. Our results showed reduced incidence of hypersensitive cell death and delayed accumulation of H(2)O(2) in wheat leaves treated with cytochalasin A compared to the control. We also found that the TaPRO profilin gene exhibited significantly different expression levels in host leaves when comparing compatible and incompatible interactions. Real-time PCR analysis revealed that the expression transcript of TaPRO was lower at each time point in incompatible interactions when compared to compatible ones, and the largest difference between the two interactions occurred at 12 h post-inoculation. Both pharmacological and gene expression results collectively support the notion that the compromise of the actin microfilament is linked to the compatible interaction between the stripe rust fungus and the leaves of its wheat host.

  5. Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.

    Directory of Open Access Journals (Sweden)

    Luis Vidali

    2009-05-01

    Full Text Available Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments.In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore.Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.

  6. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    Science.gov (United States)

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

    Science.gov (United States)

    Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

    2016-01-01

    The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

  8. A function for filamentous alpha-smooth muscle actin: Retardation of motility in human breast fibroblasts

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-Sm actin has been found also to be expressed transiently in cer...... of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and beta1-integrin. We propose that an important function of filamentous alpha-sm actin is to immobilize the cells....

  9. SPARC Interacts with Actin in Skeletal Muscle in Vitro and in Vivo

    DEFF Research Database (Denmark)

    Jørgensen, Louise H; Jepsen, Pia Lørup; Boysen, Anders

    2017-01-01

    to actin. This interaction is present in regenerating myofibers of patients with Duchenne muscular dystrophy, polymyositis, and compartment syndrome. Analysis of the α-, β-, and γ-actin isoforms in SPARC knockout myoblasts reveals a changed expression pattern with dominance of γ-actin. In SPARC knockout...... stimulation protocol, we find a defective force recovery. Therefore, SPARC appears to be an important modulator of the actin cytoskeleton, implicating maintenance of muscular function. This direct interaction with actin suggests a new role of SPARC during tissue remodeling....

  10. The conserved Tarp actin binding domain is important for chlamydial invasion.

    Directory of Open Access Journals (Sweden)

    Travis J Jewett

    2010-07-01

    Full Text Available The translocated actin recruiting phosphoprotein (Tarp is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

  11. Cross-reacting material 197 (CRM197) affects actin cytoskeleton of endothelial cells.

    Science.gov (United States)

    Özerman Edis, Bilge; Varol, Başak; Hacıosmanoğlu, Ebru; Ünlü, Ayhan; Bektaş, Muhammet

    2017-10-01

    CRM197, cross-reacting material 197, is a mutant of diphtheria toxin (DTx). CRM197 is used in pharmacology as a carrier protein. It has been recently shown that CRM197 causes breakdown in actin filaments. In order to show intracellular localization of CRM197 and visualize cell structure via actin cytoskeleton, endothelial cells were cultured and subjected to CRM197 in vitro. To address the interaction between CRM197 and actin both experimental and theoretical studies were carried out. Colocalization of CRM197 with actin filaments was determined by immunofluorescence microscopy. Following 24-hour incubation, the loss of cell-cell contact between cells was prominent. CRM197 was shown to bind to G-actin by gel filtration chromatography, and this binding was confirmed by Western blot analysis of eluted samples obtained following chromatography. Based on crystal structure, docked model of CRM197-actin complex was generated. Molecular dynamics simulation revealed that Lys42, Cys218, Cys233 of CRM197 interacts with Gly197, Arg62 and Ser60 of G-actin, respectively. CRM197 binding to G-actin, colocalization of CRM197 with actin filament, and actin cytoskeleton rearrangement resulting in the loss of cell-cell contact show that actin comes into sight as target molecule for CRM197.

  12. Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

    Science.gov (United States)

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E

    2013-10-15

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Colonna, Cecilia; Cortegano, Miguel; Calvo, María; Martínez, Susana E; Egea, Gustavo

    2007-08-07

    Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.

  14. ADF/cofilin-mediated actin retrograde flow directs neurite formation in the developing brain.

    Science.gov (United States)

    Flynn, Kevin C; Hellal, Farida; Neukirchen, Dorothee; Jacob, Sonja; Tahirovic, Sabina; Dupraz, Sebastian; Stern, Sina; Garvalov, Boyan K; Gurniak, Christine; Shaw, Alisa E; Meyn, Liane; Wedlich-Söldner, Roland; Bamburg, James R; Small, J Victor; Witke, Walter; Bradke, Frank

    2012-12-20

    Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Opposing effects of actin signaling and LFA-1 on establishing the affinity threshold for inducing effector T-cell responses in mice

    Czech Academy of Sciences Publication Activity Database

    Palmer, E.; Drobek, Aleš; Štěpánek, Ondřej

    2016-01-01

    Roč. 46, č. 8 (2016), s. 1887-1901 ISSN 0014-2980 R&D Projects: GA ČR GJ16-09208Y Institutional support: RVO:68378050 Keywords : Actin cytoskeleton * Antigen affinity treshold * LFA-1 * Rap1 * Rho-family GTPases * T-cell receptor signaling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.227, year: 2016

  16. The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei

    Czech Academy of Sciences Publication Activity Database

    Maruyama, E.O.; Hori, T.; Tanabe, H.; Kitamura, H.; Matsuda, R.; Tone, S.; Hozák, Pavel; Habermann, F.A.; von Hase, J.; Cremer, C.; Fukagawa, T.; Harata, M.

    2012-01-01

    Roč. 125, č. 16 (2012), s. 3739-3744 ISSN 0021-9533 R&D Projects: GA MŠk LC545; GA MŠk LH12143 Institutional support: RVO:68378050 Keywords : actin-related protein * histone variant * nuclear organization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.877, year: 2012

  17. The plant formin AtFH4 interacts with both actin and microtubules, and contains a newly identified microtubule-binding domain

    Czech Academy of Sciences Publication Activity Database

    Deeks, M.J.; Fendrych, Matyáš; Smertenko, A.; Bell, K.S.; Oparka, K.; Cvrčková, F.; Žárský, Viktor; Hussey, P.J.

    2010-01-01

    Roč. 123, č. 8 (2010), s. 1209-1215 ISSN 0021-9533 R&D Projects: GA MŠk(CZ) LC06004; GA ČR GAP305/10/0433 Institutional research plan: CEZ:AV0Z50380511 Keywords : Actin regulating proteins * Membrane * Microtubule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.290, year: 2010

  18. Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression

    Czech Academy of Sciences Publication Activity Database

    Deeks, M.J.; Cvrčková, F.; Machesky, M. L.; Mikitova, V.; Ketelaar, T.; Žárský, Viktor; Davies, B.; Hussey, P.J.

    2005-01-01

    Roč. 168, č. 3 (2005), s. 529-540 ISSN 0028-646X R&D Projects: GA ČR GA204/02/1461; GA ČR GA204/05/0268 Institutional research plan: CEZ:AV0Z50380511 Keywords : actin * Arabidopsis * cytoskeleton Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.285, year: 2005

  19. Substituted 2-hydroxy-N-(arylalkyl)benzamide sensitizes cancer cells to metabolic stress by disrupting actin cytoskeleton and inhibiting autophagic flux

    Czech Academy of Sciences Publication Activity Database

    Pachnikova, G.; Uldrijan, S.; Imramovský, A.; Kryštof, Vladimír; Slaninová, I.

    2016-01-01

    Roč. 37, DEC (2016), s. 70-78 ISSN 0887-2333 Institutional support: RVO:61389030 Keywords : hepatocellular-carcinoma cells * sorafenib * apoptosis * death * maturation * membrane * melanoma * Actin * Autophagy * Melanoma * Metabolic stress * Sorafenib * Substituted 2-hydroxy-N-(arylalkyl)benzamide Subject RIV: CE - Biochemistry Impact factor: 2.866, year: 2016

  20. Prevalence of actinic skin lesions in patients with basal cell carcinoma of the head: a case-control study.

    Science.gov (United States)

    Chinem, Valquíria Pessoa; Miot, Hélio Amante

    2012-01-01

    To evaluate the prevalence of actinic skin lesions in patients with basal cell carcinoma of the head. A case-control study was carried out. Cases were patients with primary, solid basal cell carcinoma of the head, less than two centimeters in diameter; and as controls, patients with other dermatoses. Constitutional and behavioral variables were analyzed, as well as actinic lesions. One hundred twenty cases and 360 controls were evaluated. Facial milia (OR = 2.3), leukoderma punctata of the upper limbs (OR = 2.9), and cutis rhomboidalis nuchae (OR = 1.8) were associated with neoplasms regardless of other variables, suggesting a risk phenotype. There was also association with light hair and eye color phenotypes, family genetics, and cumulative sun exposure. Sunburn, smoking, and alcoholism were not identified as risk factors. The use of sunscreens showed no evidence of protection; however, the control group consisted of dermatology patients who are often prescribed sunscreens. Actinic lesions were more prevalent in patients with solid basal cell carcinoma of the head than in controls, especially milia, cutis rhomboidalis nuchae, and leukoderma punctata, regardless of other known risk factors.

  1. A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants.

    Science.gov (United States)

    Thyssen, Gregory N; Fang, David D; Turley, Rickie B; Florane, Christopher B; Li, Ping; Mattison, Christopher P; Naoumkina, Marina

    2017-04-01

    Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single-celled trichomes that comprise cotton lint fibers. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li 1 short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F-actin in Li 1 plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F-actin did not uniformly decorate the nuclear envelope. While wild-type lint fiber trichome cells contained long longitudinal actin cables, the short Li 1 fiber cells accumulated disoriented transverse cables. The polymerization-defective Gly65Val allele in Li 1 plants likely disrupts processive elongation of F-actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li 1 mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  2. Actin microfilaments in presumptive statocytes of root caps and coleoptiles

    Science.gov (United States)

    White, R. G.; Sack, F. D.

    1990-01-01

    Rhodamine-phalloidin was used to determine the distribution of actin microfilament bundles (mfb) in cells thought to be the site of gravity perception (statocytes) in coleoptiles and root caps of Zea mays and Hordeum vulgare. In coleoptile cells, amyloplasts were usually observed in close proximity to thick mfb, which often appeared to divide into finer mfb adjacent to individual amyloplasts. The nucleus in these cells was surrounded by an extensive network of mfb, which were connected to thicker transvacuolar mfb. Columella cells of the root cap contained an extensive reticulum of fine mfb throughout the protoplast, but lacked the much thicker mfb seen in coleoptile cells. The distribution and extent of mfb observed in fixed cells correlates with patterns of streaming and amyloplast movement seen in living cells. A possible role for actin mfb in the perception of gravity is discussed.

  3. Osmosensation in vasopressin neurons: changing actin density to optimize function.

    Science.gov (United States)

    Prager-Khoutorsky, Masha; Bourque, Charles W

    2010-02-01

    The proportional relation between circulating vasopressin concentration and plasma osmolality is fundamental for body fluid homeostasis. Although changes in the sensitivity of this relation are associated with pathophysiological conditions, central mechanisms modulating osmoregulatory gain are unknown. Here, we review recent data that sheds important light on this process. The cell autonomous osmosensitivity of vasopressin neurons depends on cation channels comprising a variant of the transient receptor potential vanilloid 1 (TRPV1) channel. Hyperosmotic activation is mediated by a mechanical process where sensitivity increases in proportion with actin filament density. Moreover, angiotensin II amplifies osmotic activation by a rapid stimulation of actin polymerization, suggesting that neurotransmitter-induced changes in cytoskeletal organization in osmosensory neurons can mediate central changes in osmoregulatory gain. (c) 2009 Elsevier Ltd. All rights reserved.

  4. Dynamics and Morphology of Microvilli Driven by Actin Polymerization

    Science.gov (United States)

    Gov, Nir S.

    2006-07-01

    Many different cell types have dynamic protrusions, called microvilli, on their surface. We model these structures as arising from the balance between the force of actin polymerization and the restoring force of the membrane. From this simple model we calculate the distribution function of microvilli heights for several cells. We further describe the phase diagram and the resulting morphology of the microvilli aggregates on the cell surface.

  5. Actinic inspection of multilayer defects on EUV masks

    International Nuclear Information System (INIS)

    Barty, A; Liu, Y; Gullikson, E; Taylor, J S; Wood, O

    2005-01-01

    The production of defect-free mask blanks, and the development of techniques for inspecting and qualifying EUV mask blanks, remains a key challenge for EUV lithography. In order to ensure a reliable supply of defect-free mask blanks, it is necessary to develop techniques to reliably and accurately detect defects on un-patterned mask blanks. These inspection tools must be able to accurately detect all critical defects whilst simultaneously having the minimum possible false-positive detection rate. There continues to be improvement in high-speed non-actinic mask blank inspection tools, and it is anticipated that these tools can and will be used by industry to qualify EUV mask blanks. However, the outstanding question remains one of validating that non-actinic inspection techniques are capable of detecting all printable EUV defects. To qualify the performance of non-actinic inspection tools, a unique dual-mode EUV mask inspection system has been installed at the Advanced Light Source (ALS) synchrotron at Lawrence Berkeley National Laboratory. In high-speed inspection mode, whole mask blanks are scanned for defects using 13.5-nm wavelength light to identify and map all locations on the mask that scatter a significant amount of EUV light. In imaging, or defect review mode, a zone plate is placed in the reflected beam path to image a region of interest onto a CCD detector with an effective resolution on the mask of 100-nm or better. Combining the capabilities of the two inspection tools into one system provides the unique capability to determine the coordinates of native defects that can be used to compare actinic defect inspection with visible light defect inspection tools under commercial development, and to provide data for comparing scattering models for EUV mask defects

  6. Myosin lever arm directs collective motion on cellular actin network.

    Science.gov (United States)

    Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-03-18

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions.

  7. Memory Dynamics in Cross-linked Actin Networks

    Science.gov (United States)

    Scheff, Danielle; Majumdar, Sayantan; Gardel, Margaret

    Cells demonstrate the remarkable ability to adapt to mechanical stimuli through rearrangement of the actin cytoskeleton, a cross-linked network of actin filaments. In addition to its importance in cell biology, understanding this mechanical response provides strategies for creation of novel materials. A recent study has demonstrated that applied stress can encode mechanical memory in these networks through changes in network geometry, which gives rise to anisotropic shear response. Under later shear, the network is stiffer in the direction of the previously applied stress. However, the dynamics behind the encoding of this memory are unknown. To address this question, we explore the effect of varying either the rigidity of the cross-linkers or the length of actin filament on the time scales required for both memory encoding and over which it later decays. While previous experiments saw only a long-lived memory, initial results suggest another mechanism where memories relax relatively quickly. Overall, our study is crucial for understanding the process by which an external stress can impact network arrangement and thus the dynamics of memory formation.

  8. Treadmilling of actin filaments via Brownian dynamics simulations

    Science.gov (United States)

    Guo, Kunkun; Shillcock, Julian; Lipowsky, Reinhard

    2010-10-01

    Actin polymerization is coupled to the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi). Therefore, each protomer within an actin filament can attain three different nucleotide states corresponding to bound ATP, ADP/Pi, and ADP. These protomer states form spatial patterns on the growing (or shrinking) filaments. Using Brownian dynamics simulations, the growth behavior of long filaments is studied, together with the associated protomer patterns, as a function of ATP-actin monomer concentration, CT, within the surrounding solution. For concentrations close to the critical concentration CT=CT,cr, the filaments undergo treadmilling, i.e., they grow at the barbed and shrink at the pointed end, which leads to directed translational motion of the whole filament. The corresponding nonequilibrium states are characterized by several global fluxes and by spatial density and flux profiles along the filaments. We focus on a certain set of transition rates as deduced from in vitro experiments and find that the associated treadmilling (or turnover) rate is about 0.08 monomers per second.

  9. How cellular membrane properties are affected by the actin cytoskeleton.

    Science.gov (United States)

    Lemière, J; Valentino, F; Campillo, C; Sykes, C

    2016-11-01

    Lipid membranes define the boundaries of living cells and intracellular compartments. The dynamic remodelling of these membranes by the cytoskeleton, a very dynamic structure made of active biopolymers, is crucial in many biological processes such as motility or division. In this review, we present some aspects of cellular membranes and how they are affected by the presence of the actin cytoskeleton. We show that, in parallel with the direct study of membranes and cytoskeleton in vivo, biomimetic in vitro systems allow reconstitution of biological processes in a controlled environment. In particular, we show that liposomes, or giant unilamellar vesicles, encapsulating a reconstituted actin network polymerizing at their membrane are suitable models of living cells and can be used to decipher the relative contributions of membrane and actin on the mechanical properties of the cellular interface. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. The Evolution of the Actin Binding NET Superfamily

    Directory of Open Access Journals (Sweden)

    Tim eHawkins

    2014-06-01

    Full Text Available The arabidopsis Networked protein superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in arabidopsis which group into 4 distinct clades or subfamilies. NET homologues are absent from the genomes of metazoa and fungi, furthermore in Plantae NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single subfamily of the NET proteins are found encoded in the club moss genome; an extant species of the earliest vascular plants. Gymnosperms have examples from subfamilies 4 and 3 with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 subfamilies, the NET1 and pollen-expressed NET2 subfamilies are only found as independent sequences in angiosperms. This is consistent with the divergence of reproductive actin. The four subfamilies are conserved across monocots and eudicots with the numbers of members of each clade expanding at this point due in part to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants they have continued to develop and diversify in a manner which has mirrored the divergence and complexity of plant species through evolution in the ‘March of Progress’.

  11. Ethanol increases p190RhoGAP activity, leading to actin cytoskeleton rearrangements.

    Science.gov (United States)

    Selva, Javier; Egea, Gustavo

    2011-12-01

    We previously reported that cells chronically exposed to ethanol show alterations in actin cytoskeleton organization and dynamics in primary cultures of newborn rat astrocytes, a well-established in vitro model for foetal alcohol spectrum disorders. These alterations were attributed to a decrease in the cellular levels of active RhoA (RhoA-GTP), which in turn was produced by an increase in the total RhoGAP activity. We here provide evidence that p190RhoGAPs are the main factors responsible for such increase. Thus, in astrocytes chronically exposed to ethanol we observe: (i) an increase in p190A- and p190B-associated RhoGAP activity; (ii) a higher binding of p190A and p190B to RhoA-GTP; (iii) a higher p120RasGAP-p190A RhoGAP complex formation; and (iv) the recruitment of both p190RhoGAPs to the plasma membrane. The simultaneous silencing of both p190 isoforms prevents the actin rearrangements and the total RhoGAP activity increase triggered both by ethanol. Therefore, our data directly points p190RhoGAPs as ethanol-exposure molecular targets on glial cells of the CNS. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  12. Amiloride-insensitive sodium channels are directly regulated by actin cytoskeleton dynamics in human lymphoma cells.

    Science.gov (United States)

    Sudarikova, Anastasia V; Tsaplina, Olga A; Chubinskiy-Nadezhdin, Vladislav I; Morachevskaya, Elena A; Negulyaev, Yuri A

    2015-05-22

    Sodium influx mediated by ion channels of plasma membrane underlies fundamental physiological processes in cells of blood origin. However, little is known about the single channel activity and regulatory mechanisms of sodium-specific channels in native cells. In the present work, we used different modes of patch clamp technique to examine ion channels involved in Na-transporting pathway in U937 human lymphoma cells. The activity of native non-voltage-gated sodium (NVGS) channels with unitary conductance of 10 pS was revealed in cell-attached, inside-out and whole-cell configurations. NVGS channel activity is directly controlled by submembranous actin cytoskeleton. Specifically, an activation of sodium channels in U937 cells in response to microfilament disassembly was demonstrated on single-channel and integral current level. Inside-out experiments showed that filament assembly on cytoplasmic membrane surface caused fast inactivation of the channels. Biophysical characteristics of NVGS channels in U937 cells were similar to that of epithelial sodium channels (ENaCs). However, we found that amiloride, a known inhibitor of DEG/ENaC, did not block NVGS channels in U937 cells. Whole-cell current measurements revealed no amiloride-sensitive component of membrane current. Our data show that cortical actin structures represent the main factor that controls the activity of amiloride-insensitive ENaC-like channels in human lymphoma cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Cdc42/N-WASP signaling links actin dynamics to pancreatic β cell delamination and differentiation

    Science.gov (United States)

    Kesavan, Gokul; Lieven, Oliver; Mamidi, Anant; Öhlin, Zarah Löf; Johansson, Jenny Kristina; Li, Wan-Chun; Lommel, Silvia; Greiner, Thomas Uwe; Semb, Henrik

    2014-01-01

    Delamination plays a pivotal role during normal development and cancer. Previous work has demonstrated that delamination and epithelial cell movement within the plane of an epithelium are associated with a change in cellular phenotype. However, how this positional change is linked to differentiation remains unknown. Using the developing mouse pancreas as a model system, we show that β cell delamination and differentiation are two independent events, which are controlled by Cdc42/N-WASP signaling. Specifically, we show that expression of constitutively active Cdc42 in β cells inhibits β cell delamination and differentiation. These processes are normally associated with junctional actin and cell-cell junction disassembly and the expression of fate-determining transcription factors, such as Isl1 and MafA. Mechanistically, we demonstrate that genetic ablation of N-WASP in β cells expressing constitutively active Cdc42 partially restores both delamination and β cell differentiation. These findings elucidate how junctional actin dynamics via Cdc42/N-WASP signaling cell-autonomously control not only epithelial delamination but also cell differentiation during mammalian organogenesis. PMID:24449844

  14. Antibodies to filamentous actin (F-actin) in type 1 autoimmune hepatitis.

    Science.gov (United States)

    Granito, A; Muratori, L; Muratori, P; Pappas, G; Guidi, M; Cassani, F; Volta, U; Ferri, A; Lenzi, M; Bianchi, F B

    2006-03-01

    To evaluate the diagnostic significance of anti-filamentous actin antibodies (A-FAA) assessed with a commercial ELISA in comparison with immunofluorescence reactivity and patterns of anti-smooth muscle antibodies (SMA); and to correlate A-FAA positivity with clinical, immunogenetic, laboratory, and histological features in patients with autoimmune hepatitis type 1 (AIH-1). We studied 78 consecutive untreated AIH-1 patients and 160 controls: 22 with autoimmune hepatitis type 2 (AIH-2), 51 with hepatitis C, 17 with coeliac disease (CD), 20 with primary biliary cirrhosis (PBC) and 50 blood donors. SMA was evaluated by indirect immunofluorescence (IIF) on frozen sections of rat tissues, and A-FAA with a modified commercial ELISA. SMA was detected by IIF in 61 (78%) of 78 AIH-1 patients, of whom 47 (60%) had the SMA-T/G and 14 (18%) the SMA-V pattern. Of the pathological controls, 32 (20%) had the SMA-V pattern (25 with hepatitis C, 2 with AIH-2, 2 with PBC, 3 with CD). A-FAA were present in 55 AIH-1 patients (70.5%; 46 with SMA-T/G, 7 with SMA-V, and 2 SMA-negative), and in 10 controls (6%), of whom five had hepatitis C, two AIH-2, two PBC and one CD. The association between A-FAA and the SMA-T/G pattern was statistically significant (p<0.0001). A-FAA levels were higher in SMA-T/G positive than SMA-V positive AIH-1 patients and controls (p<0.0001). A-FAA positivity was significantly associated with higher gamma-globulin and IgG levels, but did not correlate with other considered parameters. The modified A-FAA ELISA strictly correlates with the SMA-T/G pattern and is a reliable and operator independent assay for AIH-1. Detection of A-FAA, even if devoid of prognostic relevance, may be useful when interpretative doubts of standard IIF arise.

  15. Antibodies to filamentous actin (F‐actin) in type 1 autoimmune hepatitis

    Science.gov (United States)

    Granito, A; Muratori, L; Muratori, P; Pappas, G; Guidi, M; Cassani, F; Volta, U; Ferri, A; Lenzi, M; Bianchi, F B

    2006-01-01

    Aims To evaluate the diagnostic significance of anti‐filamentous actin antibodies (A‐FAA) assessed with a commercial ELISA in comparison with immunofluorescence reactivity and patterns of anti‐smooth muscle antibodies (SMA); and to correlate A‐FAA positivity with clinical, immunogenetic, laboratory, and histological features in patients with autoimmune hepatitis type 1 (AIH‐1). Methods We studied 78 consecutive untreated AIH‐1 patients and 160 controls: 22 with autoimmune hepatitis type 2 (AIH‐2), 51 with hepatitis C, 17 with coeliac disease (CD), 20 with primary biliary cirrhosis (PBC) and 50 blood donors. SMA was evaluated by indirect immunofluorescence (IIF) on frozen sections of rat tissues, and A‐FAA with a modified commercial ELISA. Results SMA was detected by IIF in 61 (78%) of 78 AIH‐1 patients, of whom 47 (60%) had the SMA‐T/G and 14 (18%) the SMA‐V pattern. Of the pathological controls, 32 (20%) had the SMA‐V pattern (25 with hepatitis C, 2 with AIH‐2, 2 with PBC, 3 with CD). A‐FAA were present in 55 AIH‐1 patients (70.5%; 46 with SMA‐T/G, 7 with SMA‐V, and 2 SMA‐negative), and in 10 controls (6%), of whom five had hepatitis C, two AIH‐2, two PBC and one CD. The association between A‐FAA and the SMA‐T/G pattern was statistically significant (p<0.0001). A‐FAA levels were higher in SMA‐T/G positive than SMA‐V positive AIH‐1 patients and controls (p<0.0001). A‐FAA positivity was significantly associated with higher γ‐globulin and IgG levels, but did not correlate with other considered parameters. Conclusion The modified A‐FAA ELISA strictly correlates with the SMA‐T/G pattern and is a reliable and operator independent assay for AIH‐1. Detection of A‐FAA, even if devoid of prognostic relevance, may be useful when interpretative doubts of standard IIF arise. PMID:16505279

  16. The role of renewable liquid transportation fuels in Canada's climate action plan: Pros and cons, and stages of development of ethanol, biodiesel, and thermal depolymerization oil

    International Nuclear Information System (INIS)

    Coxworth, E.

    2003-04-01

    The feasibility of using ethanol from grain as a partial replacement for gasoline is examined. Although ethanol is widely seen as a desirable renewable transportation fuel, there are concerns about the amount of fossil fuel energy used in its production, both at the farm and the factory level. Indeed, some people claim that there is, in fact, a net energy loss when ethanol is produced from grain, not to mention the concerns about the use of grain for fuel instead of food in a world where millions of people go hungry every day of their life. The emission from ethanol plants and the cost of producing ethanol from grain are related issues that cause concern. The report urges examination of alternatives to fermentation of plant materials such as gasification, and materials other than corn, such as woody material or hay which, although more complex and not yet commercially developed, are showing promise, and deserve further attention. Other renewable liquid transportation fuels such as biodiesel from canola oil, and thermal depolymerization oils that can be derived from a wide variety of waste renewable organic materials, are also suggested as potential fuel sources. Both of these appear promising and require testing to determine implications of further developing these technologies as replacement transportation fuels. The report contains a bibliography of 37 items for ethanol, 12 items for biodiesel, and two items for thermal depolymerization oil

  17. Depolymerization of coal by O2 oxidation followed by acid hydrolysis; Sanso sanka-kasui bunkai ni yoru sekitan no teionkai jugo

    Energy Technology Data Exchange (ETDEWEB)

    Aizawa, S.; Hayashi, J.; Kumagai, H.; Chiba, T. [Hokkaido University, Sapporo (Japan). Center for Advanced Research of Energy Technology; Morooka, S. [Kyushu University, Fukuoka (Japan). Faculty of Engineering

    1996-10-28

    With an objective to elucidate characteristics of oxygen addition to coal, and characteristics of solvent extraction by means of depolymerization, experiments were performed on oxygen oxidation and acid hydrolysis of brown coals. Coals used for the experiments are Morwell (MW), Yallourn (YL) , South Banko (SB) and Wyoming (WY) coals. Test samples were suspended in weak alkaline aqueous solution, and then oxygen was blown into them with pressure kept at atmospheric pressure. After a lapse of a predetermined time, the samples were cooled, and made as acidic as pH 1.3 in hydrochloric acid, followed by acid hydrolysis. Oxygen consumption increased with the reaction time, and with the MW coal, one mol oxygen reacted to 11 mols of coal. Spectral analysis on the YL and WY coal experiments revealed that aliphatic carbon combined with aromatic carbon or ether group has turned to peroxide, whose C-C or C-O bond was broken down as a result of acid hydrolysis of the peroxide, producing oxygen containing compounds. As a result of the depolymerization, the rate of extraction by using DMF, DMSO and methanol/THF mixed solvent increased to 90% or higher. Proportion of bond and cutting-off affects largely collapse of the cross-link structure. The carbon conversion to volatiles was at most 4%. 1 ref., 10 figs.

  18. Isolation of beta-mannanase from Cocos nucifera Linn haustorium and its application in the depolymerization of beta-(1,4)-linked D-mannans.

    Science.gov (United States)

    Soumya, Rema S; Abraham, Emilia T

    2010-05-01

    Beta-mannanase was extracted from coconut (Cocos nucifera Linn) haustorium and purified through ammonium sulfate precipitation and sepharose 6B-lectin affinity chromatography. Coconut beta-mannanase is an acidic protein with a pI of 3.75. The molecular mass of coconut beta-mannanase (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was found to be 44 kDa and was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The optimum temperature and pH for enzyme activity was 70 degrees C and 5.2. The enzyme was used for the preparation of neutraceutical dietary supplement from galactomannans of guar gum and tender coconut kernel having a beta-(1,4)-linked D-mannose backbone. Depolymerized guar gum has 92% of oligosaccharides with a degree of polymerization of 3 and 7. Tender coconut kernel has a degree of polymerization of 9-39 oligosaccharides along with disaccharides and trisaccharides. Hence this mannanase will be useful to depolymerize beta-(1,4)-linked D-mannose polysaccharides from most plant sources to produce prebiotics in a cost-effective technique.

  19. The role of actin in root hair morphogenesis : studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug

    NARCIS (Netherlands)

    Miller, D.D.; Ruijter, de N.C.A.; Bisseling, T.; Emons, A.M.C.

    1999-01-01

    Root hairs develop from bulges on root epidermal cells and elongate by tip growth, in which Golgi vesicles are targeted, released and inserted into the plasma membrane on one side of the cell. We studied the role of actin in vesicle delivery and retention by comparing the actin filament

  20. Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression.

    Directory of Open Access Journals (Sweden)

    Gianina Ravenscroft

    Full Text Available Mutations in the skeletal muscle α-actin gene (ACTA1 cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G of ACTA1 (identified in a severe nemaline myopathy patient fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ~30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.

  1. Actin Nemaline Myopathy Mouse Reproduces Disease, Suggests Other Actin Disease Phenotypes and Provides Cautionary Note on Muscle Transgene Expression

    Science.gov (United States)

    Ravenscroft, Gianina; Jackaman, Connie; Sewry, Caroline A.; McNamara, Elyshia; Squire, Sarah E.; Potter, Allyson C.; Papadimitriou, John; Griffiths, Lisa M.; Bakker, Anthony J.; Davies, Kay E.; Laing, Nigel G.; Nowak, Kristen J.

    2011-01-01

    Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations. PMID:22174871

  2. Actin cytoskeleton and small heat shock proteins: how do they interact?

    Science.gov (United States)

    Mounier, Nicole; Arrigo, André-Patrick

    2002-01-01

    Actin and small heat shock proteins (sHsps) are ubiquitous and multifaceted proteins that exist in 2 reversible forms, monomers and multimers, ie, the microfilament of the cytoskeleton and oligomers of the sHsps, generally, supposed to be in a spherical and hollow form. Two situations are described in the literature, where the properties of actin are modulated by sHsps; the actin polymerization is inhibited in vitro by some sHsps acting as capping proteins, and the actin cytoskeleton is protected by some sHsps against the disruption induced by various stressful conditions. We propose that a direct actin-sHsp interaction occurs to inhibit actin polymerization and to participate in the in vivo regulation of actin filament dynamics. Protection of the actin cytoskeleton would result from an F-actin–sHsp interaction in which microfilaments would be coated by small oligomers of phosphorylated sHsps. Both proteins share common structural motives suggesting direct binding sites, but they remain to be demonstrated. Some sHsps would behave with the actin cytoskeleton as actin-binding proteins capable of either capping a microfilament when present as a nonphosphorylated monomer or stabilizing and protecting the microfilament when organized in small, phosphorylated oligomers. PMID:12380684

  3. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    Science.gov (United States)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  4. FIMBRIN1 is involved in lily pollen tube growth by stabilizing the actin fringe.

    Science.gov (United States)

    Su, Hui; Zhu, Jinsheng; Cai, Chao; Pei, Weike; Wang, Jiaojiao; Dong, Huaijian; Ren, Haiyun

    2012-11-01

    An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.

  5. Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum

    International Nuclear Information System (INIS)

    Schwartz, M.A.; Luna, E.J.

    1986-01-01

    The binding of native, 125 I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. The authors conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins

  6. Altered cell mechanics from the inside: dispersed single wall carbon nanotubes integrate with and restructure actin.

    Science.gov (United States)

    Holt, Brian D; Shams, Hengameh; Horst, Travis A; Basu, Saurav; Rape, Andrew D; Wang, Yu-Li; Rohde, Gustavo K; Mofrad, Mohammad R K; Islam, Mohammad F; Dahl, Kris Noel

    2012-05-23

    With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs) are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  7. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  8. The 5’cap of Tobacco Mosaic Virus (TMV) is required for virion attachment to the actin/ER network during early infection

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Tilsner, Jens; Bell, Karen

    to the motile cortical actin/ER network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actindependent RNA movement. The 5’ methylguanosine TMV cap was shown to be required for vRNA anchoring to the ER. TMV vRNA lacking the 5’cap failed to form granules...... the fluorescent vRNA pool nor co-injected GFP left the injected trichome, indicating that the synthesis of unlabelled progeny viral (v)RNA is required to initiate cell-cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3-vRNA formed granules that became anchored...... on the same ER-bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome....

  9. Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.

    Science.gov (United States)

    Rould, Mark A; Wan, Qun; Joel, Peteranne B; Lowey, Susan; Trybus, Kathleen M

    2006-10-20

    Actin filament growth and disassembly, as well as affinity for actin-binding proteins, is mediated by the nucleotide-bound state of the component actin monomers. The structural differences between ATP-actin and ADP-actin, however, remain controversial. We expressed a cytoplasmic actin in Sf9 cells, which was rendered non-polymerizable by virtue of two point mutations in subdomain 4 (A204E/P243K). This homogeneous monomer, called AP-actin, was crystallized in the absence of toxins, binding proteins, or chemical modification, with ATP or ADP at the active site. The two surface mutations do not perturb the structure. Significant differences between the two states are confined to the active site region and sensor loop. The active site cleft remains closed in both states. Minor structural shifts propagate from the active site toward subdomain 2, but dissipate before reaching the DNase binding loop (D-loop), which remains disordered in both the ADP and ATP states. This result contrasts with previous structures of actin made monomeric by modification with tetramethylrhodamine, which show formation of an alpha-helix at the distal end of the D-loop in the ADP-bound but not the ATP-bound form (Otterbein, L. R., Graceffa, P., and Dominguez, R. (2001) Science 293, 708-711). Our reanalysis of the TMR-modified actin structures suggests that the nucleotide-dependent formation of the D-loop helix may result from signal propagation through crystal packing interactions. Whereas the observed nucleotide-dependent changes in the structure present significantly different surfaces on the exterior of the actin monomer, current models of the actin filament lack any actin-actin interactions that involve the region of these key structural changes.

  10. A peek into tropomyosin binding and unfolding on the actin filament.

    Directory of Open Access Journals (Sweden)

    Abhishek Singh

    Full Text Available BACKGROUND: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. PRINCIPAL FINDINGS: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering, and chain dissociation (analyzed using circular dichroism. CONCLUSIONS: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest

  11. The association between osteoporotic hip fractures and actinic lesions as a biomarker for cumulative sun exposure in older people-a retrospective case-control study in Argentina.

    Science.gov (United States)

    Perroud, H A; Dagatti, M S; Amigot, B; Levit, G P; Tomat, M F; Morosano, M E; Masoni, A M; Pezzotto, S M

    2017-05-01

    The aim of this study was to analyze the association between the presence of actinic lesions (solar keratosis and non-melanoma skin cancer) and osteoporotic hip fractures in older patients. Both pathologies are common conditions in this age group. Since cumulative sun exposure is difficult to quantify, the presence of actinic lesions can be used to indirectly analyze the association between ultraviolet radiation and osteoporotic hip fractures. This was an observational case-control study. We reviewed the centralized medical records of patients with hip fracture (cases, n = 51) and patients with other diseases hospitalized in the same institution and period (controls, n = 59). The mean age of the patients was 80 ± 8.3 years (range 50-103 years). Differences in maternal hip fracture history were found between cases and controls (14.8 and 8 %, respectively; p = 0.047). Falls history in the past year was higher in cases than in controls (p < 0.0001). Actinic lesions were observed in 32.7 % of patients (prevalence rate 23.5 % in cases, 40.7 % in controls; p = 0.04). When considering patients with actinic lesions, controls have a higher FRAX score compared with cases. Although sun exposure is recommended for bone health, it represents a risk factor for actinic lesions. The presence of actinic lesions may indicate a lower osteoporotic hip fracture risk. A balance between adequate lifetime sun exposure and protection against its adverse effects is required for each patient, in the context of geographic location.

  12. Effect of 0.4 mT power frequency magnetic field on F-actin assembly of CHL cells

    International Nuclear Information System (INIS)

    Chu Keping; Cai Zhiyin; Zhang Yukun; Xia Nuohong

    2007-01-01

    Objective: To investigate the effect of 0.4mT power frequency magnetic field on the microfilament (F- actin) assembly of Chinese hamster lung (CHL) cells. Methods: F-actin were marked with immunohistochemical method, then observed under a confocal microscope. The content of ECFRs in the preparation of the detergent-insoluble cytoskeleton was measured with Western-blotting. Results: The stress fiber's of CHL cells decreased after exposure to 0.4mT power frequency magnetic field for 30min, as well as after treatment with epidermal growth factor (ECF) of 50nM. Filopodias appeared at the periphery after exposure to magnetic field as well as treatment with EGF. The EGF receptor mass associated with the detergent-insoluble cytoskeleton increased after exposure to magnetic field as well as treatment with EGF. Conclusion: 0.4mT power frequency magnetic field induced assembly of F-actin in CHL cells. The change induced by magnetic field would be related to clustering of EGFR induced by magnetic field and passing the signal down. (authors)

  13. System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

    Science.gov (United States)

    Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

    2017-01-01

    The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

  14. Lithium preserves F-actin from the disarrangement induced by either DNase I or cytochalasin D.

    Science.gov (United States)

    DalleDonne, I; Milzani, A; Fascio, U; Ratti, A; Colombo, R

    1993-01-01

    Light scattering at 546 nm, which is mainly related to the presence of rodlike particles longer than 50 nm, showed that Li+ accelerates the formation of actin filaments. Intermolecular cross-linking with N,N'-1,4-phenylene-bismaleimide proved that the observed enhancement in the light-scattering intensity is caused by the increase in the concentration of actin oligomers, which gradually elongate to form longer filaments. DNase-I-related F-actin disassembly was reduced in the presence of lithium ions, as demonstrated by fluorimetric and viscometric experiments. Li(+)-F-actin showed an apparently similar behaviour when exposed to cytochalasin D. We confirm that Li+ acts on actin polymerization by stabilizing actin nuclei and polymers. The stabilization of cytoskeletal polymers really appears as one of the mechanisms by which lithium ions influence some of the cell activities.

  15. The interaction between the adaptor protein APS and Enigma is involved in actin organisation

    DEFF Research Database (Denmark)

    Barres, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick

    2005-01-01

    and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin...... cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest...... that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation....

  16. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    Science.gov (United States)

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

  17. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Iman Jalilian

    Full Text Available The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm, in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  18. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  19. Actin expression is induced and three isoforms are differentially expressed during germination in Zea mays.

    Science.gov (United States)

    Díaz-Camino, Claudia; Conde, Renaud; Ovsenek, Nick; Villanueva, Marco A

    2005-02-01

    Previous analysis of actin in a dicotyledonous plant, Phaseolus vulgaris (or common bean), showed very low actin levels in cotyledons but they were concentrated in the embryo axis. Upon imbibition, actin expression increased 5-fold and a maximum of four actin isoforms were observed, two of them transient and two major ones were steadily expressed. In this work, analysis of the actin expression in a monocotyledonous plant, Zea mays (or maize), and over a longer period of germination/growth, showed that striking similarities exist. Actin is present in all the seed components, but it is mainly concentrated in the embryo axis. The expression of maize actin was induced during post-imbibition at both the protein and mRNA levels. Sharp increases in actin appeared from 24-48 h and again from 72-96 h. A more modest and steady actin mRNA increase in expression was observed; however, it did not appear as dramatic as in the case of common bean due to the presence of readily detectable amounts of message in the dry maize seed. The isoform distribution in the dry seed showed a pattern of at least three isovariants of pIs approximately 5.0, 5.1, and 5.2, which were differentially expressed at the various post-imbibition times analysed. Two of the actin isoforms at 48 h post-imbibition cross-reacted with a phosphotyrosine-specific antibody and they are the product of three expressed genes as shown by in vitro translation assays. These data indicate that maize actin protein and mRNA expression is induced upon the trigger of germination, and the isoform expression kinetics and patterns resemble those from bean, suggesting that, in both species, actin expression at these early germination/growth stages is a highly regulated event.

  20. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    Science.gov (United States)

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-01-01

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

  1. A mathematical model of actin filament turnover for fitting FRAP data.

    Science.gov (United States)

    Halavatyi, Aliaksandr A; Nazarov, Petr V; Al Tanoury, Ziad; Apanasovich, Vladimir V; Yatskou, Mikalai; Friederich, Evelyne

    2010-03-01

    A novel mathematical model of the actin dynamics in living cells under steady-state conditions has been developed for fluorescence recovery after photobleaching (FRAP) experiments. As opposed to other FRAP fitting models, which use the average lifetime of actins in filaments and the actin turnover rate as fitting parameters, our model operates with unbiased actin association/dissociation rate constants and accounts for the filament length. The mathematical formalism is based on a system of stochastic differential equations. The derived equations were validated on synthetic theoretical data generated by a stochastic simulation algorithm adapted for the simulation of FRAP experiments. Consistent with experimental findings, the results of this work showed that (1) fluorescence recovery is a function of the average filament length, (2) the F-actin turnover and the FRAP are accelerated in the pr