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Sample records for alpha-smooth muscle actin

  1. Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis.

    OpenAIRE

    Vyalov, S. L.; Gabbiani, G.; Kapanci, Y.

    1993-01-01

    The majority of fibroblasts in alveolar septa are characterized by the presence of cytoplasmic bundles of microfilaments that contain cytoplasmic actin isoforms; these cells have been named contractile interstitial cells or V-type myofibroblasts. In the rat, they express desmin as intermediate filament protein. In this study, we explored the possibility that modulation and replication of such septal fibroblasts result in the appearance of alpha-smooth muscle (alpha-SM) actin-positive myofibro...

  2. Alpha smooth muscle actin in the cycling ovary - an immunohistochemical study.

    Science.gov (United States)

    Hirschberg, Ruth M; Plendl, Johanna; Kaessmeyer, Sabine

    2012-01-01

    In the ovary with its cyclically developing and regressing functional bodies and the associated intense neovascularisation and remodelling, alpha-smooth muscle actin (SMA) immunolocalisation has been frequently used as a marker to establish vessel hierarchy, in angiogenesis studies, or in studies characterising ovarian neoplasms in various species. The present study aims at detection of alpha-SMA-immunolocalisation within all structural components of the cycling bovine ovary in order to complement the hitherto available data. 27 ovaries, mainly of dairy cows ranging from 23 to 118 months of age and displaying all major stages of follicle and corpora lutea development, were collected at the abattoir and subjected to routine HE and trichrome staining as well as alpha-SMA immunohistochemistry. For this purpose, the specimens were pooled to form groups of the respective stage of corpus luteum development. The ovarian stroma displayed a notable alpha-SMA-reactivity, particularly surrounding the functional bodies. The study revealed specialised vascular modifications such as multi-directionally arranged vascular smooth muscle layers, vascular sphincters and distinct epitheloid modifications of the media in ovarian arteries. Alpha-SMA-reactivity of the microcirculation within corpora lutea of various stages allowed inferences on respective angiogenic properties. The findings were discussed focussing on functional interpretations. PMID:22538540

  3. Significance of alpha smooth muscle actin expression in traumatic painful neuromas: a pilot study in rats

    Science.gov (United States)

    Weng, Weidong; Zhao, Bin; Lin, Dingshen; Gao, Weiyang; Li, Zhijie; Yan, Hede

    2016-01-01

    Treatment of painful neuromas remains a challenge and the mechanism of neuroma-associated pain is not yet fully understood. In this study, we aimed to observe the expression of alpha smooth muscle actin (α-SMA) in traumatic neuromas and to investigate its possible roles in the cause of neuropathic pain in a rat model. The rat sciatic nerve was used and the experiment was divided into two parts. In part I, our results showed significantly higher levels of α-SMA and the pain marker c-fos in the autotomy group than in the no-autotomy group. In part II, the expression of α-SMA in neuromas was down- and up-regulated using SB-431542 and GW9662, respectively. A significant correlation between autotomy scores and the expression level of α-SMA was found (R = 0.957; p neuroma-associated pain, either as a direct cause of pain or as an indirect marker of existence of local mechanical stimuli. Our findings may provide new insights into the development of new treatment modalities for the management of intractable painful neuromas. PMID:27021914

  4. alpha-Smooth muscle actin-expressing cells and lubricin in periprosthetic tissue.

    Science.gov (United States)

    Funakoshi, Tadanao; Martin, Scott D; Wolf, Bryce T; Schmid, Thomas M; Thornhill, Thomas S; Spector, Myron

    2010-05-01

    The objective of the study was to evaluate the distributions of (1) cells expressing the contractile actin isoform, alpha-smooth muscle actin (alpha-SMA) and (2) a lubricating and antiadhesion glycoprotein, lubricin, in the tissue around loose joint replacement prostheses in human subjects. Periprostehtic tissue resected at revision arthroplasty of noncemented glenoid components of total shoulder arthroplasties was obtained from 10 patients. Samples of periprosthetic tissue were stained with monoclonal antibodies to alpha-SMA and lubricin. alpha-SMA was found in cells, principally of fibroblast morphology, in many of the fields of view (FOVs) in samples from all patients. Moderate correlations were observed between the percentage of FOVs containing alpha-SMA-expressing cells and the percentages of FOVs containing polyethylene (R(2) = 0.79) and metallic (R(2) = 0.75) particles. Lubricin was identified (1) as a discrete layer on the surface, (2) within the extracellular matrix, and (3) intracellularly. These lubricin-positive features were found in samples from all patients. Strong correlations were noted between the percentages of FOVs with matrix and intracellular lubricin staining (R(2) = 0.97) and between the percentages of FOVs with surface and matrix staining for lubricin (R(2) = 0.96). Having established the presence of alpha-SMA and lubricin in periprosthetic tissue, hypotheses regarding their role in the development and persistence of periprosthetic tissue can be synthesized for future study: for example, alpha-SMA-enabled contracture of the fibrous periprosthetic tissue results in its densification, and lubricin-coated surfaces interfere with integrative repair processes necessary for resorption and remodeling.

  5. P0525 : N-Acetylated alpha smooth muscle actin levels are increased in hepatic fibrosis but decreased in hepatocellular carcinoma

    DEFF Research Database (Denmark)

    Nielsen, M.J.; Nielsen, Signe Holm; Hansen, N.U.B.;

    2015-01-01

    Alpha Smooth Muscle Actin (a-SMA) is upregulated together with extracellular matrix (ECM) during activation of Hepatic Stellate Cells (HSCs) in fibrosis. Histone deacetylase (HDAC) remove acetylations and regulate the expression of genes, which is associated with cancers. There is a close...... relationship between cirrhosis and hepatocellular carcinoma (HCC), and markers enabling identification of patients in risk of developing HCC with cirrhosis is a major unmet clinical need. We developed an ELISA for the assessment of acetylated a-SMA (Aca- SMA) in serum. The objective was to investigate...

  6. 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

    reactions. Here, we show that the presence of alpha-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without alpha-sm actin revealed migratory restraint in alpha-sm actin-positive fibroblasts. Electroporation...

  7. 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)

  8. Collagen gel contraction serves to rapidly distinguish epithelial- and mesenchymal-derived cells irrespective of alpha-smooth muscle actin expression

    DEFF Research Database (Denmark)

    Nielsen, Helga Lind; Gudjonsson, Thorarinn; Villadsen, René;

    2004-01-01

    Mesenchymal-like cells in the stroma of breast cancer may arise as a consequence of plasticity within the epithelial compartment, also referred to as epithelial-mesenchymal transition, or by recruitment of genuine mesenchymal cells from the peritumoral stroma. Cells of both the epithelial compart...... under these conditions did not augment contractility. It is concluded that epithelial-derived mesenchymal-like cells are functionally defective within a connective tissue environment irrespective of an apparent contractile phenotype.......Mesenchymal-like cells in the stroma of breast cancer may arise as a consequence of plasticity within the epithelial compartment, also referred to as epithelial-mesenchymal transition, or by recruitment of genuine mesenchymal cells from the peritumoral stroma. Cells of both the epithelial...... compartment and the stromal compartment express alpha smooth muscle actin (alpha-sm actin) as part of a myoepithelial or a myofibroblastic differentiation program, respectively. Moreover, because both epithelial- and mesenchymal-derived cells are nontumorigenic, other means of discrimination are warranted...

  9. Rapid disappearance of the medial epithelial seam during palatal fusion occurs by multifocal breakdown that is preceded by expression of alpha smooth muscle actin in the epithelium.

    Science.gov (United States)

    Gibbins, J R; Brent, S; Srivastava, M; Garibotto, N; Tazawa, Y M; Cameron, A; Hunter, N

    2000-02-01

    Breakdown of the medial epithelial seam (MES) is essential to allow bridging of the mesenchyme during palatal fusion. Evidence exists for three mechanisms for this breakdown that are incompatible at the level of individual cells in the seam. To determine if breakdown of the seam was regionally restricted, 3-dimensional reconstructions were generated using volume rendering software from 1 micron serial sections in the sagittal plane of rat palates fixed during the process of fusion. The earliest break detected in electron micrographs was cell separation and in reconstructions was a discrete defect, with a rounded outline, nearer to the nasal than to the oral margin of the seam. Further breakdown produced a pattern of rounded defects along the nasal margin of the seam resulting in interconnected columns of cells preferentially attached to the oral epithelium. Computer generated slicing of reconstructed seams showed that groups of cells evident in cross-sections as islands at this stage of breakdown of the MES could be artifacts. Unequivocal islands of epithelial cells formed later in fusion had a rounded outline, an incomplete basal lamina and a halo of cells containing phagocytosed apoptotic debris. The pattern of breakdown indicated that the MES breaks down under tension. Laser confocal microscopy of sections and whole-mounts of palates demonstrated alpha-smooth muscle actin preferentially localized in the epithelial cells of the palatal shelves immediately before and during formation of the seam. Expression in epithelial cells of the isoform of actin normally restricted to smooth muscle cells engaged in tonic contraction supported an interpretation that the epithelial cells of the seam may be capable of generating tension during the palatal fusion event. PMID:10794080

  10. Thomsen-Friedenreich (T) antigen as marker of myoepithelial and basal cells in the parotid gland, pleomorphic adenomas and adenoid cystic carcinomas. An immunohistological comparison between T and sialosyl-T antigens, alpha-smooth muscle actin and cytokeratin 14

    DEFF Research Database (Denmark)

    Therkildsen, M H; Mandel, U; Christensen, M;

    1995-01-01

    -known markers of normal MEC/basal cells (i.e. alpha-smooth muscle actin and cytokeratin 14) with T (Thomsen-Friedenreich) antigen and its sialylated derivative: sialosyl-T antigen,) in 17 normal parotid glands and in two tumour types with MEC participation (i.e pleomorphic adenomas (PA) and adenoid cystic...... was the only marker of cells in solid undifferentiated areas of adenoid cystic carcinomas. Our study supports the view, that modified "myoepithelial" cells in the tumours consist of a mixture of basal cells and myoepithelial cells. None of the investigated structures was in itself an ideal marker...

  11. Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I

    Directory of Open Access Journals (Sweden)

    Stephanie Kern

    2014-01-01

    Full Text Available We previously reported that a restrictive N-terminal truncation of cardiac troponin I (cTnI-ND is up-regulated in the heart in adaptation to hemodynamic stresses. Over-expression of cTnI-ND in the hearts of transgenic mice revealed functional benefits such as increased relaxation and myocardial compliance. In the present study, we investigated the subsequent effect on myocardial remodeling. The alpha-smooth muscle actin (α-SMA isoform is normally expressed in differentiating cardiomyocytes and is a marker for myocardial hypertrophy in adult hearts. Our results show that in cTnI-ND transgenic mice of between 2 and 3 months of age (young adults, a significant level of α-SMA is expressed in the heart as compared with wild-type animals. Although blood vessel density was increased in the cTnI-ND heart, the mass of smooth muscle tissue did not correlate with the increased level of α-SMA. Instead, immunocytochemical staining and Western blotting of protein extracts from isolated cardiomyocytes identified cardiomyocytes as the source of increased α-SMA in cTnI-ND hearts. We further found that while a portion of the up-regulated α-SMA protein was incorporated into the sarcomeric thin filaments, the majority of SMA protein was found outside of myofibrils. This distribution pattern suggests dual functions for the up-regulated α-SMA as both a contractile component to affect contractility and as possible effector of early remodeling in non-hypertrophic, non-failing cTnI-ND hearts.

  12. Construction of lentivector containing alpha-smooth muscle actin promoter by multisite Gateway technology%利用多位点Gateway技术构建小鼠平滑肌肌动蛋白α基因启动子慢病毒载体

    Institute of Scientific and Technical Information of China (English)

    袁晓峰; 项鹏; 李伟强; 胡晓俊; 彭朝权

    2012-01-01

    背景:平滑肌肌动蛋白α基因是相对局限于在血管平滑肌细胞中表达的少数几个基因之一,公认是血管平滑肌细胞表型转化的标志.目的:利用多位点Gateway技术构建慢病毒载体pLVpuro/平滑肌肌动蛋白α控制绿色荧光蛋白基因的表达.方法:设计合成含有attB位点的小鼠平滑肌肌动蛋白α基因启动子引物,构建pUp-平滑肌肌动蛋白α;通过LR反应将pUp-平滑肌肌动蛋白α和pDown-绿色荧光蛋白(含att位点的绿色荧光蛋白入门克隆)连接到目的载体pDEST-puromycin,得到pLVpuro/平滑肌肌动蛋白α-绿色荧光蛋白表达载体;经PCR和测序鉴定,将载体质粒瞬时转染C2C12细胞系,并且用免疫荧光染色检测基因的表达.结果与结论:成功构建pLVpuro/平滑肌肌动蛋白α-绿色荧光蛋白报告基因载体,测序结果表明启动子序列正确;细胞转染实验以及免疫荧光检测证实构建的报告基因载体可以反映平滑肌肌动蛋白α基因的表达情况.%BACKGROUND: Alpha-smooth muscle actin gene is one of several genes expressed in smooth muscle cells and has been recognized as the marker for smooth muscle cell phenotype transformation. OBJECTIVE: To construct a recombinant pLVpuro/aSMA-hrGFP lentiviral vector by multisite Gateway technology. METHODS: Primers containing attB sites were designed and used to amplify the alpha-smooth muscle actin (aSMA) promoter fragment by PCR from the plasmid containing the mouse aSMA promoter sequence (SMP8-Cre). By the BP recombination reaction, the attB flanked PCR product containing aSMA promoter sequence was cloned to an attP-containing pDONR P4P1r donor vector to create an entry clone, pUp-aSMA. Finally, pUp-aSMA and pDown-hrGFP were shuttled into the destination vector pDEST-puromycin by LR recombination reaction to generate pLVpuro/aSMA-hrGFP. The expression vector was confirmed by PCR and gene sequencing. Then this expression vector was transferred into the C2C12 cell

  13. Effect of botulinum toxin type A on the expression of substance P, calcitionin gene-related peptide,transforming growth factor beta-1 and alpha smooth muscle actin A in wound healing in rats%肉毒毒素A对大鼠皮肤及创面组织中SP、CGRP、TGF-β1和α-SMA的影响

    Institute of Scientific and Technical Information of China (English)

    王琳; 邰宁正; 范志宏

    2009-01-01

    Objective To investigate the effect of botulinum toxin type A on the expression of substance P(SP),calcitionin gene-related peptide(CGRP), transforming growth factor beta-1 (TGF-β1) and alpha smooth muscle actin A(α-SMA) in wound healing. Methods 60 rats were randomly divided into group C (control)、 group L (low-dose) and group H (high-dose), with 20 rats in each group. The wound-healing model was established by excision of four full-thickness skin(1 cm×1 cm, around the injection site) on the back of all SD rats on the 7th day after BTA injection. The wound size was measured and the expression of SP, CGRP, TGF-β1 and α-SMA in wound granulation tissue was assayed by immanohistocbemical staining and computerized image analysis before operation, and 3 days, 7 days and 14 days after operation. Results All the wounds healed 14 days after operation. The wound size in L and H group was not significantly different with that in C group on the 3rd day and 7th day after operation. The positive immuno-staining of SP, CGRP, TGF-β1 and α-SMA in group L and H was significantly weaker than those in C group. Meanwhile, the positive immuno-staining of all above substances in H group was weaker than those in L group significantly. Conclusions Botulinum toxin type A can decrease the expression of SP, CGRP, TGF-β1 and α-SMA in wound healing in a dose-dependent manner with no effect on the healing time.%目的 探讨肉毒毒素A对周围神经末梢释放的感觉神经肽P物质、降钙素基因相关肽及创面组织表达TGF-β1和α-平滑肌肌动蛋白的影响. 方法 60只SD大鼠随机分为空白对照组(C组)、低剂量组(L组)和高剂量组(H组),每组加只大鼠.肉毒毒素A预注射后7 d,在鼠背部建立以4个注射点为中心的创面模型,创面面积1 cm×1 cm.于术前及术后3、7、14 d,测量创面面积,免疫组织化学和Axioplan 2 Imaging图像分析软件检测多聚甲醛灌注后创面P物质、降钙素基因相关肽以及创

  14. Changes in keratins and alpha-smooth muscle actin during three-dimensional reconstitution of eccrine sweat glands.

    Science.gov (United States)

    Li, Haihong; Li, Xuexue; Zhang, Bingna; Zhang, Mingjun; Chen, Wenlong; Tang, Shijie; Fu, Xiaobing

    2016-07-01

    We have examined the changes of keratins and alpha-SMA at various time points in order to investigate the development and differentiation of eccrine sweat gland cells during the course of three-dimensional (3D) reconstitution. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 1, 2, 4, 6, 8, 14, 21, 28, 35, and 42 days post-implantation, Matrigel plugs were removed and immunostained. We found that during 3D reconstitution, keratin and alpha-SMA expression changed in a time-dependent manner. At day 1, all cells stained positively for keratin isoforms K5, K14, and K15, with the staining intensity of K15 being weak and K5 and K14 being strong, but none of the cells displayed K7, K8, or alpha-SMA. As time progressed, spheroid-like structures formed with the inner layer acquiring K7 and K8, but losing K5 and K14 expression, and the outer layer acquiring alpha-SMA expression, but losing K15 expression. K8 expression was first noted at day 14, and K7 and alpha-SMA at day 21. The loss of K15 expression was first noted at day 14, K14 at day 21, and K5 at day 28. At 28, 35, and 42 days, the spheroid-like structures could be distinguished, by immunohistochemistry, as having secretory coil-like and coiled duct-like structures. We conclude that the changes in expression of keratins and alpha-SMA in 3D-reconstituted eccrine sweat glands are similar to those of native eccrine sweat glands, indicating that the 3D reconstitution of sweat glands provides an excellent model for studying the development, cytodifferentiation, and regulation of eccrine sweat glands. PMID:26837225

  15. Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and alpha-Smooth Muscle Actin Expression of Retinal Muller Cells

    NARCIS (Netherlands)

    Bu, Shao-Chong; Kuijer, Roel; van der Worp, Roelofje J.; van Putten, Sander M.; Wouters, Olaf; Li, Xiao-Rong; Hooymans, Johanna M. M.; Los, Leonoor I.

    2015-01-01

    PURPOSE. The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Muller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigi

  16. Immunoreactivity for alpha-smooth muscle actin characterizes a potentially aggressive subgroup of little basal cell carcinomas

    Directory of Open Access Journals (Sweden)

    G Faa

    2009-06-01

    Full Text Available Basal cell carcinoma (BCC is a very common malignant skin tumor that rarely metastatizes, but is often locally aggressive. Several factors, like large size (more than 3 cm, exposure to ultraviolet rays, histological variants, level of infiltration and perineural or perivascular invasion, are associated with a more aggressive clinical course. These morphological features seem to be more determinant in mideface localized BCC, which frequently show a significantly higher recurrence rate. An immunohistochemical profile, characterized by reactivity of tumor cells for p53, Ki67 and alpha-SMA has been associated with a more aggressive behaviour in large BCCs. The aim of this study was to verify if also little (less than 3 cm basal cell carcinomas can express immunohistochemical markers typical for an aggressive behaviour.

  17. Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's - derived fibroblasts

    OpenAIRE

    Johnson Sandra; Baratz Mark E; Gallo Phillip H; Satish Latha; Kathju Sandeep

    2011-01-01

    Abstract Background Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic A...

  18. Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin.

    Science.gov (United States)

    Khaitlina, S; Antropova, O; Kuznetsova, I; Turoverov, K; Collins, J H

    1999-08-01

    In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin. Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin. Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin. When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected. At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences. Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin. Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin. We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer. The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed. PMID:10415117

  19. Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

    Science.gov (United States)

    Wong, J Z; Woodcock-Mitchell, J; Mitchell, J; Rippetoe, P; White, S; Absher, M; Baldor, L; Evans, J; McHugh, K M; Low, R B

    1998-05-01

    In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

  20. Molecular mechanical differences between isoforms of contractile actin in the presence of isoforms of smooth muscle tropomyosin.

    OpenAIRE

    Lennart Hilbert; Genevieve Bates; Roman, Horia N.; Jenna L Blumenthal; Zitouni, Nedjma B.; Apolinary Sobieszek; Mackey, Michael C.; Anne-Marie Lauzon

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for [Formula: see text]-actin ([Formula: see text...

  1. Release of α-actin into serum after skeletal muscle damage

    Science.gov (United States)

    Martinez-Amat, A; Boulaiz, H; Prados, J; Marchal, J; Padial, P; Caba, O; Rodriguez-Serrano, F; Aranega, A

    2005-01-01

    Objective: The skeletal muscle protein α-actin was investigated in the serum of subjects with severe skeletal muscle damage to assess its utility as a reliable and predictive marker of muscle damage. Methods: Serum samples were obtained from 33 healthy controls and 33 patients with severe skeletal muscle damage, defined by a total creatine kinase value of >500 IU/l (Rosalki method). Troponin I, troponin T, and myoglobin concentrations were determined by immunoassay and α-actin concentrations by Western blot and densitometry. Results: The mean serum concentration of α-actin in controls and patients with skeletal muscle damage was 600.9 and 1968.51 ng/ml, respectively, a statistically significant difference. Sera of patients with muscle damage showed higher levels of α-actin than of troponin or myoglobin. No significant difference in troponin I levels was observed between the groups. Conclusions: According to these results, α-actin was the most significant skeletal muscle damage marker analysed and may be an ideal candidate for the identification of all types of myofibre injury, including sports injuries. Our findings support the use of α-actin as a marker alongside other currently used biological proteins. PMID:16244192

  2. Intensification of the 5.9-nm actin layer line in contracting muscle.

    Science.gov (United States)

    Matsubara, I; Yagi, N; Miura, H; Ozeki, M; Izumi, T

    According to the cross-bridge model of muscle contraction, an interaction of myosin heads with interdigitating actin filaments produces tension. Although X-ray equatorial diffraction patterns of active (contracting) muscle show that the heads are in the vicinity of the actin filaments, structural proof of actual attachment of heads to actin during contraction has been elusive. We show here that during contraction of frog skeletal muscle, the 5.9-nm layer line arising from the genetic helix of actin is intensified by as much as 56% of the change which occurs when muscle enters rigor, using a two-dimensional X-ray detector. This provides strong structural evidence that myosin heads do in fact attach during contraction. PMID:6334236

  3. Skeletal muscle-specific ablation of gamma(cyto-actin does not exacerbate the mdx phenotype.

    Directory of Open Access Journals (Sweden)

    Kurt W Prins

    Full Text Available We previously documented a ten-fold increase in gamma(cyto-actin expression in dystrophin-deficient skeletal muscle and hypothesized that increased gamma(cyto-actin expression may participate in an adaptive cytoskeletal remodeling response. To explore whether increased gamma(cyto-actin fortifies the cortical cytoskeleton in dystrophic skeletal muscle, we generated double knockout mice lacking both dystrophin and gamma(cyto-actin specifically in skeletal muscle (ms-DKO. Surprisingly, dystrophin-deficient mdx and ms-DKO mice presented with comparable levels of myofiber necrosis, membrane instability, and deficits in muscle function. The lack of an exacerbated phenotype in ms-DKO mice suggests gamma(cyto-actin and dystrophin function in a common pathway. Finally, because both mdx and ms-DKO skeletal muscle showed similar levels of utrophin expression and presented with identical dystrophies, we conclude utrophin can partially compensate for the loss of dystrophin independent of a gamma(cyto-actin-utrophin interaction.

  4. The vascular smooth muscle alpha-actin gene is reactivated during cardiac hypertrophy provoked by load.

    OpenAIRE

    Black, F M; Packer, S E; Parker, T G; Michael, L H; Roberts, R; R J Schwartz; Schneider, M D

    1991-01-01

    Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; howeve...

  5. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

    Directory of Open Access Journals (Sweden)

    Shenping Wu

    Full Text Available Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  6. Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's - derived fibroblasts

    Directory of Open Access Journals (Sweden)

    Johnson Sandra

    2011-05-01

    Full Text Available Abstract Background Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA and increased production of extracellular matrix (ECM components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-β1-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis. Methods Fibroblasts derived from areas of Dupuytren's contracture cord (DC, from adjacent and phenotypically normal palmar fascia (PF, and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT were treated with TGF-β1 (2 ng/ml and/or forskolin (10 μM (a known stimulator of cAMP. Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis. Results The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA, type I (COL1A2 and type III collagen (COL3A1, and connective tissue growth factor (CTGF were all significantly increased in DC- and in PF-derived cells compared to CT-derived fibroblasts. The TGF-β1 stimulation of α-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-β1 stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types. Conclusion In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.

  7. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    Science.gov (United States)

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-07-06

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

  8. Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.

    Science.gov (United States)

    Guo, Dong-Chuan; Pannu, Hariyadarshi; Tran-Fadulu, Van; Papke, Christina L; Yu, Robert K; Avidan, Nili; Bourgeois, Scott; Estrera, Anthony L; Safi, Hazim J; Sparks, Elizabeth; Amor, David; Ades, Lesley; McConnell, Vivienne; Willoughby, Colin E; Abuelo, Dianne; Willing, Marcia; Lewis, Richard A; Kim, Dong H; Scherer, Steve; Tung, Poyee P; Ahn, Chul; Buja, L Maximilian; Raman, C S; Shete, Sanjay S; Milewicz, Dianna M

    2007-12-01

    The major function of vascular smooth muscle cells (SMCs) is contraction to regulate blood pressure and flow. SMC contractile force requires cyclic interactions between SMC alpha-actin (encoded by ACTA2) and the beta-myosin heavy chain (encoded by MYH11). Here we show that missense mutations in ACTA2 are responsible for 14% of inherited ascending thoracic aortic aneurysms and dissections (TAAD). Structural analyses and immunofluorescence of actin filaments in SMCs derived from individuals heterozygous for ACTA2 mutations illustrate that these mutations interfere with actin filament assembly and are predicted to decrease SMC contraction. Aortic tissues from affected individuals showed aortic medial degeneration, focal areas of medial SMC hyperplasia and disarray, and stenotic arteries in the vasa vasorum due to medial SMC proliferation. These data, along with the previously reported MYH11 mutations causing familial TAAD, indicate the importance of SMC contraction in maintaining the structural integrity of the ascending aorta.

  9. Human congenital myopathy actin mutants cause myopathy and alter Z-disc structure in Drosophila flight muscle.

    Science.gov (United States)

    Sevdali, Maria; Kumar, Vikash; Peckham, Michelle; Sparrow, John

    2013-03-01

    Over 190 mutations in the human skeletal muscle α-actin gene, ACTA1 cause congenital actin myopathies. We transgenically expressed six different mutant actins, G15R, I136M, D154N, V163L, V163M and D292V in Drosophila indirect flight muscles and investigated their effects in flies that express one wild type and one mutant actin copy. All the flies were flightless, and the IFMs showed incomplete Z-discs, disorganised actin filaments and 'zebra bodies'. No differences in levels of sarcomeric protein expression were observed, but tropomodulin staining was somewhat disrupted in D164N, V163L, G15R and V163M heterozygotes. A single copy of D292V mutant actin rescued the hypercontractile phenotypes caused by TnI and TnT mutants, suggesting that the D292V mutation interferes with thin filament regulation. Our results show that expression of actin mutations homologous to those in humans in the indirect flight muscles of Drosophila disrupt sarcomere organisation, with somewhat similar phenotypes to those observed in humans. Using Drosophila to study actin mutations may help aid our understanding of congential myopathies caused by actin mutations.

  10. Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

    OpenAIRE

    Trus, B L; Steven, A C; McDowall, A W; M. Unser; Dubochet, J; Podolsky, R J

    1989-01-01

    For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In ...

  11. Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Daniel Reis Waisberg

    2012-09-01

    Full Text Available OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of inter leu kin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling

  12. Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction

    Science.gov (United States)

    Koopmans, Tim; Kumawat, Kuldeep; Halayko, Andrew J; Gosens, Reinoud

    2016-01-01

    A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma. PMID:27468699

  13. TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

    Directory of Open Access Journals (Sweden)

    Yinglong Sa

    2015-04-01

    Full Text Available Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1 has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1, Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

  14. Smooth muscle actin isoforms: a tug of war between contraction and compliance.

    Science.gov (United States)

    Arnoldi, Richard; Hiltbrunner, Anita; Dugina, Vera; Tille, Jean-Christophe; Chaponnier, Christine

    2013-01-01

    In higher vertebrates, smooth muscle (SM) contains two tissue-specific actin isoforms: α-SMA and γ-SMA, which predominate in vascular and visceral SM, respectively. Whether α-SMA has been extensively studied and recognized for its contractile activity in SM and SM-like cells such as myofibroblasts, myoepithelial and myoid cells, the distribution and role of γ-SMA remained largely unknown. We developed a new specific monoclonal antibody against γ-SMA and confirmed that γ-SMA predominates in the visceral system and is minor in the vascular system, although more expressed in highly compliant veins than in stiff arteries. Contrary to α-SMA, γ-SMA is absent from myofibroblasts in vitro, and in fibrotic diseases in vivo. We raised the hypothesis that, whereas α-SMA is responsible for the "contractile" activity, γ-SMA would be involved in the "compliance" of SM and SM-like cells. Several models support this hypothesis, namely veins vs. arteries and the physiological modifications occurring in the uterus and mammary glands during pregnancy and lactation. Our results suggest that, in addition to enteric smooth muscles, γ-SMA is expressed in all the tissues submitted to an important dilation including veins, gravid uterus, and lactating mammary glands. The hypothesis of two complementary mechanical roles for the two SMA isoforms is sustained by their different intracellular distributions and by functional assays. PMID:23915964

  15. Characterization of vascular smooth muscle cell phenotype in long-term culture.

    Science.gov (United States)

    Absher, M; Woodcock-Mitchell, J; Mitchell, J; Baldor, L; Low, R; Warshaw, D

    1989-02-01

    Studies of bovine carotid artery smooth muscle cells, during long-term in vitro subcultivation (up to 100 population doublings), have revealed phenotypic heterogeneity among cells, as characterized by differences in proliferative behavior, cell morphology, and contractile-cytoskeletal protein profiles. In vivo, smooth muscle cells were spindle-shaped and expressed desmin and alpha-smooth muscle actin (50% of total actin) as their predominant cytoskeletal and contractile proteins. Within 24 h of culture, vimentin rather than desmin was the predominant intermediate filament protein, with little change in alpha-actin content. Upon initial subcultivation, all cells were flattened and fibroblastic in appearance with a concomitant fivefold reduction in alpha-actin content, whereas the beta and gamma nonmuscle actins predominated. In three out of four cell lines studied, fluctuations in proliferative activity were observed during the life span of the culture. These spontaneous fluctuations in proliferation were accompanied by coordinated changes in morphology and contractile-cytoskeletal protein profiles. During periods of enhanced proliferation a significant proportion of cells reverted to their original spindle-shaped morphology with a simultaneous increase in alpha-actin content (20 to 30% of total actin). These results suggest that in long-term culture smooth muscle cells undergo spontaneous modulations in cell phenotype and may serve as a useful model for studying the regulation of intracellular protein expression.

  16. Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. I. Smooth muscle tumors.

    OpenAIRE

    Schürch, W.; Skalli, O.; Seemayer, T. A.; Gabbiani, G.

    1987-01-01

    A series of 3 benign and 10 malignant smooth muscle (SM) neoplasms and of 2 malignant fibrous histiocytomas was examined by light microscopy, transmission electron microscopy, two-dimensional gel electrophoresis (2D-GE) and indirect immunofluorescence, using polyclonal monospecific or monoclonal antibodies to desmin, vimentin, cytokeratin, alpha-SM and alpha-sarcomeric (alpha-SR) actins. Benign neoplasms displayed typical light-microscopic features of SM, whereas leiomyosarcomas demonstrated ...

  17. Temperature change does not affect force between single actin filaments and HMM from rabbit muscles.

    OpenAIRE

    Kawai, M.; Kawaguchi, K; M. Saito; Ishiwata, S

    2000-01-01

    The temperature dependence of sliding force, velocity, and unbinding force was studied on actin filaments when they were placed on heavy meromyosin (HMM) attached to a glass surface. A fluorescently labeled actin filament was attached to the gelsolin-coated surface of a 1-microm polystyrene bead. The bead was trapped by optical tweezers, and HMM-actin interaction was performed at 20-35 degrees C to examine whether force is altered by the temperature change. Our experiments demonstrate that sl...

  18. Role of active contraction and tropomodulins in regulating actin filament length and sarcomere structure in developing zebrafish skeletal muscle

    Directory of Open Access Journals (Sweden)

    Lise eMazelet

    2016-03-01

    Full Text Available Whilst it is recognised that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1ts25 which lacks functional voltage-gated calcium channels (dihydropyridine receptors in the muscle and pharmacological immobilisation of embryos with a reversible anaesthetic (Tricaine, allowed the study of paralysis (in mutants and anaesthetised fish and recovery of movement (reversal of anaesthetic treatment. The effect of paralysis in early embryos (aged between 17-24 hours post fertilisation, hpf on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localisation of the actin capping proteins Tropomodulin 1 &4 (Tmod in fish aged from 17hpf until 42hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post fertilisation (dpf. Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralysed fish by 42hpf. In conclusion, myofibril organisation is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localisation of Tmod1 to its sarcomeric

  19. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Dybboe, Rie; Hansen, Christina Neigaard;

    2015-01-01

    [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied...... of β-actin and GAPDH was lower in older men compared with young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting...... and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology....

  20. Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Michael J Herr

    Full Text Available The most prevalent cardiovascular diseases arise from alterations in vascular smooth muscle cell (VSMC morphology and function. Tetraspanin CD9 has been previously implicated in regulating vascular pathologies; however, insight into how CD9 may regulate adverse VSMC phenotypes has not been provided. We utilized a human model of aortic smooth muscle cells to understand the consequences of CD9 deficiency on VSMC phenotypes. Upon knocking down CD9, the cells developed an abnormally small and rounded morphology. We determined that this morphological change was due to a lack of typical parallel actin arrangement. We also found similar total RhoA but decreased GTP-bound (active RhoA levels in CD9 deficient cells. As a result, cells lacking a full complement of CD9 were less contractile than their control treated counterparts. Upon restoration of RhoA activity in the CD9 deficient cells, the phenotype was reversed and cell contraction was restored. Conversely, inhibition of RhoA activity in the control cells mimicked the CD9-deficient cell phenotype. Thus, alteration in CD9 expression was sufficient to profoundly disrupt cellular actin arrangement and endogenous cell contraction by interfering with RhoA signaling. This study provides insight into how CD9 may regulate previously described vascular smooth muscle cell pathophysiology.

  1. Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells.

    Science.gov (United States)

    Tunduguru, Ragadeepthi; Chiu, Tim T; Ramalingam, Latha; Elmendorf, Jeffrey S; Klip, Amira; Thurmond, Debbie C

    2014-11-15

    Skeletal muscle accounts for ∼ 80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1(-/-) knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell.

  2. An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

    Science.gov (United States)

    Whitesell, Thomas R; Kennedy, Regan M; Carter, Alyson D; Rollins, Evvi-Lynn; Georgijevic, Sonja; Santoro, Massimo M; Childs, Sarah J

    2014-01-01

    Mural cells of the vascular system include vascular smooth muscle cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma), which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

  3. An α-smooth muscle actin (acta2/αsma zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Thomas R Whitesell

    Full Text Available Mural cells of the vascular system include vascular smooth muscle cells (SMCs and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma, which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

  4. Interaction of formin FH2 with skeletal muscle actin. EPR and DSC studies.

    Science.gov (United States)

    Kupi, Tünde; Gróf, Pál; Nyitrai, Miklós; Belágyi, József

    2013-10-01

    Formins are highly conserved proteins that are essential in the formation and regulation of the actin cytoskeleton. The formin homology 2 (FH2) domain is responsible for actin binding and acts as an important nucleating factor in eukaryotic cells. In this work EPR and DSC were used to investigate the properties of the mDia1-FH2 formin fragment and its interaction with actin. MDia1-FH2 was labeled with a maleimide spin probe (MSL). EPR results suggested that the MSL was attached to a single SH group in the FH2. In DSC and temperature-dependent EPR experiments we observed that mDia1-FH2 has a flexible structure and observed a major temperature-induced conformational change at 41 °C. The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments. In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible. Considering the complex molecular mechanisms underlying the cellular roles of formins this internal flexibility of the dimers is probably important for manifestation of their biological functions.

  5. 103Pd radioactive stent inhibits biliary duct restenosis and reduces smooth muscle actin expression during duct healing in dogs

    Institute of Scientific and Technical Information of China (English)

    Gui-Jin He; Qin-Yi Gao; Shu-He Xu; Hong Gao; Tao Jiang; Xian-Wei Dai; Kai Ma

    2006-01-01

    BACKGROUND: This study was designed to assess the expression of smooth muscle actin (SMA) in the healing process after implanting a 103Pd radioactive stent in the biliary duct, and to discuss the function and signiifcance of this stent in preventing biliary stricture formation. METHODS:A model of biliary injury in dogs was made and then a 103Pd radioactive stent was positioned in the biliary duct. The expression and distribution of SMA were assessed in the anastomotic tissue 30 days after implantation of the stent. RESULTS:SMA expression was less in the 103Pd stent group than in the ordinary stent group. The 103Pd stent inhibited scar contracture and anastomotic stenosis. CONCLUSION:The 103Pd stent can reduce the expression of SMA in the healing process and inhibit scar contracture and anastomotic stenosis in the dog biliary duct.

  6. Suppression of α Smooth Muscle Actin Accumulation by Bovine Fetal Dermal Collagen Matrix in Full Thickness Skin Wounds.

    Science.gov (United States)

    Lineaweaver, William; Bush, Katie; James, Kenneth

    2015-06-01

    The suppression of elements associated with wound contracture and unfavorable scarring is a potentially important strategy in clinical wound management. In this study, the presence of α smooth muscle actin (αSMA), a protein involved in wound contraction, was analyzed in a series of wounds in which bovine fetal collagen (BFC) acellular dermal matrix (PriMatrix) was used in staged split thickness skin graft procedures. The results obtained through histological and quantitative image analyses of incidental biopsies from these wounds demonstrated a suppression of αSMA in the wound regions occupied by assimilated BFC relative to increased levels of αSMA found in other areas of the wound. The αSMA levels found in assimilated BFC were similar to αSMA levels in uninjured human dermis. These findings suggest a mechanism by which application of BFC could decrease contraction of full thickness skin wounds.

  7. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  8. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    International Nuclear Information System (INIS)

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β1 (TGF-β1)-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β1

  9. Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction

    NARCIS (Netherlands)

    Koopmans, Tim; Kumawat, Kuldeep; Halayko, Andrew J; Gosens, Reinoud

    2016-01-01

    A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an

  10. Regulation of actin dynamics by wnt-5a : Implications for human airway smooth muscle contraction

    NARCIS (Netherlands)

    Koopmans, Tim; Kumawat, Kuldeep; Menzen, Mark; Halayko, Andrew; Gosens, Reinoud

    2016-01-01

    An important pathophysiological feature of asthma is airway hyperresponsiveness (AHR), characterized by exaggerated bronchoconstriction in which the airway smooth muscle (ASM) is fundamentally involved. How the ASM in asthmatics differs from that in non-asthmatics is a current focus for research. We

  11. Histone demethylase retinoblastoma binding protein 2 regulates the expression of α-smooth muscle actin and vimentin in cirrhotic livers

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Q. [Department of Microbiology, Key Laboratory for Experimental Teratology of the Chinese Ministry of Education, School of Medicine, Shandong University, Jinan (China); Wang, L.X. [Department of Pharmacology, School of Medicine, Shandong University, Jinan (China); Zeng, J.P. [Department of Biochemistry, School of Medicine, Shandong University, Jinan (China); Liu, X.J.; Liang, X.M.; Zhou, Y.B. [Department of Microbiology, Key Laboratory for Experimental Teratology of the Chinese Ministry of Education, School of Medicine, Shandong University, Jinan (China)

    2013-09-06

    Liver cirrhosis is one of the most common diseases of Chinese patients. Herein, we report the high expression of a newly identified histone 3 lysine 4 demethylase, retinoblastoma binding protein 2 (RBP2), and its role in liver cirrhosis in humans. The siRNA knockdown of RBP2 expression in hepatic stellate cells (HSCs) reduced levels of α-smooth muscle actin (α-SMA) and vimentin and decreased the proliferation of HSCs; and overexpression of RBP2 increased α-SMA and vimentin levels. Treatment with transforming growth factor β (TGF-β) upregulated the expression of RBP2, α-SMA, and vimentin, and the siRNA knockdown of RBP2 expression attenuated TGF-β-mediated upregulation of α-SMA and vimentin expression and HSC proliferation. Furthermore, RBP2 was highly expressed in cirrhotic rat livers. Therefore, RBP2 may participate in the pathogenesis of liver cirrhosis by regulating the expression of α-SMA and vimentin. RBP2 may be a useful marker for the diagnosis and treatment of liver cirrhosis.

  12. Lubricin and smooth muscle α-actin-containing myofibroblasts in the pseudomembranes around loose hip and knee prostheses.

    Science.gov (United States)

    Cheriyan, Thomas; Ready, John E; Brick, Gregory W; Martin, Scott D; Martin, Tamara L; Schmid, Thomas M; Padera, Robert F; Spector, Myron

    2013-03-01

    The objective was to evaluate the presence and distribution of the lubricating and anti-adhesion glycoprotein lubricin and cells containing the contractile isoform smooth muscle α-actin (SMA) in pseudomembranes around loose hip prostheses. Periprosthetic tissue was obtained at revision arthroplasty of eight aseptic, loose hip implants, and for comparison three loose knee prostheses. Immunohistochemical analysis was performed in 3 zones: zone 1, within 300μm of the edge of the implant-tissue interface; zone 2, between zones 1 and 3; zone 3, within 300μm of the resected/trimmed edge. The presence of lubricin was extensive in all samples: (1) as a discrete layer at the implant-tissue interface; (2) within the extracellular matrix (ECM); (3) intracellularly. There was significantly more high grade (>50%) lubricin surface staining at the implant-tissue interface compared with the resected edge. While there was also a significant effect of location of high grade ECM lubricin staining, there was no significant effect of implant type (i.e. hip versus knee). All but two hip pseudomembrane samples showed the presence of many SMA-containing cells. There was a significant effect of location on the number of SMA-expressing cells, but not of implant type. These findings might explain why the management of loose prosthesis is so challenging.

  13. Conservation and divergence between cytoplasmic and muscle-specific actin capping proteins: insights from the crystal structure of cytoplasmic Cap32/34 from Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Eckert Christian

    2012-06-01

    Full Text Available Abstract Background Capping protein (CP, also known as CapZ in muscle cells and Cap32/34 in Dictyostelium discoideum, plays a major role in regulating actin filament dynamics. CP is a ubiquitously expressed heterodimer comprising an α- and β-subunit. It tightly binds to the fast growing end of actin filaments, thereby functioning as a “cap” by blocking the addition and loss of actin subunits. Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles. Results To elucidate structural and functional differences between cytoplasmic and sarcomercic CP variants, we have solved the atomic structure of Cap32/34 (32 = β- and 34 = α-subunit from the cellular slime mold Dictyostelium at 2.2 Å resolution and compared it to that of chicken muscle CapZ. The two homologs display a similar overall arrangement including the attached α-subunit C-terminus (α-tentacle and the flexible β-tentacle. Nevertheless, the structures exhibit marked differences suggesting considerable structural flexibility within the α-subunit. In the α-subunit we observed a bending motion of the β-sheet region located opposite to the position of the C-terminal β-tentacle towards the antiparallel helices that interconnect the heterodimer. Recently, a two domain twisting attributed mainly to the β-subunit has been reported. At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ. Conclusions The structure of Cap32/34 from Dictyostelium discoideum allowed a detailed analysis and comparison between the cytoplasmic and sarcomeric variants of CP. Significant structural flexibility could particularly be found within the

  14. The correlation of in vivo burn scar contraction with the level of α-smooth muscle actin expression.

    Science.gov (United States)

    Wang, Xue-Qing; Kravchuk, Olena; Winterford, Clay; Kimble, Roy M

    2011-12-01

    This study describes the direct association of in vivo burn scar contraction with the level of α-smooth muscle actin (α-SMA) in scar tissue, in a porcine burn model. The expression of α-SMA was investigated in 100 biopsies from 44 6-week old burn scars and in 85 biopsies from 16 2-week old burn wounds. Statistical analysis showed that the levels of α-SMA in 6-week old scars were significantly negatively correlated to scar size (r=-0.68) and the higher levels of α-SMA were observed in smaller scars. Moreover, α-SMA was also found to be significantly positively correlated to re-epithelialisation time (r=0.57) and scar thickness (r=0.58) and higher levels of α-SMA were detected in thicker scars with delayed wound closure. Further statistical analysis revealed that scar contraction can be explained best by the level of α-SMA expression and partially by scar thickness. Other variables, such as different dressings and individual pig, may also partly contribute to scar contraction. At week 2 after-burn, the level of α-SMA expression in 16 burn wounds was significantly related to the depth of burns and wound healing outcome. To our knowledge, this is the first study to provide in vivo evidence of the association of α-SMA expression with scar contraction, scar thickness, re-epithelialisation time and the depth of burn in a large animal burn model with scars similar to human hypertrophic scar.

  15. Heterozygous de novo and inherited mutations in the smooth muscle actin (ACTG2) gene underlie megacystis-microcolon-intestinal hypoperistalsis syndrome.

    Science.gov (United States)

    Wangler, Michael F; Gonzaga-Jauregui, Claudia; Gambin, Tomasz; Penney, Samantha; Moss, Timothy; Chopra, Atul; Probst, Frank J; Xia, Fan; Yang, Yaping; Werlin, Steven; Eglite, Ieva; Kornejeva, Liene; Bacino, Carlos A; Baldridge, Dustin; Neul, Jeff; Lehman, Efrat Lev; Larson, Austin; Beuten, Joke; Muzny, Donna M; Jhangiani, Shalini; Gibbs, Richard A; Lupski, James R; Beaudet, Arthur

    2014-03-01

    Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a rare disorder of enteric smooth muscle function affecting the intestine and bladder. Patients with this severe phenotype are dependent on total parenteral nutrition and urinary catheterization. The cause of this syndrome has remained a mystery since Berdon's initial description in 1976. No genes have been clearly linked to MMIHS. We used whole-exome sequencing for gene discovery followed by targeted Sanger sequencing in a cohort of patients with MMIHS and intestinal pseudo-obstruction. We identified heterozygous ACTG2 missense variants in 15 unrelated subjects, ten being apparent de novo mutations. Ten unique variants were detected, of which six affected CpG dinucleotides and resulted in missense mutations at arginine residues, perhaps related to biased usage of CpG containing codons within actin genes. We also found some of the same heterozygous mutations that we observed as apparent de novo mutations in MMIHS segregating in families with intestinal pseudo-obstruction, suggesting that ACTG2 is responsible for a spectrum of smooth muscle disease. ACTG2 encodes γ2 enteric actin and is the first gene to be clearly associated with MMIHS, suggesting an important role for contractile proteins in enteric smooth muscle disease.

  16. Heterozygous de novo and inherited mutations in the smooth muscle actin (ACTG2 gene underlie megacystis-microcolon-intestinal hypoperistalsis syndrome.

    Directory of Open Access Journals (Sweden)

    Michael F Wangler

    2014-03-01

    Full Text Available Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS is a rare disorder of enteric smooth muscle function affecting the intestine and bladder. Patients with this severe phenotype are dependent on total parenteral nutrition and urinary catheterization. The cause of this syndrome has remained a mystery since Berdon's initial description in 1976. No genes have been clearly linked to MMIHS. We used whole-exome sequencing for gene discovery followed by targeted Sanger sequencing in a cohort of patients with MMIHS and intestinal pseudo-obstruction. We identified heterozygous ACTG2 missense variants in 15 unrelated subjects, ten being apparent de novo mutations. Ten unique variants were detected, of which six affected CpG dinucleotides and resulted in missense mutations at arginine residues, perhaps related to biased usage of CpG containing codons within actin genes. We also found some of the same heterozygous mutations that we observed as apparent de novo mutations in MMIHS segregating in families with intestinal pseudo-obstruction, suggesting that ACTG2 is responsible for a spectrum of smooth muscle disease. ACTG2 encodes γ2 enteric actin and is the first gene to be clearly associated with MMIHS, suggesting an important role for contractile proteins in enteric smooth muscle disease.

  17. Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

    OpenAIRE

    Motawea, Hanaa K. B.; Selvi C. Jeyaraj; Eid, Ali H; Mitra, Srabani; Unger, Nicholas T.; Ahmed, Amany A. E.; Flavahan, Nicholas A.; Chotani, Maqsood A.

    2013-01-01

    The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expressio...

  18. Involvement of Rac1 and the actin cytoskeleton in insulin- and contraction-stimulated intracellular signaling and glucose uptake in mature skeletal muscle

    DEFF Research Database (Denmark)

    Sylow, Lykke

    Type 2 Diabetes affects ~10 % of western adults and is associated with poor organ sensitivity to insulin that is secreted following a meal. Insulin resistance, particularly in the liver, fat, and skeletal muscle, is a key event in the pathogenesis of Type 2 Diabetes and contributes to hyperinsuli......Type 2 Diabetes affects ~10 % of western adults and is associated with poor organ sensitivity to insulin that is secreted following a meal. Insulin resistance, particularly in the liver, fat, and skeletal muscle, is a key event in the pathogenesis of Type 2 Diabetes and contributes...... understood. The aim of the current PhD was therefore to investigate the involvement of Rac1 and the actin cytoskeleton in the regulation of insulin- and contraction-stimulated glucose uptake in mature skeletal muscle. The central findings of this PhD thesis was that Rac1 was activated by both insulin...... mice. This leads to the conclusion that Rac1 is a regulator of insulin- and contraction-stimulated glucose uptake in skeletal muscle. Furthermore, this PhD thesis identifies a novel mechanism by which skeletal muscle insulin resistance may develop....

  19. Actinic Cheilitis

    Science.gov (United States)

    ... actinic cheilitis. Overview Actinic cheilitis, sometimes known as "farmer's lip" or "sailor's lip," is a precancerous condition ... Last Updated: 22 Dec 2008 Information for other ages: Table of Contents: Overview Who's At Risk Signs ...

  20. Actinic keratosis

    Science.gov (United States)

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... laser treatment called photodynamic therapy Chemical peels Skin creams such as 5-fluorouracil (5-FU) and imiquimod

  1. Pentosan polysulfate decreases prostate smooth muscle proliferation and extracellular matrix turnover.

    Science.gov (United States)

    Elliot, S J; Zorn, B H; McLeod, D G; Moul, J W; Nyberg, L; Striker, L J; Striker, G E

    2003-01-01

    Benign prostatic hyperplasia (BPH) involves proliferation of smooth muscle cells and increased deposition of extracellular matrix (ECM). We recently found that pentosan polysulfate (PPS) has marked effects on growth and ECM of smooth muscle cells derived from vascular tissues. We examined smooth muscle cells cultured from human prostates and the effects of PPS on their growth and ECM production. Fragments of surgical prostatectomy specimens were diced, digested with collagenase (0.01%), and placed in culture medium supplemented with 20% fetal bovine serum. Outgrowths of elongated cells were characterized by light microscopic examination and immunohistochemical techniques by the presence of F-actin, alpha-smooth muscle actin, and myosin, which is a characteristic of smooth muscle cells. Two independent isolates were propagated, and growth curves and ECM production were assessed in the presence and absence of PPS (10 or 100 microg/ml). PPS decreased cell number beginning at day 1 and throughout the incubation period, up to 4 days. The amount of the ECM degradative enzymes, metallo-proteinases MMP-9 and MMP-2, was examined by zymography. PPS did not alter the amount of MMP-2 in the supernatants but MMP-9 was increased 234.4 +/- 17.23-fold over control cells. Tissue inhibitor of MMP (TIMPS), examined by reverse zymography, increased 200% over control. The amount of alpha I type (IV) and alpha I type (I) collagen released in the supernatant, measured by ELISA, significantly decreased in PPS-treated cultures. In conclusion, we found that the administration of PPS decreased proliferation as well as ECM production in prostate smooth muscle. Since smooth muscle proliferation and ECM are involved in the pathophysiology of BPH, PPS may have therapeutic potential.

  2. Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication,α smooth muscle actin expression and type I collagen synthesis in vitro

    Institute of Scientific and Technical Information of China (English)

    Yang Cheng; Jian Ping; Huai-Dong Xu; Hai-Jun Fu; Zhao-Hui Zhou

    2006-01-01

    AIM: To study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and α smooth muscle actin (α SMA) expression, type I,collagen synthesis in HSC-T6 cells.METHODS: The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to β actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by β actin.RESULTS: In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P = 0.043; HBeAg, P =0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P =0.041). In HSC-T6 cells the mRNA and protein expression levels of α SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P = 0.042; respectively); The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with thosein the oxymatrine group (mRNA, P < 0.01; protein, P <0.01; respectively).CONCLUSION: OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against α SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.

  3. Smooth-muscle-like cells derived from human embryonic stem cells support and augment cord-like structures in vitro.

    Science.gov (United States)

    Vo, Elaine; Hanjaya-Putra, Donny; Zha, Yuanting; Kusuma, Sravanti; Gerecht, Sharon

    2010-06-01

    Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers--including alpha-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain--to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.

  4. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

    DEFF Research Database (Denmark)

    Galliano, M F; Huet, C; Frygelius, J;

    2000-01-01

    differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in...

  5. Polymorphism in the alpha cardiac muscle actin 1 gene is associated to susceptibility to chronic inflammatory cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Amanda Farage Frade

    Full Text Available AIMS: Chagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America, and may lead to a life-threatening inflammatory dilated, chronic Chagas cardiomyopathy (CCC. One third of T. cruzi-infected individuals progress to CCC while the others remain asymptomatic (ASY. A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Since mutations in multiple sarcomeric genes, including alpha-cardiac actin (ACTC1 have been involved in hereditary dilated cardiomyopathy, we investigated the involvement of the ACTC1 gene in CCC pathogenesis. METHODS AND RESULTS: We conducted a proteomic and genetic study on a Brazilian study population. The genetic study was done on a main cohort including 118 seropositive asymptomatic subjects and 315 cases and the replication was done on 36 asymptomatic and 102 CCC cases. ACTC1 protein and mRNA levels were lower in myocardial tissue from patients with end-stage CCC than those found in hearts from organ donors. Genotyping a case-control cohort of CCC and ASY subjects for all informative single nucleotide polymorphism (SNP in the ACTC1 gene identified rs640249 SNP, located at the 5' region, as associated to CCC. Associations are borderline after correction for multiple testing. Correlation and haplotype analysis led to the identification of a susceptibility haplotype. Functional assays have shown that the rs640249A/C polymorphism affects the binding of transcriptional factors in the promoter regions of the ACTC1 gene. Confirmation of the detected association on a larger independent replication cohort will be useful. CONCLUSIONS: Genetic variations at the ACTC1 gene may contribute to progression to chronic Chagas Cardiomyopathy among T. cruzi-infected patients, possibly by modulating transcription factor binding to ACTC1 promoter regions.

  6. X-ray diffraction indicates that active cross-bridges bind to actin target zones in insect flight muscle.

    OpenAIRE

    Tregear, R T; Edwards, R J; Irving, T C; Poole, K J; Reedy, M C; Schmitz, H.; Towns-Andrews, E; Reedy, M K

    1998-01-01

    We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. ...

  7. Expression analysis of a-smooth muscle actin and tenascin-C in the periodontal ligament under orthodontic loading or in vitro culture

    Institute of Scientific and Technical Information of China (English)

    Hui Xu; Ding Bai; L-Bruno Ruest; Jian Q Feng; Yong-Wen Guo; Ye Tian; Yan Jing; Yao He; Xiang-Long Han

    2015-01-01

    a-smooth muscle actin (a-SMA) and tenascin-C are stress-induced phenotypic features of myofibroblasts. The expression levels of these two proteins closely correlate with the extracellular mechanical microenvironment. We investigated how the expression of a-SMA and tenascin-C was altered in the periodontal ligament (PDL) under orthodontic loading to indirectly reveal the intrinsic mechanical microenvironment in the PDL. In this study, we demonstrated the synergistic effects of transforming growth factor-b1 (TGF-b1) and mechanical tensile or compressive stress on myofibroblast differentiation from human periodontal ligament cells (hPDLCs). The hPDLCs under higher tensile or compressive stress significantly increased their levels of a-SMA and tenascin-C compared with those under lower tensile or compressive stress. A similar trend was observed in the tension and compression areas of the PDL under continuous light or heavy orthodontic load in rats. During the time-course analysis of expression, we observed that an increase in a-SMA levels was matched by an increase in tenascin-C levels in the PDL under orthodontic load in vivo. The time-dependent variation of a-SMA and tenascin-C expression in the PDL may indicate the time-dependent variation of intrinsic stress under constant extrinsic loading.

  8. Amelioration of glomerulosclerosis with all-trans retinoic acid is linked to decreased plasminogen activator inhibitor-1 and α-smooth muscle actin

    Institute of Scientific and Technical Information of China (English)

    Xia LIU; Lei L(U); Bei-bei TAO; Ai-ling ZHOU; Yi-chun ZHU

    2011-01-01

    Aim:To examine the effects of all-trans retinoic acid (atRA) on renal morphology and function as well as on renal plasminogen activator inhibitor-1 (PAI-1) expression and plasmin activity in rats with 5/6 nephrectomy.Methods:Adult male Sprague Dawley rats were given 5/6 nephrectomy or sham operation. Renal function was measured 2 weeks later. The nephrectomized rats were assigned to groups matched for proteinuria and treated with vehicle or atRA (5 or 10 mg/kg by gastric gavage once daily) for the next 12 weeks. Rats with sham operation were treated with vehicle. At the end of the treatments,kidneys were collected for histological examination, Western blot analysis, and enzymatic activity measurements.Results:The 5/6 nephrectomy promoted hypertension, renal dysfunction, and glomerulosclerosis. These changes were significantly reduced in the atRA-treated group. The expressions of PAI-1 and α-smooth muscle actin (α-SMA) were significantly increased in the vehicle-treated nephrectomized rats. Treatment with atRA significantly reduced the expressions of PAI-1 and α-SMA. However, piasmin activity remained unchanged following atRA treatment.Conclusion:Treatment with atRA ameliorates glomerulosclerosis and improves renal function in rats with 5/6 nephrectomy. This is associated with a decrease in PAI-1 and α-SMA, but not with a change in plasmin activity.

  9. Actinic reticuloid

    Energy Technology Data Exchange (ETDEWEB)

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  10. Comparing the expression of myoepithelial cell markers CD10 and smooth muscle actin with the estrogen receptor status in the invasive carcinoma breast: An immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Gaurav Arora

    2013-01-01

    Full Text Available Background: Breast cancer is the most frequent cancer in females throughout the world. Around 20% of breast carcinomas are estrogen receptor alpha (ER-negative. Thus, theoretically this negativity could be either the result of down-regulation of ER expression in the tumor cells, or the result of the tumor being derived from cells which normally lack that expression. Normal basal, including myoepithelial cells of the breast is ER-negative. CD10 and smooth muscle actin (SMA are used as markers for the demonstration of these basal cells. Aims: To compare the expression of positive staining for CD10 and SMA in ER-negative and ER-positive invasive breast carcinomas. Materials and Methods: The study was performed on 40 paraffin-embedded tissues of already diagnosed cases of invasive breast carcinomas with known ER status, i.e., thirty ER-negative and ten ER-positive cases. Expression of CD10 and SMA was demonstrated using avidin-biotin-peroxidase complex (ABC technique. Tumor was considered to be positive for both markers only when more than 10% of tumor cells were stained positive. Results: Overall, CD10 tumor cell staining was seen in eight, 23.3% (7/30 ER-negative cases and in 10% (1/10 ER-positive cases. Also the staining intensity was considered to be strong. SMA tumor cell staining was seen in only 6.7% (2/30 ER-negative cases and the staining intensity was considered to be moderate. Percentages of positively stained tumor cells varied between 13% to 72% and 23% to 45% for CD10 and SMA, respectively. Conclusion: CD10 is a better marker when compared to SMA, as it is expressed in more number of cases and gives strong positivity in tumor cells. Higher expression of CD10 and SMA is correlated with higher tumor grade and ER negativity.

  11. Time-resolved x-ray diffraction studies on the intensity changes of the 5.9 and 5.1 nm actin layer lines from frog skeletal muscle during an isometric tetanus using synchrotron radiation

    International Nuclear Information System (INIS)

    Time-resolved x-ray diffraction studies have been made on the 5.9- and 5.1-nm actin layer lines from frog skeletal muscles during an isometric tetanus at 6 degrees C, using synchrotron radiation. The integrated intensities of these actin layer lines were found to increase during a tetanus by 30-50% for the 5.9-nm reflection and approximately 70% for the 5.1-nm reflection of the resting values. The intensity increase of both reflections was greater than that taking place in the transition from rest to rigor state. The intensity change of the 5.9-nm reflection preceded those of the myosin 42.9-nm off-meridional reflection and of the equatorial reflections, as well as the isometric tension development. The intensity profile of the 5.9-nm layer line during contraction was found to be different from that observed in the rigor state

  12. Immuno-Expression of Endoglin and Smooth Muscle Actin in the Vessels of Brain Metastases. Is There a Rational for Anti-Angiogenic Therapy?

    Directory of Open Access Journals (Sweden)

    Valeria Barresi

    2014-04-01

    Full Text Available Despite ongoing clinical trials, the efficacy of anti-angiogenic drugs for the treatment of brain metastases (BM is still questionable. The lower response rate to anti-angiogenic therapy in the presence of BM than in metastatic disease involving other sites suggests that BM may be insensitive to these drugs, although the biological reasons underlining this phenomenon are still to be clarified. With the aim of assessing whether the targets of anti-angiogenic therapies are actually present in BM, in the present study, we analyzed the microvessel density (MVD, a measure of neo-angiogenesis, and the vascular phenotype (mature vs. immature in the tumor tissue of a series of BM derived from different primary tumors. By using immunohistochemistry against endoglin, a specific marker for newly formed vessels, we found that neo-angiogenesis widely varies in BM depending on the site of the primary tumor, as well as on its histotype. According to our results, BM from lung cancer displayed the highest MVD counts, while those from renal carcinoma had the lowest. Then, among BM from lung cancer, those from large cell and adenocarcinoma histotypes had significantly higher MVD counts than those originating from squamous cell carcinoma (p = 0.0043; p = 0.0063. Of note, MVD counts were inversely correlated with the maturation index of the endoglin-stained vessels, reflected by the coverage of smooth muscle actin (SMA positive pericytes (r = −0.693; p < 0.0001. Accordingly, all the endoglin-positive vessels in BM from pulmonary squamous cell carcinoma and renal carcinoma, displayed a mature phenotype, while vessels with an immature phenotype were found in highly vascularized BM from pulmonary large cell and adenocarcinoma. The low MVD and mature phenotype observed in BM from some primary tumors may account for their low sensitivity to anti-angiogenic therapies. Although our findings need to be validated in correlative studies with a clinical response, this should

  13. Actin gene family in Branchiostoma belched

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    Actin is a highly conserved cytoskeletal protein that is found in essentially all eukaryotic cells,which plays a paramount role in several basic functions of the organism, such as the maintenance of cellshape, cell division, cell mobility and muscle contraction. However, little is known about actin gene family inChinese amphioxus (Branchiostoma belcheri). Here we systemically analyzed the actin genes family inBranchiostoma belched and found that amphioxus contains 33 actin genes. These genes have undergoneextensive expansion through tandem duplications by phylogenetic analysis. In addition, we also providedevidence indicating that actin genes have divergent functions by specializing their EST data in both Bran-chiostoma belched and Branchiostoma florida. Our results provided an alternative explanation for the evolu-tion of actin genes, and gave new insights into their functional roles.

  14. 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.

  15. Sarcomeric Pattern Formation by Actin Cluster Coalescence

    Science.gov (United States)

    Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

    2012-01-01

    Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

  16. Sarcomeric pattern formation by actin cluster coalescence.

    Directory of Open Access Journals (Sweden)

    Benjamin M Friedrich

    Full Text Available Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells.

  17. 1周跑台训练对大鼠骨骼肌α-actin基因表达的影响%Effects of Treadmill Training for One Week on α-actin Gene Expression of Rat Skeletal Muscle

    Institute of Scientific and Technical Information of China (English)

    于新凯; 田野

    2001-01-01

    It was well known that the damage of skeletal muscle occurred and exercise performance declined after one prolonged eccentric exercise.But there were few studies on the adaptation to the continuous eccentric exercise in skeletal muscle.The purpose of this study was to investigate the effects of downhill running once(group B) and for one week(group C ) on α-actin gene expression of rat gastrocnemius muscle.Serum CK and LDH were determined, too.The results showed that the training had obvious effects on α-actin gene expression of rat gastrocnemius muscle in group B and C.In group B, serum CK and LDH recovered obviously 7 days after training, but not to the normal value.In group C, it recovered to the normal.These results suggest that the recovery of the damage after first training had been accelerated by the continuous eccentric exercise, the subjects gradually adapted to exercise.%离心运动可造成骨骼肌损伤,但缺乏对连续离心运动的适应性研究。本研究观察1次(B组)和1周(C组)下坡跑训练对大鼠腓肠肌肌动蛋白基因表达的影响及血清CK、LDH的变化。结果显示:1次和1周训练后大鼠腓肠肌肌动蛋白基因表达都出现明显升高,B组大鼠血清CK、LDH运动后7天仍显著高于对照组,C组已恢复正常。结果提示:连续离心运动可加速一次离心运动后肌肉损伤的恢复,机体对运动逐渐产生适应。

  18. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    OpenAIRE

    Bekyarova, T. I.; Reedy, M C; Baumann, B. A. J.; Tregear, R T; Ward, A; Krzic, U.; Prince, K.M.; Perz-Edwards, R. J.; Reconditi, M.; Gore, D.; Irving, T C; Reedy, M K

    2008-01-01

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the “steric blocking” mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca2+ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence...

  19. The Structural Basis of Actin Organization by Vinculin and Metavinculin.

    Science.gov (United States)

    Kim, Laura Y; Thompson, Peter M; Lee, Hyunna T; Pershad, Mihir; Campbell, Sharon L; Alushin, Gregory M

    2016-01-16

    Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5-Å-resolution cryo-electron microscopy reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal "strap" and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1' helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform that suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner, providing a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies. PMID:26493222

  20. Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    An actin gene (CfACT1) was cloned by using RT-PCR, 3' and 5'RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1535 bp, which contains a long 3' un-translated region of 436bp and 59bp of a 5' un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACT1 is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACT1 was cloned and sequenced. Only one intron was detected:it was located between codons 42 and 43 and different from vertebrate actin genes.

  1. 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.

  2. Effect of cardiac muscle collagen and actin on myocardiopathological change of diabetes mellitus%心肌胶原蛋白和肌动蛋白对糖尿病心肌病变的研究

    Institute of Scientific and Technical Information of China (English)

    吴伟; 张锦; 邱阳

    2005-01-01

    目的研究糖尿病大鼠不同病程心肌胶原蛋白和骨架蛋白含量的变化,阐明两者对糖尿病心肌病变发生的作用.方法制造糖尿病大鼠心肌模型随机分组.氯胺T法测定羟脯氨酸含量,代表心肌胶原总含量.心肌免疫组织化学染色测定心肌胶原蛋白(Collagen Ⅰ、Collagen Ⅲ)和心肌型α肌动蛋白(α-actin)及转化生长因子β1(TGF-β1)平均积分光密度(IOD).心肌病理改变的光镜和透射电镜观察.结果糖尿病痛程6个月组心肌胶原总含量明显高于病程3个月以内组(P<0.01).病程3个月之后Ⅰ型胶原蛋白表达伴随TGF-β1的表达开始较健康鼠明显增加(P<0.01).α-actin蛋白表达较健康鼠明显减少(P<O.01).糖尿病鼠心肌横切面可见粗大胶原纤维相互连接成网状,排列紊乱,分布不匀.心肌细胞心肌型α-actin蛋白分布不均匀,着色呈浅黄色,心肌纵切面可见α-actin蛋白表达主要分布于心肌肌膜处.病程3个月后大鼠心肌细胞核皱缩,线粒体肿胀、模糊,闰盘不连续,α-actin蛋白表达明显减少,有糖原沉积现象.结论Ⅰ型心肌胶原蛋白呈现持续性增加是糖尿病鼠心肌纤维化的主要原因.心肌细胞核皱缩,线粒体肿胀、模糊,闰盘不连续,糖原沉积和心肌型actin表达减少是糖尿病心肌病病理基础.%[Objective] To investigate the content changes of myocardial collagenprotein and actin at different stages of diabetes mellitus(DM) rats. So as to illustrate that they play a role in myocardial changes of DM. [Methods] Cardiac muscle model of DM rats was grouped randomly. The hydroxy proline contents were measured with the chloramine T method. The type Ⅰ collagen, type Ⅲ collagen,transforming factor beta1 (TGF-beta1) and cardiactype α-actin were determined by myocardial immunehistochemical stain. Myocardiopathological change were observed with light microscope and transmission electron microscope. [Results] The collagen contents

  3. An unconventional form of actin in protozoan hemoflagellate, Leishmania.

    Science.gov (United States)

    Kapoor, Prabodh; Sahasrabuddhe, Amogh A; Kumar, Ashutosh; Mitra, Kalyan; Siddiqi, Mohammad Imran; Gupta, Chhitar M

    2008-08-15

    Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs. PMID:18539603

  4. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Juliane Brun

    Full Text Available The use of mesenchymal stromal cells (MSCs differentiated toward a smooth muscle cell (SMC phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2, transgelin (TAGLN, calponin (CNN1, and smooth muscle myosin heavy chain (SM-MHC; MYH11 according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion

  5. Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.

    OpenAIRE

    Chaen, S; Oiwa, K; Shimmen, T; Iwamoto, H; Sugi, H

    1989-01-01

    To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass micro...

  6. Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus and other eukaryote species revealed by nucleotide and amino acid sequence analyses

    Directory of Open Access Journals (Sweden)

    Andréia B. Poletto

    2008-01-01

    Full Text Available Actin-encoding cDNAs of Nile tilapia (Oreochromis niloticus were isolated by RT-PCR using total RNA samples of different tissues and further characterized by nucleotide sequencing and in silico amino acid (aa sequence analysis. Comparisons among the actin gene sequences of O. niloticus and those of other species evidenced that the isolated genes present a high similarity to other fish and other vertebrate actin genes. The highest nucleotide resemblance was observed between O. niloticus and O. mossambicus a-actin and b-actin genes. Analysis of the predicted aa sequences revealed two distinct types of cytoplasmic actins, one cardiac muscle actin type and one skeletal muscle actin type that were expressed in different tissues of Nile tilapia. The evolutionary relationships between the Nile tilapia actin genes and diverse other organisms is discussed.

  7. Visualization of endothelial actin cytoskeleton in the mouse retina.

    Directory of Open Access Journals (Sweden)

    Alessia Fraccaroli

    Full Text Available Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs, orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs, enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.

  8. Control of actin-based motility through localized actin binding

    International Nuclear Information System (INIS)

    A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it, if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disc. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young’s modulus of the actin network and can explain several aspects of actin-based motility. (paper)

  9. Inhibitory effects of doxycycline on argyrophilic nucleolar organizing regions and a-smooth muscle actin expression in proliferative bovine corneal myofibroblasts in vitro%多西环素对体外培养的牛角膜肌成纤维细胞核仁组成区嗜银蛋白及平滑肌肌动蛋白表达的抑制作用

    Institute of Scientific and Technical Information of China (English)

    刘曼丽; 邹文进; 黄明汉; 赵静博; 付馨余; 王松

    2013-01-01

    合成,各浓度多西环素组细胞荧光染色的灰度值分别为189.90±7.48、140.20±7.79、113.20±8.98、98.00±3.50、85.50±4.99,差异有统计学意义(F=761.79,P=0.00).结论 在10 ~ 80 mg/L浓度范围内,多西环素可抑制体外培养的牛角膜肌成纤维细胞的AgNOR增生,呈剂量-时间-效应依赖性;在药物浓度达到60 mg/L时,与120 mg/L地塞米松拥有相当的作用效果.多西环素同时具有抑制细胞合成α-SMA的作用,与地塞米松比较,对抑制角膜瘢痕的形成更具研究价值.%Objective To investigate the effect of doxycycline on the nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms.Methods Cell culture and identification:bovine corneal fibroblasts were cultured after the stroma was incubated in 1.0 and 2.0 g/L type Ⅰ collagenase in two stages.Isolated cells were plated at mantaryay culture flask in 10% of BSA RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry.The cells staining positive for Vimentin and α-SMA indicated the presence of corneal myofibroblasts.Bovine corneal myofibroblasts were treated with different concentrations of doxycycline (10,20,40,60,80 mg/L),a bland control group and the dexamethasone group (120 mg/L) were set up,each group had 30 cases.The argyrophilic nucleolar organizing regions (AgNOR) staining and the immunohistochemistry for α-SMA were performed when the cells were treated for 24 hours and 48 hours.The AgNOR count (Ag-c),AgNOR area (Ag-a) and the expression of α-SMA in the bovine corneal myofibroblasts among each experiment group and control group were compared using one-way ANOVA,further pairwise comparisons using Independent-Samples t test.Results Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal myofibroblasts

  10. Cloning and Characterization of an Abalone (Haliotis discus hannai) Actin Gene

    Institute of Scientific and Technical Information of China (English)

    MA Hongming; XU Wei; MAI Kangsen; LIUFU Zhiguo; CHEN Hong

    2004-01-01

    An actin encoding gene was cloned by using RT-PCR, 3' RACE and 5' RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3' untranslated region of 307 base pairs and 79 base pairs of 5' untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin(β-actin)than to human muscle actin.

  11. Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

    Science.gov (United States)

    Kumatani, T; Sakurai-Ozato, N; Miyawaki, N; Yokota, E; Shimmen, T; Terashima, I; Takagi, S

    2006-11-01

    In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

  12. Myocardial repair with long-term and low-dose administration of a nitric oxide synthesis inhibitor. Myofibroblasts, type III collagen and fibronectin

    Directory of Open Access Journals (Sweden)

    Pessanha Mônica Gomes

    1999-01-01

    Full Text Available OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.

  13. Transgenic overexpression of γ-cytoplasmic actin protects against eccentric contraction-induced force loss in mdx mice

    Directory of Open Access Journals (Sweden)

    Baltgalvis Kristen A

    2011-10-01

    Full Text Available Abstract Background γ-cytoplasmic (γ-cyto actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The purpose of this study was to determine whether further elevation of γ-cyto actin levels improve or exacerbate the dystrophic phenotype of mdx mice. Methods We transgenically overexpressed γ-cyto actin, specifically in skeletal muscle of mdx mice (mdx-TG, and compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different ages. We investigated the mechanism by which γ-cyto actin provides protection from force loss by studying the role of calcium channels and stretch-activated channels in isolated skeletal muscles and muscle fibers. Analysis of variance or independent t-tests were used to detect statistical differences between groups. Results Levels of γ-cyto actin in mdx-TG skeletal muscle were elevated 200-fold compared to mdx skeletal muscle and incorporated into thin filaments. Overexpression of γ-cyto actin had little effect on most parameters of mdx muscle pathology. However, γ-cyto actin provided statistically significant protection against force loss during eccentric contractions. Store-operated calcium entry across the sarcolemma did not differ between mdx fibers compared to wild-type fibers. Additionally, the omission of extracellular calcium or the addition of streptomycin to block stretch-activated channels did not improve the force-generating capacity of isolated extensor digitorum longus muscles from mdx mice during eccentric contractions. Conclusions The data presented in this study indicate that upregulation of γ-cyto actin in dystrophic skeletal muscle can attenuate force loss during eccentric contractions and that the mechanism is independent of activation of stretch-activated channels and the accumulation of extracellular calcium.

  14. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

    Science.gov (United States)

    Deng, Su; Bothe, Ingo; Baylies, Mary K

    2015-08-01

    The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

  15. 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

  16. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

    Directory of Open Access Journals (Sweden)

    Su Deng

    2015-08-01

    Full Text Available The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia, which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

  17. Actin cytoskeleton regulates Hippo signaling.

    Directory of Open Access Journals (Sweden)

    Pradeep Reddy

    Full Text Available Hippo pathway controls the organ size by modulating cell proliferation and apoptosis. However, the upstream regulation of hippo signaling by actin cytoskeleton is not clear. To elucidate the role of actin as an upstream regulator of Hippo signaling, the levels of F (filamentous-actin in cells were elevated using jasplakinolide, an actin-stabilizing drug. Induction of F-actin formation in HeLa cells resulted in decreased phosphorylation of YAP, a key effector molecule for Hippo signaling. The activated YAP is localized to the cell nucleus and YAP increase was associated with increased expression of downstream CCN growth factors CCN1/CYR61 and CCN2/CTGF. The effect of the actin-stabilizing drug was blocked when YAP levels were suppressed in YAP "knock-down" cells. In summary, using an actin-stabilizing drug we show that actin cytoskeleton is one of the upstream regulators of Hippo signaling capable of activating YAP and increasing its downstream CCN growth factors.

  18. 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.

  19. Actin-myosin network is required for proper assembly of influenza virus particles

    International Nuclear Information System (INIS)

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network

  20. Actin-myosin network is required for proper assembly of influenza virus particles

    Energy Technology Data Exchange (ETDEWEB)

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  1. 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; Fath, Thomas; Schevzov, Galina; Gunning, Peter W

    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.

  2. 动力性肺动脉高压犬肺动脉PCNA及α-平滑肌肌动蛋白的表达%Expression of PCNA and α-smooth muscle actin in pulmonary arteries of dogs with dynamic pulmonary hypertension

    Institute of Scientific and Technical Information of China (English)

    崔勤; 杨景学; 赵建斌; 朱海龙

    2001-01-01

    目的探讨平滑肌细胞增殖在动力性肺动脉高压形成中的作用. 方法利用降主动脉—左下肺动脉分流建立的幼犬动力性肺动脉高压模型,取90 d肺组织病理切片,应用鼠抗人增殖细胞核抗原(PCNA)抗体、α-平滑肌细胞肌动蛋白抗体(α-actin)、及Dako二抗免疫组化检测,图像分析仪对所得结果进行分析,然后计数高倍镜下平滑肌细胞总数及PCNA细胞阳性数,计算细胞增殖指数. 结果分流犬左下肺动脉中膜α-actin免疫组化染色呈强阳性表达,右下肺动脉弱阳性,而对照组均呈阴性;分流犬左下肺动脉PCNA阳性信号表达明显多于右肺动脉,而右肺动脉及左上肺动脉仅有少量表达;增殖指数左下肺动脉内膜为33%,中膜11%,外膜7%,右下肺动脉仅为少量. 结论平滑肌细胞增殖参与实验犬动力性肺动脉高压形成过程并与肺动脉压力增高程度和肺血管重构改变一致.%AIM To explore the action of pulmonary artery smooth muscle cell proliferation in formation of dynamic pulmonary hypertension. METHODS Using the pulmonary hypertension(PH) model of shunt from descending aorta-to-left lower pulmonary artery, the sample of the lung tissue in shunt dogs was obtained on the 90th day postoperation. The examination of PCNA and a-actin was done by immunohistochemistry method using PCAN and a-actin antibodies. The slices of pulmonary tissue were observed under high power microscope, and the data were analyzed by image pattern analysor. The positive cells of PCNA and a-actin were recorded and the percentage was calculated. RESULTS Expression of a-actin in left lower pulmonary artery was strong positive and right pulmonary artery milder than that in left pulmonary, and control group was negative. The positive expression of PCNA in left lower pulmonary arteries was more than that of right lung and upper left lobe. The index of cell proliferation was 33% in intima, 11

  3. Progresses in studies of nuclear actin

    Institute of Scientific and Technical Information of China (English)

    ZHU Xiaojuan; ZENG Xianlu; SONG Zhaoxia; HAO Shui

    2004-01-01

    Actin is a protein abundant in cells. Recently, it has been proved to be universally existent in the nuclei of many cell types. Actin and actin-binding proteins, as well as actin-related proteins, are necessary for the mediation of the conformation and function of nuclear actin, including the transformation of actin between unpolymerized and polymerized, chroinatin remodeling, regulation of gene expression and RNA processing as well as RNA transportation. In this paper, we summarized the progresses in the research of nu clear actin.

  4. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  5. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    Energy Technology Data Exchange (ETDEWEB)

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K. (IIT); (EMBL); (Scripps); (Duke); (Prince); (FSU); (MRC); (U. Florence)

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  6. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development.

    Science.gov (United States)

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-08-15

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated.

  7. Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

    Science.gov (United States)

    Haralalka, Shruti; Shelton, Claude; Cartwright, Heather N; Guo, Fengli; Trimble, Rhonda; Kumar, Ram P; Abmayr, Susan M

    2014-01-01

    The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the

  8. Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

    Directory of Open Access Journals (Sweden)

    Shruti Haralalka

    Full Text Available The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs, and cell adhesion molecules Kin-of-IrreC (Kirre and Sticks-and-stones (Sns on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and

  9. Regulation of water flow by actin-binding protein-induced actin gelatin.

    OpenAIRE

    Ito, T.; Suzuki, A.; Stossel, T. P.

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by a...

  10. 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.

  11. 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.

  12. Effect of atorvastatin on expression of macrophage and smooth muscle actin in carotid atherosclerotic plaques in rabbits%阿托伐他汀对家兔颈动脉粥样硬化斑块内巨噬细胞浸润和平滑肌肌动蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    于熙滢; 曹海利; 刘雅君; 刘洋; 李晓光; 班翔; 魏林

    2011-01-01

    目的:研究阿托伐他汀对家兔颈动脉粥样硬化(AS)斑块内巨噬细胞及平滑肌肌动蛋白(SMA)表达的影响,并探讨他汀类药物稳定AS斑块的机制.方法:24只健康雄性新西兰大耳白兔随机分为对照组(n=8)和高胆固醇血症组(n=16).16只高胆固血症组的家兔喂饲高胆固醇饲料2周后,进行颈总动脉内膜球囊拉伤术,术后再随机等分为AS模型组和阿托伐他汀组[给予阿托伐他汀5 mg/(kg·d)],两组均继续喂饲高胆固醇饲料10周.喂养第12周时处死动物,取颈总动脉进行石蜡切片,用酶标法检测不同时间点血清脂质和脂蛋白;应用光学显微镜观察AS的进程;采用免疫组化染色法检测巨噬细胞浸润和SMA在斑块处的表达.结果:阿托伐他汀组的血清总胆固醇(TC)及低密度脂蛋白-胆固醇(LDL-C)的浓度明显低于AS模型组(P<0.01),颈总动脉内膜的厚度较AS模型组显著变薄[(0.49±0.072)vs.(0.66±0.08)mm,P<0.05].免疫组化染色法检测结果示,阿托伐他汀组血管壁中巨噬细胞的数量显著较模型组减少(P<0.05)而SMA的表达较AS模型组显著增多(P<0.01).结论:阿托伐他汀可能通过抑制AS斑块内巨噬细胞的浸润并增强SMA的表达,而发挥稳定斑块的作用.%AIM: To observe inflammatory macrophages and smooth muscle actin in carotid atherosclerotic plaques in rabbits and the effects of atorvastatin on them and to explore whether statins could stabilize carotid plaques and its possible mechanism.METHODS: Twenty-four rabbits were randomly divided into three groups: control group (n = 8 ) and hypercholesterolemia group (n = 16).The hypercholesterolemia group was fed a hypercholesterol diet for 2 weeks and then catheter-induced arterial wall injury was made.Rabbits in hypercholesterolemia and aortic injury group were randomized into model group (n = 8 ) and atorvastatin group 5 mg/( kg· day) for 10 weeks (n = 8 ).Carotid arteries were isolated and paraffinembedded

  13. A Short Splice Form of Xin-Actin Binding Repeat Containing 2 (XIRP2) Lacking the Xin Repeats Is Required for Maintenance of Stereocilia Morphology and Hearing Function

    OpenAIRE

    Francis, Shimon P.; Krey, Jocelyn F.; Krystofiak, Evan S.; Cui, Runjia; Nanda, Sonali; Xu, Wenhao; Kachar, Bechara; Barr-Gillespie, Peter G.; Shin, Jung-Bum

    2015-01-01

    Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-lin...

  14. Fascin regulates nuclear actin during Drosophila oogenesis.

    Science.gov (United States)

    Kelpsch, Daniel J; Groen, Christopher M; Fagan, Tiffany N; Sudhir, Sweta; Tootle, Tina L

    2016-10-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved.

  15. TIRF microscopy analysis of human Cof1, Cof2, and ADF effects on actin filament severing and turnover.

    Science.gov (United States)

    Chin, Samantha M; Jansen, Silvia; Goode, Bruce L

    2016-04-24

    Dynamic remodeling and turnover of cellular actin networks requires actin filament severing by actin-depolymerizing factor (ADF)/Cofilin proteins. Mammals express three different ADF/Cofilins (Cof1, Cof2, and ADF), and genetic studies suggest that in vivo they perform both overlapping and unique functions. To gain mechanistic insights into their different roles, we directly compared their G-actin and F-actin binding affinities, and quantified the actin filament severing activities of human Cof1, Cof2, and ADF using in vitro total internal reflection fluorescence microscopy. All three ADF/Cofilins had similar affinities for G-actin and F-actin. However, Cof2 and ADF severed filaments much more efficiently than Cof1 at both lower and higher concentrations and using either muscle or platelet actin. Furthermore, Cof2 and ADF were more effective than Cof1 in promoting "enhanced disassembly" when combined with actin disassembly co-factors Coronin-1B and actin-interacting protein 1 (AIP1), and these differences were observed on both preformed and actively growing filaments. To probe the mechanism underlying these differences, we used multi-wavelength total internal reflection fluorescence microscopy to directly observe Cy3-Cof1 and Cy3-Cof2 interacting with actin filaments in real time during severing. Cof1 and Cof2 each bound to filaments with similar kinetics, yet Cof2 induced severing much more rapidly than Cof1, decreasing the time interval between initial binding on a filament and severing at the same location. These differences in ADF/Cofilin activities and mechanisms may be used in cells to tune filament turnover rates, which can vary widely for different actin structures.

  16. Eye features in three Danish patients with multisystemic smooth muscle dysfunction syndrome

    DEFF Research Database (Denmark)

    Moller, Hans Ulrik; Fledelius, Hans C; Milewicz, Dianna M;

    2012-01-01

    A de novo mutation of the ACTA2 gene encoding the smooth muscle cell α-actin has been established in patients with multisystemic smooth muscle dysfunction syndrome associated with patent ductus arteriosus and mydriasis present at birth.......A de novo mutation of the ACTA2 gene encoding the smooth muscle cell α-actin has been established in patients with multisystemic smooth muscle dysfunction syndrome associated with patent ductus arteriosus and mydriasis present at birth....

  17. The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

    Directory of Open Access Journals (Sweden)

    Marx Stephen J

    2008-08-01

    Full Text Available Abstract Background Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait. Results Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3, but not with non-muscle alpha-actinins (actinin-1 and actinin-4. The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells, but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment. Conclusion These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin participate in sequestering parafibromin in the cytoplasmic compartment.

  18. Expression of actin genes in the arrow worm Paraspadella gotoi (Chaetognatha).

    Science.gov (United States)

    Yasuda, E; Goto, T; Makabe, K W; Satoh, N

    1997-12-01

    Arrow worms (the phylum Chaetognatha), one of the major marine planktonic animals, exhibit features characteristic to both deuterostomes and protostomes, and their ancestry therefore remains unknown. As the first step to elucidate the molecular bases of arrow worm phylogeny, physiology and embryology, we isolated cDNA clones for three different actin genes (PgAct1, PgAct2 and PgAct3) from the benthic species Paraspadella gotoi, and examined their expression patterns in adults and juveniles. The amino acid sequences of the three actins resembled each other, with identities ranging from 86% to 92%. However, the patterns of the spatial expression of the genes were independent. The PgAct1 gene might encode a cytoplasmic actin and was expressed in oogenic cells, spermatogenic cells, and cells in the ventral ganglion. The PgAct2 and PgAct3 genes encoded actins of divergent types. The former was expressed in well-developed muscle of the head (gnathic) region and trunk muscle cells, whereas the latter was expressed in muscle of the trunk and tail regions and oogenic cells. These results suggest that, similarly to other metazoans, the chaetognath contains multiple forms of actins, which are expressed in various manners in the adult and juvenile arrow worm. PMID:9520638

  19. Actin phosphorylation correlates with actin sequestration in ATP-depleted abbit renal proximal tubules

    Institute of Scientific and Technical Information of China (English)

    Yingchun Li; Yingbin Ge; Jun Du; Rong Zhou; Jin Chen; Luo Gu

    2005-01-01

    Objective: To demonstrate the relationship between actin phosphorylation and actin sequestration in ATP-depleted rabbit renal proximal tubules. Methods: Using two-dimensional electrophoreses and Western blotting to analyze the phosphorylation state of the sequestered actin in rabbit renal proximal tubules. Results: The analytical result of the sequestered actin indicated that nearly half of the actin was phosphorylated on serine residue(s). Conclusion: Result suggested a close correlation between actin sequestration and actin phosphorylation in ATP-depleted rabbit renal proximal tubules.

  20. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.

    Science.gov (United States)

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2015-03-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

  1. A Beetle Flight Muscle Displays Leg Muscle Microstructure.

    Science.gov (United States)

    Shimomura, Toshiki; Iwamoto, Hiroyuki; Vo Doan, Tat Thang; Ishiwata, Shin'ichi; Sato, Hirotaka; Suzuki, Madoka

    2016-09-20

    In contrast to major flight muscles in the Mecynorrhina torquata beetle, the third axillary (3Ax) muscle is a minor flight muscle that uniquely displays a powerful mechanical function despite its considerably small volume, ∼1/50 that of a major flight muscle. The 3Ax muscle contracts relatively slowly, and in flight strongly pulls the beating wing to attenuate the stroke amplitude. This attenuation leads to left-right turning in flight or wing folding to cease flying. What enables this small muscle to be so powerful? To explore this question, we examined the microstructure of the 3Ax muscle using synchrotron x-ray diffraction, optical microscopy, and immunoblotting analysis. We found that the 3Ax muscle has long (∼5 μm) myofilaments and that the ratio of thick (myosin) filaments to thin (actin) filaments is 1:5 or 1:6. These characteristics are not observed in the major flight muscles, which have shorter myofilaments (∼3.5 μm) with a smaller ratio (1:3), and instead are more typical of a leg muscle. Furthermore, the flight-muscle-specific troponin isoform, TnH, is not expressed in the 3Ax muscle. Since such a microstructure is suitable for generating large tension, the 3Ax muscle is appropriately designed to pull the wing strongly despite its small volume.

  2. A Beetle Flight Muscle Displays Leg Muscle Microstructure.

    Science.gov (United States)

    Shimomura, Toshiki; Iwamoto, Hiroyuki; Vo Doan, Tat Thang; Ishiwata, Shin'ichi; Sato, Hirotaka; Suzuki, Madoka

    2016-09-20

    In contrast to major flight muscles in the Mecynorrhina torquata beetle, the third axillary (3Ax) muscle is a minor flight muscle that uniquely displays a powerful mechanical function despite its considerably small volume, ∼1/50 that of a major flight muscle. The 3Ax muscle contracts relatively slowly, and in flight strongly pulls the beating wing to attenuate the stroke amplitude. This attenuation leads to left-right turning in flight or wing folding to cease flying. What enables this small muscle to be so powerful? To explore this question, we examined the microstructure of the 3Ax muscle using synchrotron x-ray diffraction, optical microscopy, and immunoblotting analysis. We found that the 3Ax muscle has long (∼5 μm) myofilaments and that the ratio of thick (myosin) filaments to thin (actin) filaments is 1:5 or 1:6. These characteristics are not observed in the major flight muscles, which have shorter myofilaments (∼3.5 μm) with a smaller ratio (1:3), and instead are more typical of a leg muscle. Furthermore, the flight-muscle-specific troponin isoform, TnH, is not expressed in the 3Ax muscle. Since such a microstructure is suitable for generating large tension, the 3Ax muscle is appropriately designed to pull the wing strongly despite its small volume. PMID:27653488

  3. Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

    OpenAIRE

    Hakim, Chady H.; Wasala, Nalinda B.; Duan, Dongsheng

    2013-01-01

    Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z disc...

  4. Reactive oxygen species (ROS)-induced actin glutathionylation controls actin dynamics in neutrophils

    Science.gov (United States)

    Sakai, Jiro; Li, Jingyu; Subramanian, Kulandayan K.; Mondal, Subhanjan; Bajrami, Besnik; Hattori, Hidenori; Jia, Yonghui; Dickinson, Bryan C.; Zhong, Jia; Ye, Keqiang; Chang, Christopher J; Ho, Ye-Shih; Zhou, Jun; Luo, Hongbo R.

    2012-01-01

    Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils. PMID:23159440

  5. Actinic Keratoses: A Comprehensive Update

    OpenAIRE

    Ibrahim, Sherrif F.; Brown, Marc D.

    2009-01-01

    Actinic keratoses are common intra-epidermal neoplasms that lie on a continuum with squamous cell carcinoma. Tightly linked to ultraviolet irradiation, they occur in areas of chronic sun exposure, and early treatment of these lesions may prevent their progression to invasive disease. A large variety of effective treatment modalities exist, and the optimal therapeutic choice is dependent on a variety of patient- and physician-associated variables. Many established and more recent approaches ar...

  6. Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila

    OpenAIRE

    1996-01-01

    Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and pre...

  7. Mesoscopic model of actin-based propulsion.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available Two theoretical models dominate current understanding of actin-based propulsion: microscopic polymerization ratchet model predicts that growing and writhing actin filaments generate forces and movements, while macroscopic elastic propulsion model suggests that deformation and stress of growing actin gel are responsible for the propulsion. We examine both experimentally and computationally the 2D movement of ellipsoidal beads propelled by actin tails and show that neither of the two models can explain the observed bistability of the orientation of the beads. To explain the data, we develop a 2D hybrid mesoscopic model by reconciling these two models such that individual actin filaments undergoing nucleation, elongation, attachment, detachment and capping are embedded into the boundary of a node-spring viscoelastic network representing the macroscopic actin gel. Stochastic simulations of this 'in silico' actin network show that the combined effects of the macroscopic elastic deformation and microscopic ratchets can explain the observed bistable orientation of the actin-propelled ellipsoidal beads. To test the theory further, we analyze observed distribution of the curvatures of the trajectories and show that the hybrid model's predictions fit the data. Finally, we demonstrate that the model can explain both concave-up and concave-down force-velocity relations for growing actin networks depending on the characteristic time scale and network recoil. To summarize, we propose that both microscopic polymerization ratchets and macroscopic stresses of the deformable actin network are responsible for the force and movement generation.

  8. From pollen actin to crop male sterility

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Actin plays an important role in the life activity of animal and plant cells. Pollen cells have plenty of actin whose structure and characteristics are very similar to the animal actin. The nucleotide sequence and amino acid sequence of plant actin gene are very similar to those of the animal gene. The content of pollen actin from male sterile plants is much more lower than that from its maintainer plants. The expression of actin gene is organ-specific during the plant development. The expression quantity of actin gene in pollen is much more higher than those from root, stem and leaf. The expression plasmid of the anti-sense actin gene was constructed, transferred to the protoplasts of wheat and tomato to inhibit the expression of actin gene in pollen and thus the male sterile plants of wheat and tomato were obtained. The actin in pollens from the transgenic plants was reduced significantly, whereas the pistil was not affected. This study might pave a new way to breeding male sterile lines for the application of hybrid vigor of wheat and tomato.

  9. 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.

  10. Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening.

    Science.gov (United States)

    Gentry, Brian S; van der Meulen, Stef; Noguera, Philippe; Alonso-Latorre, Baldomero; Plastino, Julie; Koenderink, Gijsje H

    2012-11-01

    Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

  11. Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

    Institute of Scientific and Technical Information of China (English)

    Naizhi Chen; Xiaolu Qu; Youjun Wu; Shanjin Huang

    2009-01-01

    Actin cytoskeleton undergoes rapid reorganization In response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions inplant cell biology. The pollen tube is a well characterized actin-based call morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and un-expected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.

  12. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    Science.gov (United States)

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  13. Packaging of actin into Ebola virus VLPs

    Directory of Open Access Journals (Sweden)

    Harty Ronald N

    2005-12-01

    Full Text Available Abstract The actin cytoskeleton has been implicated in playing an important role assembly and budding of several RNA virus families including retroviruses and paramyxoviruses. In this report, we sought to determine whether actin is incorporated into Ebola VLPs, and thus may play a role in assembly and/or budding of Ebola virus. Our results indicated that actin and Ebola virus VP40 strongly co-localized in transfected cells as determined by confocal microscopy. In addition, actin was packaged into budding VP40 VLPs as determined by a functional budding assay and protease protection assay. Co-expression of a membrane-anchored form of Ebola virus GP enhanced the release of both VP40 and actin in VLPs. Lastly, disruption of the actin cytoskeleton with latrunculin-A suggests that actin may play a functional role in budding of VP40/GP VLPs. These data suggest that VP40 may interact with cellular actin, and that actin may play a role in assembly and/or budding of Ebola VLPs.

  14. 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...

  15. 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

  16. 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...

  17. A method for rapidly screening functionality of actin mutants and tagged actins

    Directory of Open Access Journals (Sweden)

    Rommelaere Heidi

    2004-01-01

    Full Text Available Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three &bgr;-actin mutants that have been associated with diseases.

  18. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  19. Load fluctuations drive actin network growth

    CERN Document Server

    Shaevitz, Joshua W

    2007-01-01

    The growth of actin filament networks is a fundamental biological process that drives a variety of cellular and intracellular motions. During motility, eukaryotic cells and intracellular pathogens are propelled by actin networks organized by nucleation-promoting factors, which trigger the formation of nascent filaments off the side of existing filaments in the network. A Brownian ratchet (BR) mechanism has been proposed to couple actin polymerization to cellular movements, whereby thermal motions are rectified by the addition of actin monomers at the end of growing filaments. Here, by following actin--propelled microspheres using three--dimensional laser tracking, we find that beads adhered to the growing network move via an object--fluctuating BR. Velocity varies with the amplitude of thermal fluctuation and inversely with viscosity as predicted for a BR. In addition, motion is saltatory with a broad distribution of step sizes that is correlated in time. These data point to a model in which thermal fluctuati...

  20. Crystal structure of an archaeal actin homolog.

    Science.gov (United States)

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  1. The spatial response of nonlinear strain propagation in response to actively driven microspheres through entangled actin networks

    Science.gov (United States)

    Falzone, Tobias; Blair, Savanna; Robertson-Anderson, Rae

    2015-03-01

    The semiflexible biopolymer actin, a ubiquitous component of nearly all biological organisms, plays an important role in many mechanically-driven processes such as muscle contraction, cancer invasion and cell motility. As such, entangled actin networks, which possess unique and complex viscoelastic properties, have been the subject of much theoretical and experimental work. However, due to this viscoelastic complexity, much is still unknown regarding the correlation of the applied stress on actin networks to the induced filament strain at the molecular and micro scale. Here, we use simultaneous optical trapping and fluorescence microscopy to characterize the link between applied microscopic forces and strain propagation as a function of strain rate and concentration. Specifically, we track fiduciary markers on entangled actin filaments before, during and after actively driving embedded microspheres through the network. These measurements provide much needed insight into the molecular-level dynamics connecting stress and strain in semiflexible polymer networks.

  2. Muscle biopsy

    Science.gov (United States)

    ... the removal of a small piece of muscle tissue for examination. ... dystrophy Myopathic changes (destruction of the muscle) Necrosis (tissue death) of muscle Necrotizing vasculitis Traumatic muscle damage Polymyositis Additional conditions ...

  3. Muscle Disorders

    Science.gov (United States)

    Your muscles help you move and help your body work. Different types of muscles have different jobs. There are many problems that can affect muscles. Muscle disorders can cause weakness, pain or even ...

  4. Muscle atrophy

    Science.gov (United States)

    Muscle wasting; Wasting; Atrophy of the muscles ... There are two types of muscle atrophy: disuse and neurogenic. Disuse atrophy is caused by not using the muscles enough . This type of atrophy can often be ...

  5. Muscle Cramps

    Science.gov (United States)

    Muscle cramps are sudden, involuntary contractions or spasms in one or more of your muscles. They often occur after exercise or at night, ... to several minutes. It is a very common muscle problem. Muscle cramps can be caused by nerves ...

  6. Prokaryotic expression and characterization of a pea actin isoform (PEAcl) fused to GFP

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shaobin; REN Dongtao; XU Xiaojing; LIU Guoqin

    2004-01-01

    Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells, it is difficult to purify each actin isoform in sufficient quantities for analyzing its physicochemical properties. In the present study, a pea(Pisum Sativum L.) actin isoform (PEAc1) fused to His-tag at its amino terminus and GFP (green fluorescent protein) at its Carboxyl terminus were expressed in E. Coli in inclusion bodies. The fusion protein (PEAc1-GFP) was highly purified with the yield of above 2 mg/L culture by dissolving inclusions in 8 mol/L urea, renaturing by dialysis in a gradient of urea, and affinity binding to Ni-resin. The purified mono meric PEAc1-GFP could efficiently bind on Dnase Ⅰ and inhibit the latter's enzyme activity. PEAc1-GFP could polymerize into green fluorescent filamentous structures (F-PEAc1-GFP), which could be labeled by TRITC-phalloidin, a specific agent for observing microfilaments. The PEAc1-GFP polymerization curve was identical with that of chicken skeletal muscle actin. The critical concentration for PEAc1-GFP to polymerize into filaments is 0.24 μmol/L. The F-PEAc1-GFP could stimulate myosin Mg-ATPase activity in a protein concentration dependant manner (about 4 folds at1 mg/mL F-PEAc1-GFP). The results above show that the PEAcl fused to GFP retained the assembly characteristic of actin, indicating that gene fusion, prokaryotic expression,denaturation and renaturation, and affinity chromatography is a useful strategy for obtaining plant actin isoform proteins in a large amount.

  7. Erbium laser resurfacing for actinic cheilitis.

    Science.gov (United States)

    Cohen, Joel L

    2013-11-01

    Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interventions (eg, cryotherapy, electrosurgery, surgical vermillionectomy, laser resurfacing), each with their known advantages and disadvantages. There is little consensus as to which treatment options offer the most clinical utility given the paucity of comparative clinical data. In my practice, laser resurfacing has become an important tool for the treatment of actinic cheilitis owing to its ease of use and overall safety, tolerability, and cosmetic acceptability. Herein the use of erbium laser resurfacing is described for three actinic cheilitis presentations for which I find it particularly useful: clinically prominent actinic cheilitis, biopsy-proven actinic cheilitis, and treatment of the entire lip following complete tumor excision of squamous cell carcinoma. All patients were treated with a 2940-nm erbium laser (Sciton Profile Contour Tunable Resurfacing Laser [TRL], Sciton, Inc., Palo Alto, CA). PMID:24196339

  8. Dynamin2 organizes lamellipodial actin networks to orchestrate lamellar actomyosin.

    Directory of Open Access Journals (Sweden)

    Manisha Menon

    Full Text Available Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2's action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.

  9. 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.

  10. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    International Nuclear Information System (INIS)

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro

  11. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  12. A Comparison of Total and Intrinsic Muscle Stiffness Among Flexors and Extensors of the Ankle, Knee and Elbow

    Science.gov (United States)

    Lemoine, Sandra M.

    1997-01-01

    This study examined 3 methods that assessed muscle stiffness. Muscle stiffness has been quantified by tissue reactive force (transverse stiffness), vibration, and force (or torque) over displacement. Muscle stiffness also has two components: reflex (due to muscle sensor activity) and intrinsic (tonic firing of motor units, elastic nature of actin and myosin cross bridges, and connective tissue). This study compared three methods of measuring muscle stiffness of agonist-antagonist muscle pairs of the ankle, knee and elbow.

  13. Reactive oxygen species (ROS)-induced actin glutathionylation controls actin dynamics in neutrophils

    OpenAIRE

    Sakai, Jiro; Li, Jingyu; Subramanian, Kulandayan K.; Mondal, Subhanjan; Bajrami, Besnik; Hattori, Hidenori; Jia, Yonghui; Dickinson, Bryan C; Zhong, Jia; Ye, Keqiang; Chang, Christopher J.; Ho, Ye-Shih; Zhou, Jun; Luo, Hongbo R.

    2012-01-01

    The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin poly...

  14. Actinic review of EUV masks

    Science.gov (United States)

    Feldmann, Heiko; Ruoff, Johannes; Harnisch, Wolfgang; Kaiser, Winfried

    2010-04-01

    Management of mask defects is a major challenge for the introduction of EUV for HVM production. Once a defect has been detected, its printing impact needs to be predicted. Potentially the defect requires some repair, the success of which needs to be proven. This defect review has to be done with an actinic inspection system that matches the imaging conditions of an EUV scanner. During recent years, several concepts for such an aerial image metrology system (AIMS™) have been proposed. However, until now no commercial solution exists for EUV. Today, advances in EUV optics technology allow envisioning a solution that has been discarded before as unrealistic. We present this concept and its technical cornerstones.While the power requirement for the EUV source is less demanding than for HVM lithography tools, radiance, floor space, and stability are the main criteria for source selection. The requirement to emulate several generations of EUV scanners demands a large flexibility for the ilumination and imaging systems. New critical specifications to the EUV mirrors in the projection microscope can be satisfied using our expertise from lithographic mirrors. In summary, an EUV AIMS™ meeting production requirements seems to be feasible.

  15. Actin: its cumbersome pilgrimage through cellular compartments.

    Science.gov (United States)

    Schleicher, Michael; Jockusch, Brigitte M

    2008-06-01

    In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days' knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.

  16. Why muscle is an efficient shock absorber.

    Directory of Open Access Journals (Sweden)

    Michael A Ferenczi

    Full Text Available Skeletal muscles power body movement by converting free energy of ATP hydrolysis into mechanical work. During the landing phase of running or jumping some activated skeletal muscles are subjected to stretch. Upon stretch they absorb body energy quickly and effectively thus protecting joints and bones from impact damage. This is achieved because during lengthening, skeletal muscle bears higher force and has higher instantaneous stiffness than during isometric contraction, and yet consumes very little ATP. We wish to understand how the actomyosin molecules change their structure and interaction to implement these physiologically useful mechanical and thermodynamical properties. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch compared to those during isometric contraction at physiological temperature using synchrotron radiation. The intensities of the off-meridional layer lines and fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that upon stretch the fraction of actin-bound myosin heads is higher than during isometric contraction. On the other hand, the intensities of the actin layer lines are lower than during isometric contraction. Taken together, these results suggest that during stretch, a significant fraction of actin-bound heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. As the strong or stereo-specific myosin binding to actin is necessary for actin activation of the myosin ATPase, this finding explains the low metabolic cost of energy absorption by muscle during the landing phase of locomotion.

  17. Role of cyclic nucleotide-dependent actin cytoskeletal dynamics:Ca(2+](i and force suppression in forskolin-pretreated porcine coronary arteries.

    Directory of Open Access Journals (Sweden)

    Kyle M Hocking

    Full Text Available Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+]i and phosphorylation of myosin light chains (MLC. However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

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

    OpenAIRE

    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 ...

  19. 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.

  20. Skeletal muscle

    Science.gov (United States)

    There are approximately 650-850 muscles in the human body these include skeletal (striated), smooth and cardiac muscle. The approximation is based on what some anatomists consider separate muscle or muscle systems. Muscles are classified based on their anatomy (striated vs. smooth) and if they are v...

  1. The length–tension curve in muscle depends on lattice spacing

    OpenAIRE

    Williams, C. David; Salcedo, Mary K.; Irving, Thomas C.; Regnier, Michael; Daniel, Thomas L.

    2013-01-01

    Classic interpretations of the striated muscle length–tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing ...

  2. Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Chalmel Frédéric

    2007-08-01

    Full Text Available Abstract Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our

  3. 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 agen

  4. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

    Science.gov (United States)

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

  5. Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

    Science.gov (United States)

    Mavrakis, Manos; Azou-Gros, Yannick; Tsai, Feng-Ching; Alvarado, José; Bertin, Aurélie; Iv, Francois; Kress, Alla; Brasselet, Sophie; Koenderink, Gijsje H; Lecuit, Thomas

    2014-04-01

    Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

  6. Muscle disorder

    Science.gov (United States)

    Myopathic changes; Myopathy; Muscle problem ... Blood tests sometimes show abnormally high muscle enzymes. If a muscle disorder might also affect other family members, genetic testing may be done. When someone has symptoms and signs ...

  7. 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 GFP-m

  8. Structural Basis of Actin Filament Nucleation by Tandem W Domains

    Directory of Open Access Journals (Sweden)

    Xiaorui Chen

    2013-06-01

    Full Text Available Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl, a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.

  9. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Hidemi; Matsuura, Yoshiyuki, E-mail: matsuura.yoshiyuki@d.mbox.nagoya-u.ac.jp

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

  10. 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.

  11. Actin-dependent mechanisms in AMPA receptor trafficking

    Directory of Open Access Journals (Sweden)

    Jonathan G Hanley

    2014-11-01

    Full Text Available The precise regulation of AMPA receptor (AMPAR number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits during learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signalling pathways that modulate actin polymerization and depolymerisation. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine.

  12. The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms.

    Directory of Open Access Journals (Sweden)

    Jayabalan M Joseph

    Full Text Available Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps. To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group. According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8 as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.

  13. Insulin induces PKC-dependent proliferation of mesenteric vascular smooth muscle cells from hypertensive patients

    Institute of Scientific and Technical Information of China (English)

    Xukai WANG; Yan WANG; Chenming YANG; Ying WAN; Xianwen JI

    2006-01-01

    Background and objectives Proliferation of human vascular smooth muscle cells (VSMCs) induced by hyperinsulinemia is a very common clinical pathology. Extensive research has focused on PKC (Protein kinase C)-MAPK (mitogen-activated protein kinase)intracellular signal transduction and the phenotypic modulation accompanied by reorganization of intracellular F-actins in VSMCs.Methods DNA synthesis, signaling of ERK1/2 MAPKs, and changes in α-smooth muscle (SM) actin and F-actin were studied in hypertensive and normotensive human arterial VSMCs exposed to insulin and PMA with and without the PKC inhibitor, GF109203X.Results Differences among cell types in MAPK signaling, α-SM actin, and F-actin isoforms in VSMCs harvested from the arteries of patients with essential hypertension (EH) and normotension (NT) were identified in response to insulin treatment. Proliferation and activation of MAPK were more pronounced in EH VSMCs than in NEH VSMCs. Insulin exposure decreased expression of α-SM actin and was accompanied by rearrangement of intracellular F-actins in VSMCs, especially in the EH group. These effects were reversed by treatment with the PKC inhibitor. Conclusions Human mesenteric VSMCs of EH and NT patients differed in proliferation, MAPK signaling, and degree of changes in α-SM actin and F-actin isoforms immediately following insulin exposure in vitro.

  14. The interaction between actin and FA fragment of diphtheria toxin

    OpenAIRE

    Ünlü, A.; Bektaş, M.; Şener, S.; Nurten, R.

    2012-01-01

    Actin protein has many other cellular functions such as movement, chemotaxis, secretion and cytodiaresis. Besides, it have structural function. Actin is a motor protein that it has an important role in the movement process of toxin in the cell. It is known that F-actin gives carriage support during the endosomal process. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerisation of monomeric G-actin molecules through seque...

  15. Spontaneous actin dynamics in contractile rings

    Science.gov (United States)

    Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

    Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

  16. The actin binding protein adseverin regulates osteoclastogenesis.

    Science.gov (United States)

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  17. The actin binding protein adseverin regulates osteoclastogenesis.

    Directory of Open Access Journals (Sweden)

    Siavash Hassanpour

    Full Text Available Adseverin (Ads, a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG. Ads is induced during OCG downstream of RANK-ligand (RANKL stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

  18. Impact of weightlessness on muscle function

    Science.gov (United States)

    Tischler, M. E.; Slentz, M.

    1995-01-01

    The most studied skeletal muscles which depend on gravity, "antigravity" muscles, are located in the posterior portion of the legs. Antigravity muscles are characterized generally by a different fiber type composition than those which are considered nonpostural. The gravity-dependent function of the antigravity muscles makes them particularly sensitive to weightlessness (unweighting) resulting in a substantial loss of muscle protein, with a relatively greater loss of myofibrillar (structural) proteins. Accordingly alpha-actin mRNA decreases in muscle of rats exposed to microgravity. In the legs, the soleus seems particularly responsive to the lack of weight-bearing associated with space flight. The loss of muscle protein leads to a decreased cross-sectional area of muscle fibers, particularly of the slow-twitch, oxidative (SO) ones compared to fast-twitch glycolytic (FG) or oxidative-glycolytic (FOG) fibers. In some muscles, a shift in fiber composition from SO to FOG has been reported in the adaptation to spaceflight. Changes in muscle composition with spaceflight have been associated with decreased maximal isometric tension (Po) and increased maximal shortening velocity. In terms of fuel metabolism, results varied depending on the pathway considered. Glucose uptake, in the presence of insulin, and activities of glycolytic enzymes are increased by space flight. In contrast, oxidation of fatty acids may be diminished. Oxidation of pyruvate, activity of the citric acid cycle, and ketone metabolism in muscle seem to be unaffected by microgravity.

  19. Enhanced force generation by smooth muscle myosin in vitro.

    OpenAIRE

    VanBuren, P; Work, S S; Warshaw, D.M.

    1994-01-01

    To determine whether the apparent enhanced force-generating capabilities of smooth muscle relative to skeletal muscle are inherent to the myosin cross-bridge, the isometric steady-state force produced by myosin in the in vitro motility assay was measured. In this assay, myosin adhered to a glass surface pulls on an actin filament that is attached to an ultracompliant (50-200 nm/pN) glass microneedle. The number of myosin cross-bridge heads able to interact with a length of actin filament was ...

  20. Effects of Rimonabant on Cannabinoid Receptor 1 andα-Smooth Muscle Actin in C57 Mice with Experimental Hepatic Fibrosis%利莫那班对肝纤维化C57小鼠肝组织大麻素受体1及α-SMA表达的影响

    Institute of Scientific and Technical Information of China (English)

    叶立红; 王翀奎; 陈秀丽; 杨莉; 戴二黑

    2014-01-01

    目的:研究大麻素受体1(CB1)拮抗剂利莫那班对肝纤维化模型C57小鼠肝组织中CB1、α-平滑肌肌动蛋白(α-SMA)表达的影响,及其抗肝纤维化的作用机制。方法30只C57小鼠随机分为3组,分别为正常对照组、模型对照组及利莫那班组,每组10只。采用四氯化碳腹腔注射诱导形成小鼠肝纤维化模型。造模2周后于继续造模同时,正常对照组和模型对照组每天生理盐水灌胃,利莫那班组用利莫那班灌胃。第8周造模结束时处死小鼠,留取肝脏组织标本,分别进行HE和Masson三色染色,应用免疫组织化学方法检测肝组织中CB1和α-SMA的表达,并进行肝组织纤维化评分(S评分)。结果模型对照组和利莫那班组肝组织S评分、CB1和α-SMA阳性表达量均高于正常对照组(均P<0.05),利莫那班组均低于模型对照组(均P<0.05);正常对照组、模型对照组和利莫那班组CB1评分、α-SMA评分与S评分相互之间均呈正相关(均P<0.05)。结论肝组织CB1的激活可促进肝纤维化的形成, CB1拮抗剂利莫那班通过抑制CB1表达,进而抑制肝星状细胞的增殖和激活,从而起到抗肝纤维化的作用。%Objective To study the effect of rimonabant, cannabinoid receptor 1(CB1) antagonist, on the expressions of CB1 andα-smooth muscle actin (α-SMA) in C57 mice with experimental hepatic fibrosis, and their mechanisms in liver fibrosis progression thereof. Methods Thirty C57 mice were randomly divided into three groups, normal control group, mod-el control group and model+rimonabant group, 10 mice for each group. The mouse model of experimental hepatic fibrosis was induced by intraperitoneal injection with 10%CCl4 for two weeks. The normal saline was delivered by gavage daily in normal control group and model control group. Rimonabant was given to mice in model+rimonabant group. Mice were sacri-ficed at the end of eight weeks. Samples of

  1. 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.

  2. CNS myelin wrapping is driven by actin disassembly.

    Science.gov (United States)

    Zuchero, J Bradley; Fu, Meng-Meng; Sloan, Steven A; Ibrahim, Adiljan; Olson, Andrew; Zaremba, Anita; Dugas, Jason C; Wienbar, Sophia; Caprariello, Andrew V; Kantor, Christopher; Leonoudakis, Dmitri; Leonoudakus, Dmitri; Lariosa-Willingham, Karen; Kronenberg, Golo; Gertz, Karen; Soderling, Scott H; Miller, Robert H; Barres, Ben A

    2015-07-27

    Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility.

  3. Actin protofilament orientation in deformation of the erythrocyte membrane skeleton.

    OpenAIRE

    Picart, C.; Dalhaimer, P.; Discher, D. E.

    2000-01-01

    The red cell's spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the ...

  4. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    CERN Document Server

    Carlsson, Anders E

    2010-01-01

    The polymerization of actin via branching at a cell membrane containing nucleation-promoting factors is simulated using a stochastic-growth methodology. The polymerized-actin distribution displays three types of behavior: a) traveling waves, b) moving patches, and c) random fluctuations. Increasing actin concentration causes a transition from patches to waves. The waves and patches move by a treadmilling mechanism which does not require myosin II. The effects of downregulation of key proteins on actin wave behavior are evaluated.

  5. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    Science.gov (United States)

    Carlsson, Anders E.

    2010-06-01

    The polymerization of actin via branching at a cell membrane containing nucleation-promoting factors is simulated using a stochastic-growth methodology. The polymerized-actin distribution displays three types of behavior: (a) traveling waves, (b) moving patches, and (c) random fluctuations. Increasing actin concentration causes a transition from patches to waves. The waves and patches move by a treadmilling mechanism not involving myosin II. The effects of downregulation of key proteins on actin wave behavior are evaluated.

  6. Actin of Beta vulgaris seedlings under the clinorotation

    Science.gov (United States)

    Kozeko, L. Ye.

    We study the influence of altered gravity on actin expression in roots of Beta vulguris seedlings grown on the horizontal clinostat (2 rpm) from seed germination for three days. It is shown that the total actin quantity was not influenced. Three actin isoforms are revealed; a relative protein quantity of these isoforms was similar both in clinorotated seedlings and in ones grown in norm. This point to stable expression of actin under the altered gravity conditions.

  7. Viscoelastic properties of actin-coated membranes

    Science.gov (United States)

    Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; Mackintosh, F. C.; Chatenay, D.

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (ω=0) two-dimensional (2D) shear modulus G02D~0.5 to 5 μN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G'2D(f )~f0.85+/-0.07] and of the bending modulus (κACM(f)~f0.55+/-0.21) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  8. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    Science.gov (United States)

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  9. Actin-organising properties of the muscular dystrophy protein myotilin.

    Science.gov (United States)

    von Nandelstadh, Pernilla; Grönholm, Mikaela; Moza, Monica; Lamberg, Arja; Savilahti, Harri; Carpén, Olli

    2005-10-15

    Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between myotilin and actin. Functionally important regions in myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.

  10. Dynamics and Regulation of Actin Cytoskeleton in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Ren Haiyun

    2007-01-01

    @@ The actin cytoskeleton constituted of globular actin (G-actin) is a ubiquitous component of eukaryotic cells and plays crucial roles in diverse physiological processes in plant cells, such as cytoplasmic streaming, organelle and nucleus positioning, cell morphogenesis, cell division, tip growth, etc.

  11. 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

    2014-02-01

    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.

  12. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells

    OpenAIRE

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-01-01

    Summary Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cel...

  13. Highly extensible skeletal muscle in snakes.

    Science.gov (United States)

    Close, Matthew; Perni, Stefano; Franzini-Armstrong, Clara; Cundall, David

    2014-07-15

    Many snakes swallow large prey whole, and this process requires large displacements of the unfused tips of the mandibles and passive stretching of the soft tissues connecting them. Under these conditions, the intermandibular muscles are highly stretched but subsequently recover normal function. In the highly stretched condition we observed in snakes, sarcomere length (SL) increased 210% its resting value (SL0), and actin and myosin filaments no longer overlapped. Myofibrils fell out of register and triad alignment was disrupted. Following passive recovery, SLs returned to 82% SL0, creating a region of double-overlapping actin filaments. Recovery required recoil of intracellular titin filaments, elastic cytoskeletal components for realigning myofibrils, and muscle activation. Stretch of whole muscles exceeded that of sarcomeres as a result of extension of folded terminal tendon fibrils, stretching of extracellular elastin and independent slippage of muscle fibers. Snake intermandibular muscles thus provide a unique model of how basic components of vertebrate skeletal muscle can be modified to permit extreme extensibility.

  14. CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

    Directory of Open Access Journals (Sweden)

    Elena P Moiseeva

    Full Text Available CADM1 is a major receptor for the adhesion of mast cells (MCs to fibroblasts, human airway smooth muscle cells (HASMCs and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM. Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

  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. α-SM Actin Expression as Prognostic Indicator in IgA Nephropathy (Berger’s Disease

    Directory of Open Access Journals (Sweden)

    M. T. Pastorello

    2005-01-01

    Full Text Available Recent studies have demonstrated that α-Smooth Muscle actin expression in glomerular and tubulointerstitial compartments of renal tissue could represent a prognostic marker in several renal diseases. Our objective was to identify the prognostic value of α-SM actin actin expression on the evolution of renal damage in Primary IgA nephropathy (Berger’s Disease. 43 patients followed up from 1988 to 1999 at the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil, was studied. Clinical-laboratory data were obtained from the medical records of the patients using a protocol containing name, race, gender, origin, profession, age at clinical presentation of the disease and personal and family history. The parameters assessed in the approach to IgA nephropathy were serum creatinine, creatinine clearance, serum albumin, total serum protein, 24 hours proteinuria, glycaemia, serum sodium, potassium, calcium and phosphorus ions, analysis of urinary sediment, serum complement profile, blood count, and renal biopsy. Morphological evaluation was performed by renal biopsy using common light and immunofluorescence microscopy. Immunohistochemical studies were performed using a murine monoclonal antibody to α-SM actin. Our data showed that α-SM actin expression in the glomerular and tubulointerstitial compartments are not correlated with unfavorable clinical course of primary IgA nephropathy.

  17. Urinary bladder stone associated with seminal vesicle and prostate infection in a Copenhagen rat

    Science.gov (United States)

    Senapati, Shantibhusan; Suklabaidya, Sujit; Mallik, Hrudananda; Panda, Sabyasachi; Hota, Datteswar; Baisakh, Manas R.

    2016-01-01

    We report a very rare case of urinary bladder stone in a laboratory rat, which was associated with severe prostatitis and seminal vesiculitis. Importantly, the histopathological analysis revealed the rare variety of keratinizing desquamative squamous metaplasia of bladder, prostate, and seminal vesicle epithelium. Immunohistochemistry for alpha smooth muscle actin protein and aniline blue staining for collagen clearly showed interstitial prostate fibrosis. The detail information about these findings and subsequent discussion are provided here.

  18. Evaluation of myofibroblasts and its association with TGF-β and IFN-γ in lesions of patients with american tegumentary leishmaniasis Avaliação de miofibroblastos e sua associação com TGF-β e IFN-γ em lesões de pacientes com leishmaniose tegumentar americana

    OpenAIRE

    Agostinho Gonçalves Viana; Carlos Alberto de Carvalho Fraga; Paulo Rogério Ferreti Bonan

    2012-01-01

    BACKGROUND: Leishmaniasis is caused by protozoa of Leishmania spp. genus. It is transmitted by the bite of the sand fly insect. It is believed that 12 million people are infected with this disease and that its annual incidence is 2 million; this number is increasing. OBJECTIVES: The present study aimed to evaluate the expression of myofibroblasts through alpha smooth muscle actin labeling, and to analyze their relationship with the expression of the cytokines Interferon gama (IFN-γ) and ...

  19. Urinary bladder stone associated with seminal vesicle and prostate infection in a Copenhagen rat.

    Science.gov (United States)

    Senapati, Shantibhusan; Suklabaidya, Sujit; Mallik, Hrudananda; Panda, Sabyasachi; Hota, Datteswar; Baisakh, Manas R

    2016-01-01

    We report a very rare case of urinary bladder stone in a laboratory rat, which was associated with severe prostatitis and seminal vesiculitis. Importantly, the histopathological analysis revealed the rare variety of keratinizing desquamative squamous metaplasia of bladder, prostate, and seminal vesicle epithelium. Immunohistochemistry for alpha smooth muscle actin protein and aniline blue staining for collagen clearly showed interstitial prostate fibrosis. The detail information about these findings and subsequent discussion are provided here. PMID:27433075

  20. Myofibroblast expression in airways and alveoli is affected by smoking and COPD

    OpenAIRE

    Karvonen, Henna M; Lehtonen, Siri T.; Harju, Terttu; Sormunen, Raija T.; Lappi-Blanco, Elisa; Mäkinen, Johanna M.; Laitakari, Kirsi; Johnson, Shirley; Kaarteenaho, Riitta L

    2013-01-01

    Background Chronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD. Methods α-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistoc...

  1. Rac1 is a novel regulator of contraction-stimulated glucose uptake in skeletal muscle.

    Science.gov (United States)

    Sylow, Lykke; Jensen, Thomas E; Kleinert, Maximilian; Mouatt, Joshua R; Maarbjerg, Stine J; Jeppesen, Jacob; Prats, Clara; Chiu, Tim T; Boguslavsky, Shlomit; Klip, Amira; Schjerling, Peter; Richter, Erik A

    2013-04-01

    In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (~60-100%) and humans (~40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20-58% in extensor digitorum longus (EDL; P contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

  2. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8.

    Directory of Open Access Journals (Sweden)

    Eleanna Stamatakou

    Full Text Available Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1 and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

  3. Effect of trichloroacetic acid on the isolation of tropomyosin from sea urchin lantern muscle.

    Science.gov (United States)

    Ishimoda-Takagi, T; Ozaki, S

    1983-03-01

    Sea urchin lantern muscle tropomyosin showed two components in sodium dodecyl sulfate (SDS) gel electrophoresis in the presence of 5 M urea, although the molecular weights of these components were apparently identical. One of these components seemed to have been digested with an enzyme such as carboxypeptidase, and the tropomyosin had lost the abilities to polymerize and to bind to actin. A crude extract prepared from the lantern muscle treated with trichloroacetic acid (TCA) contained predominantly tropomyosin. Tropomyosin purified from TCA-treated lantern muscle seemed to be intact and retained the ability to bind to actin.

  4. The length-tension curve in muscle depends on lattice spacing.

    Science.gov (United States)

    Williams, C David; Salcedo, Mary K; Irving, Thomas C; Regnier, Michael; Daniel, Thomas L

    2013-09-01

    Classic interpretations of the striated muscle length-tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing is a significant force regulator, increasing the slope of muscle's force-length dependence. PMID:23843386

  5. Modeling Muscles

    Science.gov (United States)

    Goodwyn, Lauren; Salm, Sarah

    2007-01-01

    Teaching the anatomy of the muscle system to high school students can be challenging. Students often learn about muscle anatomy by memorizing information from textbooks or by observing plastic, inflexible models. Although these mediums help students learn about muscle placement, the mediums do not facilitate understanding regarding integration of…

  6. Maintaining Muscle Mitochondria Via Trans-synaptic Signaling

    OpenAIRE

    Long, Jennifer B.; Van Vactor, David

    2012-01-01

    Dominantly inherited VAPB mutations are implicated in neurodegenerative disease, including amyotrophic lateral sclerosis and spinal muscular atrophy. In the current issue, Han et al. (2012) uncover a mechanism through which the secreted VAPB MSP domain regulates actin organization and mitochondrial function in muscle cells through LAR and Robo receptor activation.

  7. Magnetic Dipoles and Quantum Coherence in Muscle Contraction

    CERN Document Server

    Hatori, K; Matsuno, K; Hatori, Kuniyuki; Honda, Hajime; Matsuno, Koichiro

    2001-01-01

    An actin filament contacting myosin molecules as a functional unit of muscle contraction induces magnetic dipoles along the filament when ATP molecules to be hydrolyzed are available there. The induced magnetic dipoles are coherent over the entire filament, though they are fluctuating altogether as constantly being subject to the ambient thermal agitations.

  8. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility

    Science.gov (United States)

    Benanti, Erin L.; Nguyen, Catherine M.; Welch, Matthew D.

    2015-01-01

    Summary Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, while their close relative B. thailandensis is nonpathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection. PMID:25860613

  9. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    Science.gov (United States)

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  10. The neuronal and actin commitment: Why do neurons need rings?

    Science.gov (United States)

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc.

  11. 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. PMID:26240174

  12. Gelsolin mediates calcium-dependent disassembly of Listeria actin tails

    Science.gov (United States)

    Larson, Laura; Arnaudeau, Serge; Gibson, Bruce; Li, Wei; Krause, Ryoko; Hao, Binghua; Bamburg, James R.; Lew, Daniel P.; Demaurex, Nicolas; Southwick, Frederick

    2005-01-01

    The role of intracellular Ca2+ in the regulation of actin filament assembly and disassembly has not been clearly defined. We show that reduction of intracellular free Ca2+ concentration ([Ca2+]i) to <40 nM in Listeria monocytogenes-infected, EGFP–actin-transfected Madin–Darby canine kidney cells results in a 3-fold lengthening of actin filament tails. This increase in tail length is the consequence of marked slowing of the actin filament disassembly rate, without a significant change in assembly rate. The Ca2+-sensitive actin-severing protein gelsolin concentrates in the Listeria rocket tails at normal resting [Ca2+]i and disassociates from the tails when [Ca2+]i is lowered. Reduction in [Ca2+]i also blocks the severing activity of gelsolin, but not actin-depolymerizing factor (ADF)/cofilin microinjected into Listeria-infected cells. In Xenopus extracts, Listeria tail lengths are also calcium-sensitive, markedly shortening on addition of calcium. Immunodepletion of gelsolin, but not Xenopus ADF/cofilin, eliminates calcium-sensitive actin-filament shortening. Listeria tail length is also calcium-insensitive in gelsolin-null mouse embryo fibroblasts. We conclude that gelsolin is the primary Ca2+-sensitive actin filament recycling protein in the cell and is capable of enhancing Listeria actin tail disassembly at normal resting [Ca2+]i (145 nM). These experiments illustrate the unique and complementary functions of gelsolin and ADF/cofilin in the recycling of actin filaments. PMID:15671163

  13. 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.

  14. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

  15. Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility

    OpenAIRE

    Gallo, Gianluca; Yee, Hal F.; Letourneau, Paul C.

    2002-01-01

    Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon ...

  16. Lamin A/C and emerin regulate MKL1/SRF activity by modulating actin dynamics

    Science.gov (United States)

    Ho, Chin Yee; Jaalouk, Diana E.; Vartiainen, Maria K.; Lammerding, Jan

    2013-01-01

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization. PMID:23644458

  17. Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics.

    Science.gov (United States)

    Ho, Chin Yee; Jaalouk, Diana E; Vartiainen, Maria K; Lammerding, Jan

    2013-05-23

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna(-/-)) and Lmna(N195K/N195K) mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna(-/-) and Lmna(N195K/N195K) mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

  18. Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.

    Science.gov (United States)

    Li, Jia; Chen, Shu; Cleary, Rachel A; Wang, Ruping; Gannon, Olivia J; Seto, Edward; Tang, Dale D

    2014-08-01

    Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction.

  19. Adhesive F-actin waves: a novel integrin-mediated adhesion complex coupled to ventral actin polymerization.

    Directory of Open Access Journals (Sweden)

    Lindsay B Case

    Full Text Available At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These "adhesive F-actin waves" require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization.

  20. Actin-Dynamics in Plant Cells: The Function of Actin Perturbing Substances Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins

    Science.gov (United States)

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

    This chapter will give an overview of the most common F-actin perturbing substances, that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement or when apoptosis has to be induced. These substances can be divided into two major subclasses – F-actin stabilizing and polymerizing substances like jasplakinolide, chondramides and F-actin severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane permeable F-actin stabilizing and polymerizing agent, which may even have anti-cancer activities. Cytochalasins, derived from fungi show an F-actin severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges, however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin- and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given. PMID:26498789

  1. Identification of sucrose synthase as an actin-binding protein

    Science.gov (United States)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  2. Steric effects induce geometric remodeling of actin bundles in filopodia

    CERN Document Server

    Dobramysl, Ulrich; Erban, Radek

    2016-01-01

    Filopodia are ubiquitous fingerlike protrusions, spawned by many eukaryotic cells, to probe and interact with their environments. Polymerization dynamics of actin filaments, comprising the structural core of filopodia, largely determine their instantaneous lengths and overall lifetimes. The polymerization reactions at the filopodial tip require transport of G-actin, which enter the filopodial tube from the filopodial base and diffuse toward the filament barbed ends near the tip. Actin filaments are mechanically coupled into a tight bundle by cross-linker proteins. Interestingly, many of these proteins are relatively short, restricting the free diffusion of cytosolic G-actin throughout the bundle and, in particular, its penetration into the bundle core. To investigate the effect of steric restrictions on G-actin diffusion by the porous structure of filopodial actin filament bundle, we used a particle-based stochastic simulation approach. We discovered that excluded volume interactions result in partial and the...

  3. A Robust Actin Filaments Image Analysis Framework.

    Science.gov (United States)

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-08-01

    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 osteoblasts grown in

  4. A Robust Actin Filaments Image Analysis Framework

    Science.gov (United States)

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    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 osteoblasts

  5. Formins: Bringing new insights to the organization of actin cytoskeleton

    Institute of Scientific and Technical Information of China (English)

    GUO Chunqing; REN Haiyun

    2006-01-01

    The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attracted much attention as they can nucleate unbranched actin filament from the profilin bound actin pool in vivo. In recent years, a number of formin family members from different organisms have been reported, and their characteristics are known more clearly, although some questions are still to be clarified. Here, we summarize the structures, functions and nucleation mechanisms of different formin family proteins, intending to compare them and give some new clues to the study of formins.

  6. Immunocytochemical identification of actin in mitochondria of Physarum polycephalum

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Mitochondria isolated from the plasmodia of Physarum polycephalum Schw. are reacted with rabbit anti-actin antibody, and detected with FITC-conjugated sheep anti-rabbit IgG antibody. The results of indirect immunofluorescence show that actin exists in the mitochondria. Western blot analysis confirms the existence of actin in the protein preparation of the mitochondria. The indirect immunoelectron microscopic observation using the same antibodies verifies further that actin is the constituents of mitochondria, and it is dispersively distributed in the mitochondria of P. polycephalum.

  7. Correlative nanoscale imaging of actin filaments and their complexes.

    Science.gov (United States)

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E; Reisler, Emil; Gimzewski, James K

    2013-07-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  8. 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 in...

  9. Visualization of Actin Cytoskeletal Dynamics in Fixed and Live Drosophila Egg Chambers.

    Science.gov (United States)

    Groen, Christopher M; Tootle, Tina L

    2015-01-01

    Visualization of actin cytoskeletal dynamics is critical for understanding the spatial and temporal regulation of actin remodeling. Drosophila oogenesis provides an excellent model system for visualizing the actin cytoskeleton. Here, we present methods for imaging the actin cytoskeleton in Drosophila egg chambers in both fixed samples by phalloidin staining and in live egg chambers using transgenic actin labeling tools.

  10. A two-segment model for thin filament architecture in skeletal muscle.

    Science.gov (United States)

    Gokhin, David S; Fowler, Velia M

    2013-02-01

    Correct specification of myofilament length is essential for efficient skeletal muscle contraction. The length of thin actin filaments can be explained by a novel 'two-segment' model, wherein the thin filaments consist of two concatenated segments, which are of either constant or variable length. This is in contrast to the classic 'nebulin ruler' model, which postulates that thin filaments are uniform structures, the lengths of which are dictated by nebulin. The two-segment model implicates position-specific microregulation of actin dynamics as a general principle underlying actin filament length and stability.

  11. Muscle-specific androgen receptor deletion shows limited actions in myoblasts but not in myofibers in different muscles in vivo.

    Science.gov (United States)

    Rana, Kesha; Chiu, Maria W S; Russell, Patricia K; Skinner, Jarrod P; Lee, Nicole K L; Fam, Barbara C; Zajac, Jeffrey D; MacLean, Helen E

    2016-08-01

    The aim of this study was to investigate the direct muscle cell-mediated actions of androgens by comparing two different mouse lines. The cre-loxP system was used to delete the DNA-binding activity of the androgen receptor (AR) in mature myofibers (MCK mAR(ΔZF2)) in one model and the DNA-binding activity of the AR in both proliferating myoblasts and myofibers (α-actin mAR(ΔZF2)) in another model. We found that hind-limb muscle mass was normal in MCK mAR(ΔZF2) mice and that relative mass of only some hind-limb muscles was reduced in α-actin mAR(ΔZF2) mice. This suggests that myoblasts and myofibers are not the major cellular targets mediating the anabolic actions of androgens on male muscle during growth and development. Levator ani muscle mass was decreased in both mouse lines, demonstrating that there is a myofiber-specific effect in this unique androgen-dependent muscle. We found that the pattern of expression of genes including c-myc, Fzd4 and Igf2 is associated with androgen-dependent changes in muscle mass; therefore, these genes are likely to be mediators of anabolic actions of androgens. Further research is required to identify the major targets of androgen actions in muscle, which are likely to include indirect actions via other tissues.

  12. Adhesion and Fusion of Muscle Cells Are Promoted by Filopodia.

    Science.gov (United States)

    Segal, Dagan; Dhanyasi, Nagaraju; Schejter, Eyal D; Shilo, Ben-Zion

    2016-08-01

    Indirect flight muscles (IFMs) in Drosophila are generated during pupariation by fusion of hundreds of myoblasts with larval muscle templates (myotubes). Live observation of these muscles during the fusion process revealed multiple long actin-based protrusions that emanate from the myotube surface and require Enabled and IRSp53 for their generation and maintenance. Fusion is blocked when formation of these filopodia is compromised. While filopodia are not required for the signaling process underlying critical myoblast cell-fate changes prior to fusion, myotube-myoblast adhesion appears to be filopodia dependent. Without filopodia, close apposition between the cell membranes is not achieved, the cell-adhesion molecule Duf is not recruited to the myotube surface, and adhesion-dependent actin foci do not form. We therefore propose that the filopodia are necessary to prime the heterotypic adhesion process between the two cell types, possibly by recruiting the cell-adhesion molecule Sns to discrete patches on the myoblast cell surface.

  13. Interactions between the Yeast SM22 Homologue Scp1 and Actin Demonstrate the Importance of Actin Bundling in Endocytosis* S⃞

    OpenAIRE

    Gheorghe, Dana M.; Aghamohammadzadeh, Soheil; Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Winder, Steve J.; Ayscough, Kathryn R.

    2008-01-01

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that all...

  14. Poorly Understood Aspects of Striated Muscle Contraction

    Directory of Open Access Journals (Sweden)

    Alf Månsson

    2015-01-01

    Full Text Available Muscle contraction results from cyclic interactions between the contractile proteins myosin and actin, driven by the turnover of adenosine triphosphate (ATP. Despite intense studies, several molecular events in the contraction process are poorly understood, including the relationship between force-generation and phosphate-release in the ATP-turnover. Different aspects of the force-generating transition are reflected in the changes in tension development by muscle cells, myofibrils and single molecules upon changes in temperature, altered phosphate concentration, or length perturbations. It has been notoriously difficult to explain all these events within a given theoretical framework and to unequivocally correlate observed events with the atomic structures of the myosin motor. Other incompletely understood issues include the role of the two heads of myosin II and structural changes in the actin filaments as well as the importance of the three-dimensional order. We here review these issues in relation to controversies regarding basic physiological properties of striated muscle. We also briefly consider actomyosin mutation effects in cardiac and skeletal muscle function and the possibility to treat these defects by drugs.

  15. Systems analysis of biological networks in skeletal muscle function.

    Science.gov (United States)

    Smith, Lucas R; Meyer, Gretchen; Lieber, Richard L

    2013-01-01

    Skeletal muscle function depends on the efficient coordination among subcellular systems. These systems are composed of proteins encoded by a subset of genes, all of which are tightly regulated. In the cases where regulation is altered because of disease or injury, dysfunction occurs. To enable objective analysis of muscle gene expression profiles, we have defined nine biological networks whose coordination is critical to muscle function. We begin by describing the expression of proteins necessary for optimal neuromuscular junction function that results in the muscle cell action potential. That action potential is transmitted to proteins involved in excitation-contraction coupling enabling Ca(2+) release. Ca(2+) then activates contractile proteins supporting actin and myosin cross-bridge cycling. Force generated by cross-bridges is transmitted via cytoskeletal proteins through the sarcolemma and out to critical proteins that support the muscle extracellular matrix. Muscle contraction is fueled through many proteins that regulate energy metabolism. Inflammation is a common response to injury that can result in alteration of many pathways within muscle. Muscle also has multiple pathways that regulate size through atrophy or hypertrophy. Finally, the isoforms associated with fast muscle fibers and their corresponding isoforms in slow muscle fibers are delineated. These nine networks represent important biological systems that affect skeletal muscle function. Combining high-throughput systems analysis with advanced networking software will allow researchers to use these networks to objectively study skeletal muscle systems. PMID:23188744

  16. Cross-bridge model of muscle contraction. Quantitative analysis.

    OpenAIRE

    Eisenberg, E.; Hill, T L; Chen, Y.

    1980-01-01

    We recently presented, in a qualitative manner, a cross-bridge model of muscle contraction which was based on a biochemical kinetic cycle for the actomyosin ATPase activity. This cross-bridge model consisted of two cross-bridge states detached from actin and two cross-bridge states attached to actin. In the present paper, we attempt to fit this model quantitatively to both biochemical and physiological data. We find that the resulting complete cross-bridge model is able to account reasonably ...

  17. Actin filaments and microtubule dual-granule transport in human adhered platelets: the role of alpha-dystrobrevins.

    Science.gov (United States)

    Cerecedo, Doris; Cisneros, Bulmaro; Mondragón, Ricardo; González, Sirenia; Galván, Iván J

    2010-04-01

    Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension-activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. alpha- and beta-dystrobrevin members of the dystrophin-associated protein complex in muscle and non-muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton-disrupting drugs, quantification of soluble P-selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of alpha-dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which alpha-dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.

  18. Modulating F-actin organization induces organ growth by affecting the Hippo pathway

    OpenAIRE

    Sansores-Garcia, Leticia; Bossuyt, Wouter; Wada, Ken-Ichi; Yonemura, Shigenobu; Tao, Chunyao; Sasaki, Hiroshi; Halder, Georg

    2011-01-01

    This study identifies actin organization as an upstream regulator of the Hippo pathway: F-actin accumulation promotes Yorkie-dependent transcriptional activation. This modulation of Hippo signalling by actin regulators controls organ growth in Drosophila.

  19. 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.

  20. Filament assembly by Spire: key residues and concerted actin binding.

    Science.gov (United States)

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

  1. Actin puts the squeeze on Drosophila glue secretion.

    Science.gov (United States)

    Merrifield, Christien J

    2016-02-01

    An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.

  2. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    Science.gov (United States)

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  3. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.

    Science.gov (United States)

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  4. Location of and post-mortem changes in some cytoskeletal proteins in pork and cod muscle

    DEFF Research Database (Denmark)

    Morrison, E.H.; Bremner, Allan; Purslow, P.P.

    2000-01-01

    The cytoskeletal proteins actin, nebulin, spectrin, desmin, vinculin and talin were labelled immunohistochemically in sections of muscle from commercially available pigs and cod (Gadus morhua) taken pre-rigor and from samples stored for several days. Actin, nebulin and spectrin gave similar...... labelling in fish. Labelling for talin in pork muscle was intense at the sarcolemma but was not present in samples stored for 4 days. In contrast, the label for talin was concentrated at the myotendinous junction of the cod muscle throughout the storage period. These are the first reports of the detection...... and location of spectrin and vinculin in fish muscle and of the location of talin. The results are discussed in terms of muscle structure, function and post-mortem tenderisation. (C) 2000 Society of Chemical Industry....

  5. 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

  6. Structural Modeling and Molecular Dynamics Simulation of the Actin Filament

    Energy Technology Data Exchange (ETDEWEB)

    Splettstoesser, Thomas [University of Heidelberg; Holmes, Kenneth [Max Planck Institute, Heidelberg, Germany; Noe, Frank [DFG Research Center Matheon, FU Berlin, Germany; Smith, Jeremy C [ORNL

    2011-01-01

    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.

  7. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    Science.gov (United States)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  8. Mechanical properties of branched actin filaments

    CERN Document Server

    Razbin, Mohammadhosein; Benetatos, Panayotis; Zippelius, Annette

    2015-01-01

    Cells moving on a two dimensional substrate generate motion by polymerizing actin filament networks inside a flat membrane protrusion. New filaments are generated by branching off existing ones, giving rise to branched network structures. We investigate the force-extension relation of branched filaments, grafted on an elastic structure at one end and pushing with the free ends against the leading edge cell membrane. Single filaments are modeled as worm-like chains, whose thermal bending fluctuations are restricted by the leading edge cell membrane, resulting in an effective force. Branching can increase the stiffness considerably; however the effect depends on branch point position and filament orientation, being most pronounced for intermediate tilt angles and intermediate branch point positions. We describe filament networks without cross-linkers to focus on the effect of branching. We use randomly positioned branch points, as generated in the process of treadmilling, and orientation distributions as measur...

  9. 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.

  10. Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

    Science.gov (United States)

    Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

    2013-01-01

    The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

  11. Concentration profiles of actin-binding molecules in lamellipodia

    Science.gov (United States)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  12. Interconnection between actin cytoskeleton and plant defense signaling.

    Science.gov (United States)

    Janda, Martin; Matoušková, Jindřiška; Burketová, Lenka; Valentová, Olga

    2014-01-01

    Actin cytoskeleton is the fundamental structural component of eukaryotic cells. It has a role in numerous elementary cellular processes such as reproduction, development and also in response to abiotic and biotic stimuli. Remarkably, the role of actin cytoskeleton in plant response to pathogens is getting to be under magnifying glass. Based on microscopic studies, most of the data showed, that actin plays an important role in formation of physiological barrier in the site of infection. Actin dynamics is involved in the transport of antimicrobial compounds and cell wall fortifying components (e.g. callose) to the site of infection. Also the role in PTI (pathogen triggered immunity) and ETI (effector triggered immunity) was recently indicated. On the other hand much less is known about the transcriptome reprogramming upon changes in actin dynamics. Our recently published results showed that drugs inhibiting actin polymerization (latrunculin B, cytochalasin E) cause the induction of genes which are involved in salicylic acid (SA) signaling pathway. In this addendum we would like to highlight in more details current state of knowledge concerning the involvement of actin dynamics in plant defense signaling.

  13. Drebrin attenuates the interaction between actin and myosin-V.

    Science.gov (United States)

    Ishikawa, Ryoki; Katoh, Kaoru; Takahashi, Ayumi; Xie, Ce; Oseki, Koushi; Watanabe, Michitoshi; Igarashi, Michihiro; Nakamura, Akio; Kohama, Kazuhiro

    2007-07-27

    Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.

  14. Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

    Directory of Open Access Journals (Sweden)

    Anabelle S. Cornachione

    2014-07-01

    Full Text Available Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

  15. The STARS signaling pathway: a key regulator of skeletal muscle function.

    Science.gov (United States)

    Lamon, Séverine; Wallace, Marita A; Russell, Aaron P

    2014-09-01

    During the last decade, the striated muscle activator of Rho signaling (STARS), a muscle-specific protein, has been proposed to play an increasingly important role in skeletal muscle growth, metabolism, regeneration and stress adaptation. STARS influences actin dynamics and, as a consequence, regulates the myocardin-related transcription factor A/serum response factor (MRTF-A/SRF) transcriptional program, a well-known pathway controlling skeletal muscle development and function. Muscle-specific stress conditions, such as exercise, positively regulates, while disuse and degenerative muscle diseases are associated with a downregulation of STARS and its downstream partners, suggesting a pivotal role for STARS in skeletal muscle health. This review provides a comprehensive overview of the known role and regulation of STARS and the members of its signaling pathway, RhoA, MRTF-A and SRF, in skeletal muscle.

  16. Analysis of actinic flux profiles measured from an ozonesonde balloon

    Science.gov (United States)

    Wang, P.; Allaart, M.; Knap, W. H.; Stammes, P.

    2015-04-01

    A green light sensor has been developed at KNMI to measure actinic flux profiles using an ozonesonde balloon. In total, 63 launches with ascending and descending profiles were performed between 2006 and 2010. The measured uncalibrated actinic flux profiles are analysed using the Doubling-Adding KNMI (DAK) radiative transfer model. Values of the cloud optical thickness (COT) along the flight track were taken from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) Cloud Physical Properties (CPP) product. The impact of clouds on the actinic flux profile is evaluated on the basis of the cloud modification factor (CMF) at the cloud top and cloud base, which is the ratio between the actinic fluxes for cloudy and clear-sky scenes. The impact of clouds on the actinic flux is clearly detected: the largest enhancement occurs at the cloud top due to multiple scattering. The actinic flux decreases almost linearly from cloud top to cloud base. Above the cloud top the actinic flux also increases compared to clear-sky scenes. We find that clouds can increase the actinic flux to 2.3 times the clear-sky value at cloud top and decrease it to about 0.05 at cloud base. The relationship between CMF and COT agrees well with DAK simulations, except for a few outliers. Good agreement is found between the DAK-simulated actinic flux profiles and the observations for single-layer clouds in fully overcast scenes. The instrument is suitable for operational balloon measurements because of its simplicity and low cost. It is worth further developing the instrument and launching it together with atmospheric chemistry composition sensors.

  17. Role of actin in auxin transport and transduction of gravity

    Science.gov (United States)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  18. Actin-based dynamics during spermatogenesis and its significance

    Institute of Scientific and Technical Information of China (English)

    XIAO Xiang; YANG Wan-xi

    2007-01-01

    Actin can be found in all kinds ofeukaryotic cells, maintaining their shapes and motilities, while its dynamics in sperm cells is understood less than their nonmuscle somatic cell counterparts. Spermatogenesis is a complicated process, resulting in the production of mature sperm from primordial germ cell. Significant structural and biochemical changes take place in the seminiferous epithelium of the adult testis during spermatogenesis. It was proved that all mammalian sperm contain actin, and that F-actin may play an important role during spermatogenesis, especially in nuclear shaping. Recently a new model for sperm head elongation based on the acrosome-acroplaxome-manchette complex has been proposed. In Drosophila, F-actin assembly is supposed to be very crucial during individualization. In this mini-review, we provide an overview of the structure, function, and regulation characteristics of actin cytoskeleton, and a summary of the current status of research of actin-based structure and movement is also provided, with emphasis on the role of actins in sperm head shaping during spermiogenesis and the cell junction dynamics in the testis. Research of the Sertoli ectoplasmic specialization is in the spotlight, which is a testis-specific actin-based junction very important for the movement of germ cells across the epithelium. Study of the molecular architecture and the regulating mechanism of the Sertoli ectoplasmic specialization has become an intriguing field. All this may lead to a new strategy for male infertility and,at the same time, a novel idea may result in devising much safer contraception with high efficiency. It is hoped that the advances listed in this review would give developmental and morphological researchers a favorable investigating outline and could help to enlarge the view of new strategies and models for actin dynamics during spermatogenesis.

  19. Pathogenic microbes manipulate cofilin activity to subvert actin cytoskeleton.

    Science.gov (United States)

    Zheng, Kai; Kitazato, Kaio; Wang, Yifei; He, Zhendan

    2016-09-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are key players in controlling the temporal and spatial extent of actin dynamics, which is crucial for mediating host-pathogen interactions. Pathogenic microbes have evolved molecular mechanisms to manipulate cofilin activity to subvert the actin cytoskeletal system in host cells, promoting their internalization into the target cells, modifying the replication niche and facilitating their intracellular and intercellular dissemination. The study of how these pathogens exploit cofilin pathways is crucial for understanding infectious disease and providing potential targets for drug therapies. PMID:25853495

  20. Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy.

    Science.gov (United States)

    Li, Frank; Buck, Danielle; De Winter, Josine; Kolb, Justin; Meng, Hui; Birch, Camille; Slater, Rebecca; Escobar, Yael Natelie; Smith, John E; Yang, Lin; Konhilas, John; Lawlor, Michael W; Ottenheijm, Coen; Granzier, Henk L

    2015-09-15

    Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness. PMID:26123491

  1. HSP20 phosphorylation and airway smooth muscle relaxation

    Directory of Open Access Journals (Sweden)

    Mariam Ba

    2009-06-01

    Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

  2. An ionic-chemical-mechanical model for muscle contraction.

    Science.gov (United States)

    Manning, Gerald S

    2016-12-01

    The dynamic process underlying muscle contraction is the parallel sliding of thin actin filaments along an immobile thick myosin fiber powered by oar-like movements of protruding myosin cross bridges (myosin heads). The free energy for functioning of the myosin nanomotor comes from the hydrolysis of ATP bound to the myosin heads. The unit step of translational movement is based on a mechanical-chemical cycle involving ATP binding to myosin, hydrolysis of the bound ATP with ultimate release of the hydrolysis products, stress-generating conformational changes in the myosin cross bridge, and relief of built-up stress in the myosin power stroke. The cycle is regulated by a transition between weak and strong actin-myosin binding affinities. The dissociation of the weakly bound complex by addition of salt indicates the electrostatic basis for the weak affinity, while structural studies demonstrate that electrostatic interactions among negatively charged amino acid residues of actin and positively charged residues of myosin are involved in the strong binding interface. We therefore conjecture that intermediate states of increasing actin-myosin engagement during the weak-to-strong binding transition also involve electrostatic interactions. Methods of polymer solution physics have shown that the thin actin filament can be regarded in some of its aspects as a net negatively charged polyelectrolyte. Here we employ polyelectrolyte theory to suggest how actin-myosin electrostatic interactions might be of significance in the intermediate stages of binding, ensuring an engaged power stroke of the myosin motor that transmits force to the actin filament, and preventing the motor from getting stuck in a metastable pre-power stroke state. We provide electrostatic force estimates that are in the pN range known to operate in the cycle. PMID:27603027

  3. Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

    Science.gov (United States)

    Goryunov, Dmitry; Liem, Ronald K H

    2016-01-01

    The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues.

  4. miR-8 controls synapse structure by repression of the actin regulator enabled.

    Science.gov (United States)

    Loya, Carlos M; McNeill, Elizabeth M; Bao, Hong; Zhang, Bing; Van Vactor, David

    2014-05-01

    MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play important roles in nervous system development and physiology. However, our understanding of the strategies by which miRNAs control synapse development is limited. We find that the highly conserved miRNA miR-8 regulates the morphology of presynaptic arbors at the Drosophila neuromuscular junction (NMJ) through a postsynaptic mechanism. Developmental analysis shows that miR-8 is required for presynaptic expansion that occurs in response to larval growth of the postsynaptic muscle targets. With an in vivo sensor, we confirm our hypothesis that the founding member of the conserved Ena/VASP (Enabled/Vasodilator Activated Protein) family is regulated by miR-8 through a conserved site in the Ena 3' untranslated region (UTR). Synaptic marker analysis and localization studies suggest that Ena functions within the subsynaptic reticulum (SSR) surrounding presynaptic terminals. Transgenic lines that express forms of a conserved mammalian Ena ortholog further suggest that this localization and function of postsynaptic Ena/VASP family protein is dependent on conserved C-terminal domains known to mediate actin binding and assembly while antagonizing actin-capping proteins. Ultrastructural analysis demonstrates that miR-8 is required for SSR morphogenesis. As predicted by our model, we find that Ena is both sufficient and necessary to account for miR-8-mediated regulation of SSR architecture, consistent with its localization in this compartment. Finally, electrophysiological analysis shows that miR-8 is important for spontaneous neurotransmitter release frequency and quantal content. However, unlike the structural phenotypes, increased expression of Ena fails to mimic the functional defects observed in miR-8-null animals. Together, these findings suggest that miR-8 limits the expansion of presynaptic terminals during larval synapse development through regulation of postsynaptic actin assembly that

  5. Live cell imaging of the assembly, disassembly, and actin cable–dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae

    OpenAIRE

    Huckaba, Thomas M.; Gay, Anna Card; Pantalena, Luiz Fernando; Yang, Hyeong-Cheol; Liza A Pon

    2004-01-01

    Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex...

  6. A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK 1/2 to promote fascin-1/actin binding and filopodia stability

    Directory of Open Access Journals (Sweden)

    Jayo Asier

    2012-08-01

    Full Text Available Abstract Background Fascin-1 is an actin crosslinking protein that is important for the assembly of cell protrusions in neurons, skeletal and smooth muscle, fibroblasts, and dendritic cells. Although absent from most normal adult epithelia, fascin-1 is upregulated in many human carcinomas, and is associated with poor prognosis because of its promotion of carcinoma cell migration, invasion, and metastasis. Rac and Cdc42 small guanine triphosphatases have been identified as upstream regulators of the association of fascin-1 with actin, but the possible role of Rho has remained obscure. Additionally, experiments have been hampered by the inability to measure the fascin-1/actin interaction directly in intact cells. We investigated the hypothesis that fascin-1 is a functional target of Rho in normal and carcinoma cells, using experimental approaches that included a novel fluorescence resonance energy transfer (FRET/fluorescence lifetime imaging (FLIM method to measure the interaction of fascin-1 with actin. Results Rho activity modulates the interaction of fascin-1 with actin, as detected by a novel FRET method, in skeletal myoblasts and human colon carcinoma cells. Mechanistically, Rho regulation depends on Rho kinase activity, is independent of the status of myosin II activity, and is not mediated by promotion of the fascin/PKC complex. The p-Lin-11/Isl-1/Mec-3 kinases (LIMK, LIMK1 and LIMK2, act downstream of Rho kinases as novel binding partners of fascin-1, and this complex regulates the stability of filopodia. Conclusions We have identified a novel activity of Rho in promoting a complex between fascin-1 and LIMK1/2 that modulates the interaction of fascin-1 with actin. These data provide new mechanistic insight into the intracellular coordination of contractile and protrusive actin-based structures. During the course of the study, we developed a novel FRET method for analysis of the fascin-1/actin interaction, with potential general

  7. Single muscle fiber gene expression in human skeletal muscle: validation of internal control with exercise

    International Nuclear Information System (INIS)

    Reverse transcription and real-time PCR have become the method of choice for the detection of low-abundance mRNA transcripts obtained from small human muscle biopsy samples. GAPDH, β-actin, β-2M, and 18S rRNA are widely employed as endogenous control genes, with the assumption that their expression is unregulated and constant for given experimental conditions. The aim of this study was to determine if mRNA transcripts could be performed on isolated human single muscle fibers and to determine reliable housekeeping genes (HKGs) using quantitative gene expression protocols at rest and in response to an acute exercise bout. Muscle biopsies were obtained from the gastrocnemius of three adult males before, immediately after, and 4 h following 30 min of treadmill running at 70% of VO2max. A total of 40 single fibers (MHC I and IIa) were examined for GAPDH, β-actin, β-2M, and 18S rRNA using quantitative RT-PCR and SYBR Green detection. All analyzed single fiber segments showed ribosomal RNA (28S/18S). No degradation or additional bands below ribosomal were detected (rRNA ratio 1.5-1.8). Also, no high or low-molecular weight genomic DNA contamination was observed. For each housekeeping gene the duplicate average SD was ±0.13 with a CV of 0.58%. Stable expression of GAPDH was observed at all time points for each fiber type (MHC I and IIa). Inconsistent expression of β-actin, β-2M, and 18S rRNA was observed during the post-exercise time points for each fiber type. These data indicate that successful extraction of high quality RNA from human single muscle fibers along with quantification of mRNA of selected genes can be performed. Furthermore, exercise does influence the expression of certain HKGs with GAPDH being the most stable

  8. Antenna mechanism of length control of actin cables

    CERN Document Server

    Mohapatra, Lishibanya; Kondev, Jane

    2014-01-01

    Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This antenna mechanism involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentra...

  9. Differential requirements for actin during yeast and mammalian endocytosis.

    Science.gov (United States)

    Aghamohammadzadeh, Soheil; Ayscough, Kathryn R

    2009-08-01

    Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination. PMID:19597484

  10. Nanosecond electric pulses trigger actin responses in plant cells

    International Nuclear Information System (INIS)

    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.

  11. Roles of Cortactin, an Actin Polymerization Mediator, in Cell Endocytosis

    Institute of Scientific and Technical Information of China (English)

    Li CHEN; Zhi-Wei WANG; Jian-wei ZHU; Xi ZHAN

    2006-01-01

    Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene.Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents.Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 (SH3)-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation.Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.

  12. Tracing myoblast fusion in Drosophila embryos by fluorescent actin probes.

    Science.gov (United States)

    Haralalka, Shruti; Abmayr, Susan M

    2015-01-01

    Myoblast fusion in the Drosophila embryo is a highly elaborate process that is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs). It occurs through an asymmetric event in which actin foci assemble in the FCMs at points of cell-cell contact and direct the formation of membrane protrusions that drive fusion. Herein, we describe the approach that we have used to image in living embryos the highly dynamic actin foci and actin-rich projections that precede myoblast fusion. We discuss resources currently available for imaging actin and myogenesis, and our experience with these resources if available. This technical report is not intended to be comprehensive on providing instruction on standard microscopy practices or software utilization. However, we discuss microscope parameters that we have used in data collection, and our experience with image processing tools in data analysis.

  13. A model actin comet tail disassembling by severing

    International Nuclear Information System (INIS)

    We use a numerical simulation to model an actin comet tail as it grows from the surface of a small object (a bead) and disassembles by severing. We explore the dependence of macroscopic properties such as the local tail radius and tail length on several controllable properties, namely the bead diameter, the bead velocity, the severing rate per unit length, and the actin gel mesh size. The model predicts an F-actin density with an initial exponential decay followed by an abrupt decay at the edge of the tail, and predicts that the comet tail diameter is constant along the length of the tail. The simulation results are used to fit a formula relating the comet tail length to the control parameters, and it is proposed that this formula offers a means to extract quantitative information on the actin gel mesh size and severing kinetics from simple macroscopic measurements

  14. Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks.

    Science.gov (United States)

    Osmanagic-Myers, Selma; Rus, Stefanie; Wolfram, Michael; Brunner, Daniela; Goldmann, Wolfgang H; Bonakdar, Navid; Fischer, Irmgard; Reipert, Siegfried; Zuzuarregui, Aurora; Walko, Gernot; Wiche, Gerhard

    2015-11-15

    Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis. PMID:26519478

  15. Computational defect review for actinic mask inspections

    Science.gov (United States)

    Morgan, Paul; Rost, Daniel; Price, Daniel; Corcoran, Noel; Satake, Masaki; Hu, Peter; Peng, Danping; Yonenaga, Dean; Tolani, Vikram

    2013-04-01

    As optical lithography continues to extend into low-k1 regime, resolution of mask patterns continues to diminish. The limitation of 1.35 NA posed by water-based lithography has led to the application of various resolution enhancement techniques (RET), for example, use of strong phase-shifting masks, aggressive OPC and sub-resolution assist features, customized illuminators, etc. The adoption of these RET techniques combined with the requirements to detect even smaller defects on masks due to increasing MEEF, poses considerable challenges for a mask inspection engineer. Inspecting masks under their actinic-aerial image conditions would detect defects that are more likely to print under those exposure conditions. However, this also makes reviewing such defects in their low-contrast aerial images very challenging. On the other hand, inspecting masks under higher resolution inspection optics would allow for better viewing of defects post-inspection. However, such inspections generally would also detect many more defects, including printable and nuisance, thereby making it difficult to judge which are of real concern for printability on wafer. Often, an inspection engineer may choose to use Aerial and/or high resolution inspection modes depending on where in the process flow the mask is and the specific device-layer characteristics of the mask. Hence, a comprehensive approach is needed in handling defects both post-aerial and post-high resolution inspections. This analysis system is designed for the Applied Materials Aera™ mask inspection platform, all data reported was collected using the Aera.

  16. Polymerization of fluorescent analogue of plant actin in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg2+ and K+, the fluorescent actin analogue polymerized into filaments in vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.

  17. The core and conserved role of MAL is homeostatic regulation of actin levels.

    Science.gov (United States)

    Salvany, Lara; Muller, Julius; Guccione, Ernesto; Rørth, Pernille

    2014-05-15

    The transcription cofactor MAL is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, we provide genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics.

  18. Computational Analysis of the Transcriptional Regulation of the Actin Family

    Institute of Scientific and Technical Information of China (English)

    郑家顺; 吴加金; 孙之荣

    2002-01-01

    Transcriptional regulation is a very important regulatory step in the regulation of gene expression. Transcription factors (TFs) play an important role in controlling the temporal special specificity of gene expression. The regulation area of actin genes was analyzed statistically to predict the transcription factor binding sites in the regulatory area. A group of transcription factors located in most of the sequences is believed to play an important role in co-regulating the expression of actin genes.

  19. 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

  20. Course 6: Physics of Composite Cell Membrane and Actin Based Cytoskeleton

    Science.gov (United States)

    Sackmann, E.; Bausch, A. R.; Vonna, L.

    1 Architecture of composite cell membranes 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer 1.3 The actin cortex: Architecture and function 2 Physics of the actin based cytoskeleton 2.1 Actin is a living semiflexible polymer 2.2 Actin network as viscoelastic body 2.3 Correlation between macroscopic viscoelasticity and molecular 3 Heterogeneous actin gels in cells and biological function 3.1 Manipulation of actin gels 3.2 Control of organization and function of actin cortex by cell signalling 4 Micromechanics and microrheometry of cells 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways 6 On cells as adaptive viscoplastic bodies 7 Controll of cellular protrusions controlled by actin/myosin cortex

  1. Comparative mapping of the actin-binding protein 280 genes in human and mouse

    Energy Technology Data Exchange (ETDEWEB)

    Gariboldi, M.; Canzian, F.; Manenti, G.; De Gregorio, L. (Istituto Nazionale Tumori, Milan (Italy)); Maestrini, E.; Rivella, S. (Istituto di Genetica Biochimica ed Evoluzionistica, Pavia (Italy)); Chatterjee, A.; Herman, G.E. (Universita di Bari (Italy)); Archidiacono, N.; Antonacci, R. (Institute for Molecular Genetics, Houston, TX (United States)) (and others)

    1994-05-15

    Two genes encode actin-binding protein 280 isoforms. ABP-280 or filamin (FLN1) is present in the cytoskeleton of many cell types, whereas expression of FLN2 is limited to skeletal muscle and heart. FLN1 maps to human chromosome Xq28, and, by physical mapping in YAC clones, the authors have mapped the homologous murine locus (Fln1) to mouse chromosome X, in a region of syntenic homology with human chromosome X. They mapped FLN2 to human chromosome 7q32-q35 by analysis of somatic cell hybrids containing portions of chromosome 7, and, by using a mapping panel from an interspecific murine cross, they mapped the corresponding murine locus (Fln2) to murine chromosome 6 in a region homologous to human chromosome 7. 21 refs., 1 fig., 1 tab.

  2. Contractile units in disordered actomyosin bundles arise from F-actin buckling

    CERN Document Server

    Lenz, Martin; Gardel, Margaret L; Dinner, Aaron R

    2012-01-01

    Bundles of filaments and motors are central to contractility in cells. The classic example is striated muscle, where actomyosin contractility is mediated by highly organized sarcomeres which act as fundamental contractile units. However, many contractile bundles in vivo and in vitro lack sarcomeric organization. Here we propose a model for how contractility can arise in actomyosin bundles without sarcomeric organization and validate its predictions with experiments on a reconstituted system. In the model, internal stresses in frustrated arrangements of motors with diverse velocities cause filaments to buckle, leading to overall shortening. We describe the onset of buckling in the presence of stochastic actin-myosin detachment and predict that buckling-induced contraction occurs in an intermediate range of motor densities. We then calculate the size of the "contractile units" associated with this process. Consistent with these results, our reconstituted actomyosin bundles contract at relatively high motor dens...

  3. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    Science.gov (United States)

    Lomakin, Alexis J; Lee, Kun-Chun; Han, Sangyoon J; Bui, Duyen A; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-11-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient.

  4. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

    OpenAIRE

    1990-01-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is...

  5. Muscle strain (image)

    Science.gov (United States)

    A muscle strain is the stretching or tearing of muscle fibers. A muscle strain can be caused by sports, exercise, a ... something that is too heavy. Symptoms of a muscle strain include pain, tightness, swelling, tenderness, and the ...

  6. Refined distribution of myelinated trigeminal proprioceptive nerve fibres in Mueller's muscle as the mechanoreceptors to induce involuntary reflexive contraction of the levator and frontalis muscles.

    Science.gov (United States)

    Yuzuriha, Shunsuke; Matsuo, Kiyoshi; Hirasawa, Chihiro; Moriizumi, Tetsuji

    2009-11-01

    Stretching of mechanoreceptors in Mueller's muscle induces reflexive contraction of not only the levator muscle but also the frontalis muscle as two different eyelid-opening muscles. Previously, we reported that fine neural myelinated structures, acting as mechanoreceptors, were found in the proximal Mueller's muscle. Since there is a risk of misunderstanding that the middle and distal Mueller's muscle does not contain mechanoreceptors and can be invalidated or resected, the accurate distribution of myelinated trigeminal proprioceptive nerve fibres as mechanoreceptors in Mueller's muscle was refined horizontally in this study. We explored 10 whole Mueller's muscles between the levator muscle and the tarsus of the upper eyelids obtained from five Japanese cadavers. The specimens were serially sliced along the horizontal plane and stained with HE, S-100 protein to determine the presence of Schwann cells, and smooth muscle actin antibody to determine the presence of Mueller's smooth muscle fibres. Although all myelinated nerve fibres in the intermuscular connective tissues among the sympathetically innervated Mueller's multi-unit smooth muscle fibres may not correspond to the proprioceptive nerve fibres, the nerve bundles consisting of multiple myelinated nerve fibres were well distributed in the proximal Mueller's muscle, and single myelinated nerve fibres were well distributed in the middle and distal Mueller's muscle. We believe that the mechanoreceptors in Mueller's muscle consist of myelinated proprioceptive nerve fibres with nerve endings possibly attached to collagen fibres in the intermuscular connective tissues present among Mueller's smooth muscle fibres. As the myelinated nerve fibres innervate the middle and distal Mueller's muscle to a greater extent than those in the proximal Mueller's muscle, the former may be more important as mechanoreceptors than the latter and should not be invalidated or excised during surgery for treatment of blepharoptosis to

  7. Preservative solution for skeletal muscle biopsy samples

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    Kurt, Yasemin Gulcan; Kurt, Bulent; Ozcan, Omer; Topal, Turgut; Kilic, Abdullah; Muftuoglu, Tuba; Acikel, Cengizhan; Sener, Kenan; Sahiner, Fatih; Yigit, Nuri; Aydin, Ibrahim; Alay, Semih; Ekinci, Safak

    2015-01-01

    Context: Muscle biopsy samples must be frozen with liquid nitrogen immediately after excision and maintained at -80°C until analysis. Because of this requirement for tissue processing, patients with neuromuscular diseases often have to travel to centers with on-site muscle pathology laboratories for muscle biopsy sample excision to ensure that samples are properly preserved. Aim: Here, we developed a preservative solution and examined its protectiveness on striated muscle tissues for a minimum of the length of time that would be required to reach a specific muscle pathology laboratory. Materials and Methods: A preservative solution called Kurt-Ozcan (KO) solution was prepared. Eight healthy Sprague-Dawley rats were sacrificed; striated muscle tissue samples were collected and divided into six different groups. Muscle tissue samples were separated into groups for morphological, enzyme histochemical, molecular, and biochemical analysis. Statistical method used: Chi-square and Kruskal Wallis tests. Results: Samples kept in the KO and University of Wisconsin (UW) solutions exhibited very good morphological scores at 3, 6, and 18 hours, but artificial changes were observed at 24 hours. Similar findings were observed for the evaluated enzyme activities. There were no differences between the control group and the samples kept in the KO or UW solution at 3, 6, and 18 hours for morphological, enzyme histochemical, and biochemical features. The messenger ribonucleic acid (mRNA) of β-actin gene was protected up to 6 hours in the KO and UW solutions. Conclusion: The KO solution protects the morphological, enzyme histochemical, and biochemical features of striated muscle tissue of healthy rats for 18 hours and preserves the mRNA for 6 hours. PMID:26019417

  8. Preservative solution for skeletal muscle biopsy samples

    Directory of Open Access Journals (Sweden)

    Yasemin Gulcan Kurt

    2015-01-01

    Full Text Available Context : Muscle biopsy samples must be frozen with liquid nitrogen immediately after excision and maintained at -80 o C until analysis. Because of this requirement for tissue processing, patients with neuromuscular diseases often have to travel to centers with on-site muscle pathology laboratories for muscle biopsy sample excision to ensure that samples are properly preserved. Aim: Here, we developed a preservative solution and examined its protectiveness on striated muscle tissues for a minimum of the length of time that would be required to reach a specific muscle pathology laboratory. Materials and Methods: A preservative solution called Kurt-Ozcan (KO solution was prepared. Eight healthy Sprague-Dawley rats were sacrificed; striated muscle tissue samples were collected and divided into six different groups. Muscle tissue samples were separated into groups for morphological, enzyme histochemical, molecular, and biochemical analysis. Statistical method used: Chi-square and Kruskal Wallis tests. Results: Samples kept in the KO and University of Wisconsin (UW solutions exhibited very good morphological scores at 3, 6, and 18 hours, but artificial changes were observed at 24 hours. Similar findings were observed for the evaluated enzyme activities. There were no differences between the control group and the samples kept in the KO or UW solution at 3, 6, and 18 hours for morphological, enzyme histochemical, and biochemical features. The messenger ribonucleic acid (mRNA of β-actin gene was protected up to 6 hours in the KO and UW solutions. Conclusion: The KO solution protects the morphological, enzyme histochemical, and biochemical features of striated muscle tissue of healthy rats for 18 hours and preserves the mRNA for 6 hours.

  9. Actin-binding Rho activating protein is expressed in the central nervous system of normal adult rats

    Institute of Scientific and Technical Information of China (English)

    Lihua Liu; Jutta Schaper; Mingying Luo; Baolin Yang; Xiaoqiong Wu; Wu Zhu; Yinglu Guan; Weijun Cai; Kerstin Troidl; Wolfgang Schaper

    2012-01-01

    Previous studies show that actin-binding Rho activating protein (Abra) is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence.Results show ed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with pos itive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and w hite matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.

  10. Changes of smooth muscle contractile filaments in small bowel atresia

    Institute of Scientific and Technical Information of China (English)

    Stefan Gfroerer; Henning Fiegel; Priya Ramachandran; Udo Rolle; Roman Metzger

    2012-01-01

    AIM:To investigate morphological changes of intestinal smooth muscle contractile fibres in small bowel atresia patients.METHODS:Resected small bowel specimens from small bowel atresia patients (n =12) were divided into three sections (proximal,atretic and distal).Standard histology hematoxylin-eosin staining and enzyme immunohistochemistry was performed to visualize smooth muscle contractile markers α-smooth muscle actin (SMA) and desmin using conventional paraffin sections of the proximal and distal bowel.Small bowel from agematched patients (n =2) undergoing Meckel's diverticulum resection served as controls.RESULTS:The smooth muscle coat in the proximal bowel of small bowel atresia patients was thickened compared with control tissue,but the distal bowel was unchanged.Expression of smooth muscle contractile fibres SMA and desmin within the proximal bowel was slightly reduced compared with the distal bowel and control tissue.There were no major differences in the architecture of the smooth muscle within the proximal bowel and the distal bowel.The proximal and distal bowel in small bowel atresia patients revealed only minimal differences regarding smooth muscle morphology and the presence of smooth muscle contractile filament markers.CONCLUSION:Changes in smooth muscle contractile filaments do not appear to play a major role in postoperative motility disorders in small bowel atresia.

  11. Phosphoproteomic analysis of aged skeletal muscle.

    Science.gov (United States)

    Gannon, Joan; Staunton, Lisa; O'Connell, Kathleen; Doran, Philip; Ohlendieck, Kay

    2008-07-01

    One of the most important post-translational modifications is represented by phosphorylation on tyrosine, threonine and serine residues. Since abnormal phosphorylation is associated with various pathologies, it was of interest to perform a phosphoproteomic profiling of age-related skeletal muscle degeneration. We used the fluorescent phospho-specific Pro-Q Diamond dye to determine whether changes in the overall phosphorylation of the soluble skeletal muscle proteome differs significantly between young adult and senescent fibres. As an established model system of sarcopenia, we employed 30-month-old rat gastrocnemius fibres. Following the mass spectrometric identification of 59 major 2-D phosphoprotein landmark spots, the fluorescent dye staining survey revealed that 22 muscle proteins showed a differential expression pattern between 3-month- and 30-month-old muscle. Increased phosphorylation levels were shown for myosin light chain 2, tropomyosin alpha, lactate dehydrogenase, desmin, actin, albumin and aconitase. In contrast, decreased phospho-specific dye binding was observed for cytochrome c oxidase, creatine kinase and enolase. Thus, aging-induced alterations in phosphoproteins appear to involve the contractile machinery and the cytoskeleton, as well as the cytosolic and mitochondrial metabolism. This confirms that sarcopenia of old age is a complex neuromuscular pathology that is associated with drastic changes in the abundance and structure of key muscle proteins. PMID:18575773

  12. On the thermodynamics of smooth muscle contraction

    Science.gov (United States)

    Stålhand, Jonas; McMeeking, Robert M.; Holzapfel, Gerhard A.

    2016-09-01

    Cell function is based on many dynamically complex networks of interacting biochemical reactions. Enzymes may increase the rate of only those reactions that are thermodynamically consistent. In this paper we specifically treat the contraction of smooth muscle cells from the continuum thermodynamics point of view by considering them as an open system where matter passes through the cell membrane. We systematically set up a well-known four-state kinetic model for the cross-bridge interaction of actin and myosin in smooth muscle, where the transition between each state is driven by forward and reverse reactions. Chemical, mechanical and energy balance laws are provided in local forms, while energy balance is also formulated in the more convenient temperature form. We derive the local (non-negative) production of entropy from which we deduce the reduced entropy inequality and the constitutive equations for the first Piola-Kirchhoff stress tensor, the heat flux, the ion and molecular flux and the entropy. One example for smooth muscle contraction is analyzed in more detail in order to provide orientation within the established general thermodynamic framework. In particular the stress evolution, heat generation, muscle shorting rate and a condition for muscle cooling are derived.

  13. Ca2+-Dependent Regulations and Signaling in Skeletal Muscle: From Electro-Mechanical Coupling to Adaptation

    Directory of Open Access Journals (Sweden)

    Sebastian Gehlert

    2015-01-01

    Full Text Available Calcium (Ca2+ plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca2+ is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca2+ regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca2+-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca2+ ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca2+ ions in adult muscle but also highlight recent findings of critical Ca2+-dependent mechanisms essential for skeletal muscle-regulation and maintenance.

  14. DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

    Science.gov (United States)

    Molnár, Imre; Migh, Ede; Szikora, Szilárd; Kalmár, Tibor; Végh, Attila G; Deák, Ferenc; Barkó, Szilvia; Bugyi, Beáta; Orfanos, Zacharias; Kovács, János; Juhász, Gábor; Váró, György; Nyitrai, Miklós; Sparrow, John; Mihály, József

    2014-02-01

    During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

  15. DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

    Directory of Open Access Journals (Sweden)

    Imre Molnár

    2014-02-01

    Full Text Available During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

  16. Structure of a pentavalent G-actin*MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator.

    Science.gov (United States)

    Mouilleron, Stéphane; Langer, Carola A; Guettler, Sebastian; McDonald, Neil Q; Treisman, Richard

    2011-06-14

    Subcellular localization of the actin-binding transcriptional coactivator MRTF-A is controlled by its interaction with monomeric actin (G-actin). Signal-induced decreases in G-actin concentration reduce MRTF-A nuclear export, leading to its nuclear accumulation, whereas artificial increases in G-actin concentration in resting cells block MRTF-A nuclear import, retaining it in the cytoplasm. This regulation is dependent on three actin-binding RPEL motifs in the regulatory domain of MRTF-A. We describe the structures of pentavalent and trivalent G-actin•RPEL domain complexes. In the pentavalent complex, each RPEL motif and the two intervening spacer sequences bound an actin monomer, forming a compact assembly. In contrast, the trivalent complex lacked the C-terminal spacer- and RPEL-actins, both of which bound only weakly in the pentavalent complex. Cytoplasmic localization of MRTF-A in unstimulated fibroblasts also required binding of G-actin to the spacer sequences. The bipartite MRTF-A nuclear localization sequence was buried in the pentameric assembly, explaining how increases in G-actin concentration prevent nuclear import of MRTF-A. Analyses of the pentavalent and trivalent complexes show how actin loads onto the RPEL domain and reveal a molecular mechanism by which actin can control the activity of one of its binding partners.

  17. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Dov, Nadav [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel); Korenstein, Rafi, E-mail: korens@post.tau.ac.il [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel)

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  18. Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell.

    Science.gov (United States)

    Guevorkian, Karine; Manzi, John; Pontani, Léa-Lætitia; Brochard-Wyart, Françoise; Sykes, Cécile

    2015-12-15

    Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.

  19. Optogenetics to target actin-mediated synaptic loss in Alzheimer's

    Science.gov (United States)

    Zahedi, Atena; DeFea, Kathryn; Ethell, Iryna

    2013-03-01

    Numerous studies in Alzheimer's Disease (AD) animal models show that overproduction of Aβ peptides and their oligomerization can distort dendrites, damage synapses, and decrease the number of dendritic spines and synapses. Aβ may trigger synapse loss by modulating activity of actin-regulating proteins, such as Rac1 and cofilin. Indeed, Aβ1-42 oligomers can activate actin severing protein cofilin through calcineurin-mediated activation of phosphatase slingshot and inhibit an opposing pathway that suppresses cofilin phosphorylation through Rac-mediated activation of LIMK1. Excessive activation of actin-severing protein cofilin triggers the formation of a non-dynamic actin bundles, called rods that are found in AD brains and cause loss of synapses. Hence, regulation of these actin-regulating proteins in dendritic spines could potentially provide useful tools for preventing the synapse/spine loss associated with earlier stages of AD neuropathology. However, lack of spatiotemporal control over their activity is a key limitation. Recently, optogenetic advancements have provided researchers with convenient light-activating proteins such as photoactivatable Rac (PARac). Here, we transfected cultured primary hippocampal neurons and human embryonic kidney (HEK) cells with a PARac/ mCherry-containing plasmid and the mCherry-positive cells were identified and imaged using an inverted fluorescence microscope. Rac1 activation was achieved by irradiation with blue light (480nm) and live changes in dendritic spine morphology were observed using mCherry (587nm). Rac activation was confirmed by immunostaining for phosphorylated form of effector proteinP21 protein-activated kinase 1 (PAK1) and reorganization of actin. Thus, our studies confirm the feasibility of using the PA-Rac construct to trigger actin re-organization in the dendritic spines.

  20. Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms

    Directory of Open Access Journals (Sweden)

    Yu Jun

    2009-03-01

    Full Text Available Abstract Background Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking. Results We constructed a cDNA library from Aphonopelma sp. (Tarantula skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags from randomly picked clones. EST analysis showed 305 unigenes, among which 81 had more than 2 ESTs. Twenty abundant unigenes had matches to skeletal muscle-related genes including actin, myosin, tropomyosin, troponin-I, T and C, paramyosin, muscle LIM protein, muscle protein 20, a-actinin and tandem Ig/Fn motifs (found in giant sarcomere-related proteins. Matches to myosin light chain kinase and calponin were also identified. These results support the existence of both actin-linked and myosin-linked regulation in tarantula skeletal muscle. We have predicted full-length as well as partial cDNA sequences both experimentally and computationally for myosin heavy and light chains, actin, tropomyosin, and troponin-I, T and C, and have deduced the putative peptides. A preliminary analysis of the structural and functional properties was also carried out. Sequence similarities suggested multiple isoforms of most myofibrillar proteins, supporting the generality of multiple isoforms known from previous muscle sequence studies. This may be related to a mix of muscle fiber types. Conclusion The present study serves as a basis for defining the transcriptome of tarantula skeletal muscle, for future in vitro expression of tarantula proteins, and for interpreting structural and functional observations in this model species.

  1. Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

    Institute of Scientific and Technical Information of China (English)

    Chun-Hai Dong; Wei-Ping Tang; Jia-Yao Liu

    2013-01-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin,and is directly involved in the depolymerization of actin filaments.To better understand the actin binding sites of the Arabidopsis thaliana L.AtADF1,we generated mutants of AtADF1 and investigated their functions in vitro and in vivo.Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G-and F-actin binding.The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A,R137/A) form another actin binding site that is important for F-actin binding.Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L.plants overexpressing these mutants,we analyzed how these mutant proteins regulate actin organization and affect seedling growth.Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional,unless the affinity foractin monomers is also affected.The G-actin binding activity of the ADF plays an essential role in actin binding,depolymerization of actin polymers,and therefore in the control of actin organization.

  2. Interactions between the yeast SM22 homologue Scp1 and actin demonstrate the importance of actin bundling in endocytosis.

    Science.gov (United States)

    Gheorghe, Dana M; Aghamohammadzadeh, Soheil; Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Winder, Steve J; Ayscough, Kathryn R

    2008-05-30

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process. PMID:18400761

  3. State transitions of actin cortices in vitro and in vivo

    Science.gov (United States)

    Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

    Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

  4. Cortactin promotes exosome secretion by controlling branched actin dynamics.

    Science.gov (United States)

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

    2016-07-18

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

  5. 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.

  6. Antenna Mechanism of Length Control of Actin Cables.

    Directory of Open Access Journals (Sweden)

    Lishibanya Mohapatra

    2015-06-01

    Full Text Available Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This "antenna mechanism" involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control.

  7. Emerging roles of actin cytoskeleton regulating enzymes in drug addiction: Actin or reactin’?

    Science.gov (United States)

    Rothenfluh, Adrian; Cowan, Christopher W.

    2013-01-01

    Neurons rely on their cytoskeleton to give them shape and stability, and on cytoskeletal dynamics for growth and synaptic plasticity. Because drug addiction is increasingly seen as the inappropriate learning of strongly reinforcing stimuli, the role of the cytoskeleton in shaping drug memories has been of increasing interest in recent years. Does the cytoskeleton have an active role in shaping these memories, and to what extent do alterations in the cytoskeleton reflect the acute actions of drug exposure, or homeostatic reactions to the chronic exposure to drugs of abuse? Here we will review recent advances in understanding the role of the cytoskeleton in the development of drug addiction, with a focus on actin filaments, as they have been studied in greater detail. PMID:23428655

  8. Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin

    OpenAIRE

    Emma J McGhie; Hayward, Richard D.; Koronakis, Vassilis

    2001-01-01

    Pathogen-induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmon ella by non-phagocytic intestinal epithelial cells. Two Salmonella actin-binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F-actin bundling, we demonstrate that SipA stimulates substantially SipC-mediated nucleation of actin polymerization....

  9. Smooth muscle cells largely develop independently of functional hemogenic endothelium

    Directory of Open Access Journals (Sweden)

    Monika Stefanska

    2014-01-01

    Full Text Available Vascular smooth muscle cells represent a major component of the cardiovascular system. In vitro studies have shown that FLK1+ cells derived from embryonic stem (ES cells can differentiate into both endothelial and smooth muscle cells. These FLK1+ cells also contain a mesodermal precursor, the hemangioblast, able to produce endothelial, blood and smooth muscle cells. The generation of blood precursors from the hemangioblast was recently shown to occur through a transient cell population of specialised endothelium, a hemogenic endothelium. To date, the lineage relationship between this cell population and smooth muscle cell progenitors has not been investigated. In this study, we generated a reporter ES cell line in which expression of the fluorescent protein H2B-VENUS is driven by the α-smooth muscle actin (α-SMA regulatory sequences. We demonstrated that this reporter cell line efficiently trace smooth muscle development during ES cell differentiation. Although some smooth muscle cells are associated with broad endothelial development, we established that smooth muscle cells are mostly generated independently from a specialised functional hemogenic endothelium. This study provides new and important insights into hematopoietic and vascular development, which may help in driving further progress towards the development of bioengineered vascular grafts for regenerative medicine.

  10. Regional blood flow in the normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not by capillary pericytes

    OpenAIRE

    Hill, Robert A.; Tong, Lei; Yuan, Peng; Murikinati, Sasidhar; Gupta, Shobhana; Grutzendler, Jaime

    2015-01-01

    The precise regulation of cerebral blood flow is critical for normal brain function and its disruption underlies many neuropathologies. The extent to which smooth muscle-covered arterioles or pericyte-covered capillaries control vasomotion during neurovascular coupling remains controversial. We found that capillary pericytes in mice and humans do not express smooth muscle actin and are morphologically and functionally distinct from adjacent precapillary smooth muscle cells (SMCs). Using optic...

  11. Caveolin-3 promotes a vascular smooth muscle contractile phenotype

    Directory of Open Access Journals (Sweden)

    Jorge L. Gutierrez-Pajares

    2015-06-01

    Full Text Available Epidemiological studies have demonstrated the importance of cardiovascular diseases in Western countries. Among the cell types associated with a dysfunctional vasculature, smooth muscle cells are believed to play an essential role in the development of these illnesses. Vascular smooth muscle cells are key regulators of the vascular tone and also have an important function in the development of atherosclerosis and restenosis. While in the normal vasculature contractile smooth muscle cells are predominant, in atherosclerotic vascular lesions, synthetic cells migrate toward the neointima, proliferate, and synthetize extracellular matrix proteins. In the present study, we have examined the role of caveolin-3 in the regulation of smooth muscle cell phenotype. Caveolin-3 is expressed in vivo in normal arterial smooth muscle cells, but its expression appears to be lost in cultured smooth muscle cells. Our data show that caveolin-3 expression in the A7r5 smooth muscle cell line is associated with increased expression of contractility markers such as smooth muscleactin, smooth muscle myosin heavy chain but decreased expression of the synthetic phenotype markers such as p-Elk and Klf4. Moreover, we also show that caveolin-3 expression can reduce proliferation upon treatment with LDL or PDGF. Finally, we show that caveolin-3-expressing smooth muscle cells are less sensitive to apoptosis than control cells upon treatment with oxidized LDL. Taken together, our data suggest that caveolin-3 can regulate the phenotypic switch between contractile and synthetic smooth muscle cells. A better understanding of the factors regulating caveolin-3 expression and function in this cell type will permit the development of a better comprehension of the factors regulating smooth muscle function in atherosclerosis and restenosis.

  12. New Insights into Dynamic Actin-Based Chloroplast Photorelocation Movement

    Institute of Scientific and Technical Information of China (English)

    Sam-Geun Kong; Masamitsu Wada

    2011-01-01

    Chloroplast movement is essential for plants to survive under various environmental light conditions.Phototropins-plant-specific blue-light-activated receptor kinases-mediate the response by perceiving light intensity and direction.Recently,novel chloroplast actin (cp-actin) filaments have been identified as playing a pivotal role in the directional chloroplast photorelocation movement.Encouraging progress has recently been made in this field of research through molecular genetics and cell biological analyses.This review describes factors that have been identified as being involved in chloroplast movement and their roles in the regulation of cp-actin filaments,thus providing a basis for reflection on their biochemical activities and functions.

  13. Dissecting principles governing actin assembly using yeast extracts.

    Science.gov (United States)

    Michelot, Alphée; Drubin, David G

    2014-01-01

    In this chapter, we describe recent protocols that we have developed to trigger actin assembly and actin-based motility in yeast cell extracts. Our method allows for the fast preparation of yeast extracts that are competent in dynamic assembly of distinct actin filament structures of biologically appropriate protein composition. Compared to previous extract-based systems using other eukaryotic cell types, yeast provides a unique advantage for combining reconstituted assays with the preparation of extracts from genetically modified yeast strains. We present a global strategy for dissecting the functions of individual proteins, where the activities of the proteins are analyzed in systems of variable complexity, ranging from simple mixtures of pure proteins to the full complexity of a cell's cytoplasm.

  14. Formation of actin networks in microfluidic concentration gradients

    Science.gov (United States)

    Strelnikova, Natalja; Herren, Florian; Schoenenberger, Cora-Ann; Pfohl, Thomas

    2016-05-01

    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.

  15. 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.

  16. Impaired Organization and Function of Myofilaments in Single Muscle Fibers from a Mouse Model of Pompe Disease

    Energy Technology Data Exchange (ETDEWEB)

    Xu, S.; Galperin, M; Melvin, G; Horowits, R; Raben, N; Plotz, P; Yu, L

    2010-01-01

    Pompe disease, a deficiency of lysosomal acid {alpha}-glucosidase, is a disorder of glycogen metabolism that can affect infants, children, or adults. In all forms of the disease, there is progressive muscle pathology leading to premature death. The pathology is characterized by accumulation of glycogen in lysosomes, autophagic buildup, and muscle atrophy. The purpose of the present investigation was to determine if myofibrillar dysfunction in Pompe disease contributes to muscle weakness beyond that attributed to atrophy. The study was performed on isolated myofibers dissected from severely affected fast glycolytic muscle in the {alpha}-glucosidase knockout mouse model. Psoas muscle fibers were first permeabilized, so that the contractile proteins could be directly relaxed or activated by control of the composition of the bathing solution. When normalized by cross-sectional area, single fibers from knockout mice produced 6.3 N/cm{sup 2} of maximum Ca{sup 2+}-activated tension compared with 12.0 N/cm{sup 2} produced by wild-type fibers. The total protein concentration was slightly higher in the knockout mice, but concentrations of the contractile proteins myosin and actin remained unchanged. Structurally, X-ray diffraction showed that the actin and myosin filaments, normally arranged in hexagonal arrays, were disordered in the knockout muscle, and a lower fraction of myosin cross bridges was near the actin filaments in the relaxed muscle. The results are consistent with a disruption of actin and myosin interactions in the knockout muscles, demonstrating that impaired myofibrillar function contributes to weakness in the diseased muscle fibers.

  17. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

    Science.gov (United States)

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-12-01

    Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed.

  18. Filament attachment dynamics in actin-based propulsion

    CERN Document Server

    Katz, J I

    2005-01-01

    Theory and experiment have established that F-actin filaments are strongly attached to the intracellular parasites (such as Listeria) they propel with ``comet tails''. We consider the implications of these observations for propulsion. By calculating the motion produced in various models of attachment and comparing to experiment we demonstrate that the attachment must be sliding rather than hinged. By modeling experiments on ActA-coated spheres we draw conclusions regarding the interaction between F-actin and their surfaces that may also be applicable to living systems.

  19. Oral nicotinamide and actinic keratosis: a supplement success story.

    Science.gov (United States)

    Kim, Burcu; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Nicotinamide has shown potential as a safe and effective intervention for the prevention of malignant and premalignant skin lesions. Recent studies have shown that nicotinamide, in both oral and topical forms, is able to prevent ultraviolet-induced immunosuppression in humans [1,2,3] and mice [4,5]. Immunosuppression is a known factor for the progression of premalignant lesions, such as actinic keratosis [6]. Murine studies have shown that nicotinamide is also able to protect against photocarcinogenesis [4,5]. Preliminary human studies suggest that nicotinamide may help prevent skin cancers and enhance the regression of actinic keratoses.

  20. Oral nicotinamide and actinic keratosis: a supplement success story.

    Science.gov (United States)

    Kim, Burcu; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Nicotinamide has shown potential as a safe and effective intervention for the prevention of malignant and premalignant skin lesions. Recent studies have shown that nicotinamide, in both oral and topical forms, is able to prevent ultraviolet-induced immunosuppression in humans [1,2,3] and mice [4,5]. Immunosuppression is a known factor for the progression of premalignant lesions, such as actinic keratosis [6]. Murine studies have shown that nicotinamide is also able to protect against photocarcinogenesis [4,5]. Preliminary human studies suggest that nicotinamide may help prevent skin cancers and enhance the regression of actinic keratoses. PMID:25561219

  1. 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

  2. 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...

  3. 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.

  4. Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary

    Directory of Open Access Journals (Sweden)

    Lundberg Mathias

    2007-12-01

    Full Text Available Abstract Background Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation in vivo is unclear. Results We have studied glutathionylation of α- and β-actin in vitro using an enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. α- and β-actin were both glutathionylated when incubated with reduced glutathione (GSH combined with diamide as a thiol oxidant. However, β-actin was also glutathionylated by both glutathione disulfide (GSSG and GSH in the absence of diamide whereas α-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of β-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. β-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in β-actin. Conclusion We conclude that glutathionylation of β-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.

  5. Rac1- a novel regulator of contraction-stimulated glucose uptake in skeletal muscle

    DEFF Research Database (Denmark)

    Sylow, Lykke; Møller, Lisbeth L V; Kleinert, Maximilian;

    2014-01-01

    Muscle contraction stimulates muscle glucose uptake by facilitating translocation of the glucose transporter 4 from intracellular locations to the cell surface, which allows for diffusion of glucose into the myofibers. However, the intracellular mechanisms regulating this process are not well....../contraction-stimulated glucose uptake in skeletal muscle, since muscle-specific Rac1 knockout mice display reduced ex vivo contraction- and in vivo exercise-stimulated glucose uptake in skeletal muscle. The molecular mechanisms by which Rac1 regulate glucose uptake is presently unknown. However, recent studies link Rac1...... to the actin cytoskeleton, the small GTPase RalA, and/or free radical production, which have previously been shown to be regulators of glucose uptake in muscle. We propose a model in which Rac1 is activated by contraction- and exercise-induced stretch signals and that Rac1 in conjunction with other signaling...

  6. Pattern formation in polymerising actin flocks: spirals, spots and waves without nonlinear chemistry

    CERN Document Server

    Goff, Thomas Le; Marenduzzo, Davide

    2016-01-01

    We propose a model solely based on actin treadmilling and polymerisation which describes many characteristic states of actin wave formation: spots, spirals and travelling waves. In our model, as in experiments on cell recovering motility following actin depolymerisation, we choose an isotropic low density initial condition; polymerisation of actin filaments then raises the density towards the Onsager threshold where they align. We show that this alignment, in turn, destabilizes the isotropic phase and generically induces transient actin spots or spirals as part of the dynamical pathway towards a polarized phase which can either be uniform or consist of a series of actin-wave trains (flocks). Our results uncover a universal route to actin wave formation in the absence of any system specific nonlinear biochemistry, and it may help understand the mechanism underlying the observation of actin spots and waves in vivo. They also suggest a minimal setup to design similar patterns in vitro.

  7. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    Science.gov (United States)

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

  8. Contribution of elastic tissues to the mechanics and energetics of muscle function during movement.

    Science.gov (United States)

    Roberts, Thomas J

    2016-01-01

    Muscle force production occurs within an environment of tissues that exhibit spring-like behavior, and this elasticity is a critical determinant of muscle performance during locomotion. Muscle force and power output both depend on the speed of contraction, as described by the isotonic force-velocity curve. By influencing the speed of contractile elements, elastic structures can have a profound effect on muscle force, power and work. In very rapid movements, elastic mechanisms can amplify muscle power by storing the work of muscle contraction slowly and releasing it rapidly. When energy must be dissipated rapidly, such as in landing from a jump, energy stored rapidly in elastic elements can be released more slowly to stretch muscle contractile elements, reducing the power input to muscle and possibly protecting it from damage. Elastic mechanisms identified so far rely primarily on in-series tendons, but many structures within muscles exhibit spring-like properties. Actomyosin cross-bridges, actin and myosin filaments, titin, and the connective tissue scaffolding of the extracellular matrix all have the potential to store and recover elastic energy during muscle contraction. The potential contribution of these elements can be assessed from their stiffness and estimates of the strain they undergo during muscle function. Such calculations provide boundaries for the possible roles these springs might play in locomotion, and may help to direct future studies of the uses of elastic elements in muscle.

  9. Patterning the dorsal longitudinal flight muscles (DLM) of Drosophila: insights from the ablation of larval scaffolds

    Science.gov (United States)

    Fernandes, J. J.; Keshishian, H.

    1996-01-01

    The six Dorsal Longitudinal flight Muscles (DLMs) of Drosophila develop from three larval muscles that persist into metamorphosis and serve as scaffolds for the formation of the adult fibers. We have examined the effect of muscle scaffold ablation on the development of DLMs during metamorphosis. Using markers that are specific to muscle and myoblasts we show that in response to the ablation, myoblasts which would normally fuse with the larval muscle, fuse with each other instead, to generate the adult fibers in the appropriate regions of the thorax. The development of these de novo DLMs is delayed and is reflected in the delayed expression of erect wing, a transcription factor thought to control differentiation events associated with myoblast fusion. The newly arising muscles express the appropriate adult-specific Actin isoform (88F), indicating that they have the correct muscle identity. However, there are frequent errors in the number of muscle fibers generated. Ablation of the larval scaffolds for the DLMs has revealed an underlying potential of the DLM myoblasts to initiate de novo myogenesis in a manner that resembles the mode of formation of the Dorso-Ventral Muscles, DVMs, which are the other group of indirect flight muscles. Therefore, it appears that the use of larval scaffolds is a superimposition on a commonly used mechanism of myogenesis in Drosophila. Our results show that the role of the persistent larval muscles in muscle patterning involves the partitioning of DLM myoblasts, and in doing so, they regulate formation of the correct number of DLM fibers.

  10. Muscle strain treatment

    Science.gov (United States)

    Treatment - muscle strain ... Question: How do you treat a muscle strain ? Answer: Rest the strained muscle and apply ice for the first few days after the injury. Anti-inflammatory medicines or acetaminophen ( ...

  11. Ultrastructural study of muscles fibers in tick Hyalomma (Hyalomma) anatolicum anatolicum (Ixodoidea: Ixodidae).

    Science.gov (United States)

    Bughdadi, Faisal A

    2010-09-01

    In the present study, ticks were obtained from a colony maintained at 28 degrees C and 75% relative humidity in at the Department of Biology, University College Umm Al-Qura University, Saudi Arabia and the Transmission Electron Microscope technique (TEM) was used to describes the ultrastructure and description of muscle of the of ixodid tick Hyalomma (Hyalomma) anatolicum anatolicum. The results showed that muscles of the unfed ticks Hyalomma (Hyalomma) anatolicum anatolicum in longitudinal sections are spindle-shaped to cylindrical muscle fibers. In the unfed nymph Hyalomma (Hyalomma) anatolicum anatolicum skeletal and visceral muscles are distinguished according to structure, function and position. These muscles include the capitulum, dorsoventral and leg oblique muscles. All muscle fibers are ensheathed (covered by sheath) in a sarcolemma. Their muscle fibers have striated pattern of successive sarcomeres whose thick myosin filaments are surrounded by orbitals of up to 12 thin actin filaments. The cytoplasm of the epidermal cell appears largely devoted with complicated microtubules present in parallel with long axis of adjacent muscle fibers. The cell membrane invaginates into tubular system extending deeply into the sarcoplasm and closely associated to cisternae of sarcoplasmic reticulum. The tubular system and sarcoplasmic reticulum forming two-membered (dyads) are considered to be the main route of calcium ions whose movement are synchronized with the motor impulse to control muscles contraction. In the sarcoplasm two types of muscle fibers are recognized according to thickness and density and mitochondrial size, distribution and population. Both skeletal and visceral muscles are invaginated by tracheoles and innervated by nerve axons containing synaptic vesicles. The actin and myosin filaments are slightly interrupted and the tubular system sarcoplasmic reticulum is well demonstrated. PMID:21313907

  12. Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Feng Shi

    Full Text Available Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1

  13. Actin based processes that could determine the cytoplasmic architecture of plant cells

    OpenAIRE

    Honing; Emons, A.M.C.; Ketelaar, M.J.

    2007-01-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells that is likely to depend on actin-based force generation is the organisation of the cytoplasm. We compare the function of actin binding proteins of three well-studied mammalian models that depend on...

  14. Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos

    OpenAIRE

    1992-01-01

    In the syncytial blastoderm stage of Drosophila embryogenesis, dome- shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically re...

  15. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  16. Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

    OpenAIRE

    Groen, Christopher M.; Spracklen, Andrew J.; Fagan, Tiffany N.; Tootle, Tina L.

    2012-01-01

    Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. He...

  17. 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.

  18. 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

  19. Photodynamic therapy for actinic keratosis in organ transplant patients

    DEFF Research Database (Denmark)

    Basset-Seguin, N; Baumann Conzett, K; Gerritsen, M J P;

    2013-01-01

    Background The incidence of actinic keratoses (AK) and non-melanoma skin cancer (NMSC) in organ transplant recipients (OTRs) is significantly higher than in immunocompetent patients. Rates of progression and recurrence following treatment are higher too, in part due to the effects...

  20. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    Science.gov (United States)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  1. Mical links semaphorins to F-actin disassembly

    NARCIS (Netherlands)

    Hung, R.J.; Yazdani, U.; Yoon, J.; Wu, H.; Yang, T.; Gupta, N.; Huang, Z.; Berkel, van W.J.H.; Terman, J.R.

    2010-01-01

    How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer

  2. Actin-Dependent Alterations of Dendritic Spine Morphology in Shankopathies

    Science.gov (United States)

    Sarowar, Tasnuva

    2016-01-01

    Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains—the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, and βPIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiple in vitro and in vivo models. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling.

  3. Immortalized CNS pericytes are quiescent smooth muscle actin-negative and pluripotent

    OpenAIRE

    Dore-Duffy, Paula; Mehedi, Afroza; Wang, Xueqian; Bradley, Michael; Trotter, Richard; Gow, Alexander

    2011-01-01

    Despite their identification more than 100 years ago by the French scientist Charles-Marie Benjamin Rouget, microvascular pericytes have proven difficult to functionally characterize, due in part to their relatively low numbers and the lack of specific cell markers. However, recent progress is beginning to shed light on the diverse biological functions of these cells. Pericytes are thought to be involved in regulating vascular homeostasis and hemostasis as well as serving as a local source of...

  4. Relaxin stimulates MMP-2 and α-smooth muscle actin expression by human periodontal ligament cells

    NARCIS (Netherlands)

    Henneman, S.; Bildt, M.M.; Groot, J. de; Kuijpers-Jagtman, A.M.; Von den Hoff, J.W.

    2008-01-01

    The main cells in the periodontal ligament (PDL) are the fibroblasts, which play an important role in periodontal remodelling. Matrix metalloproteinases (MMPs) are largely responsible for the degradation of extracellular matrix proteins in the PDL. Previous studies have indicated that MMP production

  5. Multidrug Resistance-Related Protein 1 (MRP1) Function and Localization Depend on Cortical Actin

    NARCIS (Netherlands)

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

    2011-01-01

    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 resu

  6. Dynamic organization of actin cytoskeleton during the polarity formation and germination of pollen protoplasts

    Institute of Scientific and Technical Information of China (English)

    XU Xia; Zl Huijun; SUN Yina; REN Haiyun

    2004-01-01

    The formation of the polarity of pollen protoplast and the dynamics of actin cytoskeleton were observed by non-fixation, Alexa-Phalloidin probing and confocal laser scanning microscopy. Our results showed that the protoplast obtained from stored pollen contained numerous crystalline fusiform bodies to constitute a storage form of actin. When dormant pollen was hydrated, the actin cytoskeleton forms a fine network spreading uniformly in the protoplast. In the process of polarity formation and germination of pollen protoplast, actin filaments marshaled slowly to the brim, and then formed multilayer continuous actin filament bundles surrounding the cortical of the protoplast. When the protoplast was exposed to actin filament-disrupting drugs, such as Latrunculin A and Cytochalasin D, continuously arranged actin bundles were disturbed and in this condition, the protoplast could not germinate. But when exposed to actin filament stabiling drug-phalliodin, the dynamics of actin filaments in the protoplasts behaved normally and the protoplasts could germinate normally. These results were also confirmed by the pharmacology experiments on pollen grains. And when Latrunculin A or Cytochalasin D was washed off, the ratio of pollen germination was resumed partly. All the results above show that the dynamic organization of the actin cytoskeleton are critical in the cell polarity formation and germination of pollen protoplast, and that the reorganization of actin cytoskeleton is mainly due to the rearrangement of actin filament arrays.

  7. 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 conside

  8. Cysteine-rich protein 1 (CRP1 regulates actin filament bundling

    Directory of Open Access Journals (Sweden)

    Fraley Tamara S

    2005-12-01

    Full Text Available Abstract Background Cysteine-rich protein 1 (CRP1 is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified. Results CRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of α-actinin, CRP1 was found to stabilize the interaction of α-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with α-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts. Conclusion CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of α-actinin with actin filament bundles.

  9. Gamma-actin is involved in regulating centrosome function and mitotic progression in cancer cells.

    Science.gov (United States)

    Po'uha, Sela T; Kavallaris, Maria

    2015-01-01

    Reorganization of the actin cytoskeleton during mitosis is crucial for regulating cell division. A functional role for γ-actin in mitotic arrest induced by the microtubule-targeted agent, paclitaxel, has recently been demonstrated. We hypothesized that γ-actin plays a role in mitosis. Herein, we investigated the effect of γ-actin in mitosis and demonstrated that γ-actin is important in the distribution of β-actin and formation of actin-rich retraction fibers during mitosis. The reduced ability of paclitaxel to induce mitotic arrest as a result of γ-actin depletion was replicated with a range of mitotic inhibitors, suggesting that γ-actin loss reduces the ability of broad classes of anti-mitotic agents to induce mitotic arrest. In addition, partial depletion of γ-actin enhanced centrosome amplification in cancer cells and caused a significant delay in prometaphase/metaphase. This prolonged prometaphase/metaphase arrest was due to mitotic defects such as uncongressed and missegregated chromosomes, and correlated with an increased presence of mitotic spindle abnormalities in the γ-actin depleted cells. Collectively, these results demonstrate a previously unknown role for γ-actin in regulating centrosome function, chromosome alignment and maintenance of mitotic spindle integrity.

  10. Effects of AMPPNP on the orientation and rotational dynamics of spin-labeled muscle cross-bridges.

    OpenAIRE

    Fajer, P G; Fajer, E A; Brunsvold, N J; Thomas, D. D.

    1988-01-01

    We have used electron paramagnetic resonance (EPR) to investigate the orientation, rotational motion, and actin-binding properties of rabbit psoas muscle cross-bridges in the presence of the nonhydrolyzable nucleotide analogue, 5'-adenylylimido-diphosphate (AMPPNP). This analogue is known to decrease muscle tension without affecting its stiffness, suggesting an attached cross-bridge state different from rigor. We spin-labeled the SH1 groups on myosin heads and performed conventional EPR to ob...

  11. Statistics of actin-propelled trajectories in noisy environments.

    Science.gov (United States)

    Wen, Fu-Lai; Chen, Hsuan-Yi; Leung, Kwan-Tai

    2016-06-01

    Actin polymerization is ubiquitously utilized to power the locomotion of eukaryotic cells and pathogenic bacteria in living systems. Inevitably, actin polymerization and depolymerization proceed in a fluctuating environment that renders the locomotion stochastic. Previously, we have introduced a deterministic model that manages to reproduce actin-propelled trajectories in experiments, but not to address fluctuations around them. To remedy this, here we supplement the deterministic model with noise terms. It enables us to compute the effects of fluctuating actin density and forces on the trajectories. Specifically, the mean-squared displacement (MSD) of the trajectories is computed and found to show a super-ballistic scaling with an exponent 3 in the early stage, followed by a crossover to a normal, diffusive scaling of exponent 1 in the late stage. For open-end trajectories such as straights and S-shaped curves, the time of crossover matches the decay time of orientational order of the velocities along trajectories, suggesting that it is the spreading of velocities that leads to the crossover. We show that the super-ballistic scaling of MSD arises from the initial, linearly increasing correlation of velocities, before time translational symmetry is established. When the spreading of velocities reaches a steady state in the long-time limit, short-range correlation then yields a diffusive scaling in MSD. In contrast, close-loop trajectories like circles exhibit localized periodic motion, which inhibits spreading. The initial super-ballistic scaling of MSD arises from velocity correlation that both linearly increases and oscillates in time. Finally, we find that the above statistical features of the trajectories transcend the nature of noises, be it additive or multiplicative, and generalize to other self-propelled systems that are not necessarily actin based. PMID:27415296

  12. Statistics of actin-propelled trajectories in noisy environments

    Science.gov (United States)

    Wen, Fu-Lai; Chen, Hsuan-Yi; Leung, Kwan-tai

    2016-06-01

    Actin polymerization is ubiquitously utilized to power the locomotion of eukaryotic cells and pathogenic bacteria in living systems. Inevitably, actin polymerization and depolymerization proceed in a fluctuating environment that renders the locomotion stochastic. Previously, we have introduced a deterministic model that manages to reproduce actin-propelled trajectories in experiments, but not to address fluctuations around them. To remedy this, here we supplement the deterministic model with noise terms. It enables us to compute the effects of fluctuating actin density and forces on the trajectories. Specifically, the mean-squared displacement (MSD) of the trajectories is computed and found to show a super-ballistic scaling with an exponent 3 in the early stage, followed by a crossover to a normal, diffusive scaling of exponent 1 in the late stage. For open-end trajectories such as straights and S-shaped curves, the time of crossover matches the decay time of orientational order of the velocities along trajectories, suggesting that it is the spreading of velocities that leads to the crossover. We show that the super-ballistic scaling of MSD arises from the initial, linearly increasing correlation of velocities, before time translational symmetry is established. When the spreading of velocities reaches a steady state in the long-time limit, short-range correlation then yields a diffusive scaling in MSD. In contrast, close-loop trajectories like circles exhibit localized periodic motion, which inhibits spreading. The initial super-ballistic scaling of MSD arises from velocity correlation that both linearly increases and oscillates in time. Finally, we find that the above statistical features of the trajectories transcend the nature of noises, be it additive or multiplicative, and generalize to other self-propelled systems that are not necessarily actin based.

  13. Mechanical force-induced polymerization and depolymerization of F-actin at water/solid interfaces

    Science.gov (United States)

    Zhang, Xueqiang; Hu, Xiuyuan; Lei, Haozhi; Hu, Jun; Zhang, Yi

    2016-03-01

    Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization.Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin

  14. IGF-I and TGF-beta1 have distinct effects on phenotype and proliferation of intestinal fibroblasts.

    Science.gov (United States)

    Simmons, James G; Pucilowska, Jolanta B; Keku, Temitope O; Lund, P Kay

    2002-09-01

    Insulin-like growth factor I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) are upregulated in myofibroblasts at sites of fibrosis in experimental enterocolitis and in Crohn's disease (CD). We compared the sites of expression of IGF-I and TGF-beta1 in a rat peptidoglycan-polysaccharide (PG-PS) model of chronic granulomatous enterocolitis and fibrosis. We used the human colonic CCD-18Co fibroblast/myofibroblast cell line to test the hypothesis that TGF-beta1 and IGF-I interact to regulate proliferation, collagen synthesis, and activated phenotype typified by expression of alpha-smooth muscle actin and organization into stress fibers. IGF-I potently stimulated while TGF-beta1 inhibited basal DNA synthesis. TGF-beta1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-beta1 but not IGF-I potently stimulated expression of alpha-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-beta1 attenuated stress fiber formation without reducing alpha-smooth muscle actin expression. Stress fibers were not a prerequisite for increased collagen synthesis. TGF-beta1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-beta1 pretreatment. IGF-I potently stimulated proliferation of TGF-beta1-activated myofibroblasts without reversing activated fibrogenic phenotype. We conclude that TGF-beta1 and IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and proliferation may require sequential actions of TGF-beta1 and IGF-I, but they may act in concert to increase collagen deposition. PMID:12181198

  15. Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation

    NARCIS (Netherlands)

    Sharma, Pawan; Tran, Thai; Stelmack, Gerald L; McNeill, Karol; Gosens, Reinoud; Mutawe, Mark M; Unruh, Helmut; Gerthoffer, William T; Halayko, Andrew J

    2008-01-01

    Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. T

  16. Regulation of actin cytoskeleton architecture by Eps8 and Abi1

    Directory of Open Access Journals (Sweden)

    Miller Jeffrey R

    2005-10-01

    Full Text Available Abstract Background The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive. Results Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects. Conclusion Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development.

  17. An ongoing role for structural sarcomeric components in maintaining Drosophila melanogaster muscle function and structure.

    Directory of Open Access Journals (Sweden)

    Alexander D Perkins

    Full Text Available Animal muscles must maintain their function while bearing substantial mechanical loads. How muscles withstand persistent mechanical strain is presently not well understood. The basic unit of muscle is the sarcomere, which is primarily composed of cytoskeletal proteins. We hypothesized that cytoskeletal protein turnover is required to maintain muscle function. Using the flight muscles of Drosophila melanogaster, we confirmed that the sarcomeric cytoskeleton undergoes turnover throughout adult life. To uncover which cytoskeletal components are required to maintain adult muscle function, we performed an RNAi-mediated knockdown screen targeting the entire fly cytoskeleton and associated proteins. Gene knockdown was restricted to adult flies and muscle function was analyzed with behavioural assays. Here we analyze the results of that screen and characterize the specific muscle maintenance role for several hits. The screen identified 46 genes required for muscle maintenance: 40 of which had no previously known role in this process. Bioinformatic analysis highlighted the structural sarcomeric proteins as a candidate group for further analysis. Detailed confocal and electron microscopic analysis showed that while muscle architecture was maintained after candidate gene knockdown, sarcomere length was disrupted. Specifically, we found that ongoing synthesis and turnover of the key sarcomere structural components Projectin, Myosin and Actin are required to maintain correct sarcomere length and thin filament length. Our results provide in vivo evidence of adult muscle protein turnover and uncover specific functional defects associated with reduced expression of a subset of cytoskeletal proteins in the adult animal.

  18. Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant

    OpenAIRE

    Stevens, J. M.; Ulrich, R L; Taylor, L A; Wood, M W; DeShazer, D; M.P. Stevens; Galyov, E. E.

    2005-01-01

    Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind ...

  19. Actin-Binding Proteins from Burkholderia mallei and Burkholderia thailandensis Can Functionally Compensate for the Actin-Based Motility Defect of a Burkholderia pseudomallei bimA Mutant

    OpenAIRE

    Stevens, Joanne M; Ulrich, Ricky L.; Taylor, Lowrie A.; Wood, Michael W.; DeShazer, David; Stevens, Mark P.; Galyov, Edouard E.

    2005-01-01

    Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind ...

  20. 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.

  1. The conserved Tarp actin binding domain is important for chlamydial invasion.

    Directory of Open Access Journals (Sweden)

    Travis J Jewett

    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.

  2. Restricted morphological and behavioral abnormalities following ablation of β-actin in the brain.

    Directory of Open Access Journals (Sweden)

    Thomas R Cheever

    Full Text Available The local translation of β-actin is one mechanism proposed to regulate spatially-restricted actin polymerization crucial for nearly all aspects of neuronal development and function. However, the physiological significance of localized β-actin translation in neurons has not yet been demonstrated in vivo. To investigate the role of β-actin in the mammalian central nervous system (CNS, we characterized brain structure and function in a CNS-specific β-actin knock-out mouse (CNS-ActbKO. β-actin was rapidly ablated in the embryonic mouse brain, but total actin levels were maintained through upregulation of other actin isoforms during development. CNS-ActbKO mice exhibited partial perinatal lethality while survivors presented with surprisingly restricted histological abnormalities localized to the hippocampus and cerebellum. These tissue morphology defects correlated with profound hyperactivity as well as cognitive and maternal behavior impairments. Finally, we also identified localized defects in axonal crossing of the corpus callosum in CNS-ActbKO mice. These restricted defects occurred despite the fact that primary neurons lacking β-actin in culture were morphologically normal. Altogether, we identified novel roles for β-actin in promoting complex CNS tissue architecture while also demonstrating that distinct functions for the ubiquitously expressed β-actin are surprisingly restricted in vivo.

  3. Preliminarily Investigating the Polymorphism of Self-organized Actin Filament in Vitro by Atomic Force Microscope

    Institute of Scientific and Technical Information of China (English)

    Jun ZHANG; Yuan-Liang WANG; Xin-Yong Chen; Chuang-Long HE; Chao CHENG; Yang CAO

    2004-01-01

    With the atomic force microscope (AFM), we preliminarily investigated the large-scale structure of actin filaments formed in low concentration protein solution (5 μg/ml) via self-organization without the presence of any F-actin dynamic interfering factors (such as phalloidin) in vitro. It was found that the G-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single F-actin to giant filament in tree-like branched aggregates. The observed polymerized actin filaments, to which our most intense attention was attracted, was discretely distributed and showed obvious polymorphism distinctly different from those in the presence of phalloidin or actin binding proteins (fimbrin, gelsolin, etc.) in previous experiments. Latter structures were mainly composed of single F-actin and/or multifilaments clearly consisting of several single F-actin. The experimental results clearly demonstrated that non-interference with the F-actin intrinsic dynamics in self-organizing could lead to the polymorphism of actin filamentous structures, and further analysis implied that the disturbance of normal F-actin dynamics by many factors could prevent the emergence of structural polymorphism, more often than not, give rise to formation of specific structures instead and different interference would bring about various particular structures under certain conditions.

  4. Differentiation of Mature and Immature Vasculature

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials Needed 1. PAP pen (Abcam ab2601) - Histochoice MB Fixative (Electron Microscopy Sciences 64115-01) - 10xPBS (Fisher BP3994) - Triton X (Roche 12754423) - Bovine serum albumin (Sigma A8022-500G) - Mouse CD31 [PECAM] (BD Biosciences 555025) - Rabbit alpha smooth muscle actin (Abcam ab5694) - Alexa Fluor 555 donkey anti-mouse IgG (H L) (Invitrogen A31570) - Alexa Fluor 488 donkey anti-rabbit IgG (H L) (Invitrogen A21206) ### Solutions 1. Wash Bu...

  5. Histological observation of complete closure of anterior capsulotomy in 2 cases.

    Science.gov (United States)

    Tanaka, Sai-Ichi; Saika, Shizuya; Tamura, Manabu; Ohnishi, Yoshitaka

    2004-06-01

    We report the histological finding of complete closure of the anterior capsulotomy window in 2 cases. The cases were successfully treated with surgery after neodymium:YAG laser anterior capsulotomy failed. Histology and immunohistochemistry were performed to determine the pathogenesis. Histology revealed the presence of elongated, fibroblast-like lens epithelial cells in association with extracellular matrix accumulation, which were positive for collagen types, fibronectin, and osteopontin. The cells were labeled with anti-alpha-smooth muscle actin antibody. The finding indicates that phenotypic modulation in lens epithelial cell to contractile cell type and accumulation of matrix are involved in closure of the anterior capsulotomy window.

  6. Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction

    DEFF Research Database (Denmark)

    Burns, Jorge S; Kristiansen, Malthe; Kristensen, Lars P;

    2011-01-01

    . Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells...... of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization....

  7. The histopathology of a human mesenchymal stem cell experimental tumor model: support for an hMSC origin for Ewing's sarcoma?

    DEFF Research Database (Denmark)

    Burns, J S; Abdallah, B M; Schrøder, Henrik Daa;

    2008-01-01

    -forming potential of early passage hMSC-TERT20 cells, tumors derived from late passage cells expressed early biomarkers of osteogenesis. However, hMSC-TERT20 cells were heterogeneous for alpha smooth muscle actin (ASMA) expression and one out of six hMSC-TERT20 derived single cell clones was strongly ASMA positive....... Tumors from this ASMA+ clone had distinctive vascular qualities with hot spots of high CD34+ murine endothelial cell density, together with CD34- regions with a branching periodic acid Schiff reaction pattern. Such clone-specific differences in host vascular response provide novel models to explore...

  8. Polymorphisms in the 3' UTR in the neurocalcin delta gene affect mRNA stability, and confer susceptibility to diabetic nephropathy

    DEFF Research Database (Denmark)

    Kamiyama, Masumi; Kobayashi, Masaaki; Araki, Shin-ichi;

    2007-01-01

    Using a large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese type 2 diabetic patients, we have identified a gene encoding neurocalcin delta (NCALD) as a candidate for a susceptibility gene to diabetic nephropathy; the landmark SNP was found in the 3' UTR...... in normal kidney tissues. In an experiment using a short interfering RNA targeting NCALD, we found that silencing of the NCALD led to a considerable enhancement of cell migration, accompanied by a significant reduction in E-cadherin expression, and by an elevation of alpha smooth muscle actin expression...

  9. Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

    Directory of Open Access Journals (Sweden)

    Claudia Forero

    2000-06-01

    Full Text Available The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

  10. Endocytosis-dependent coordination of multiple actin regulators is required for wound healing.

    Science.gov (United States)

    Matsubayashi, Yutaka; Coulson-Gilmer, Camilla; Millard, Tom H

    2015-08-01

    The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form "signaling centers" along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing.

  11. Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ.

    Science.gov (United States)

    Lin, Ying-Hsi; Warren, Chad M; Li, Jieli; McKinsey, Timothy A; Russell, Brenda

    2016-08-01

    The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCɛ (dnPKCɛ) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

  12. Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

    Science.gov (United States)

    Rosenthal, Nadia A.

    1999-01-01

    upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis

  13. Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

    Science.gov (United States)

    Carlsson, Anders

    Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

  14. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  15. Actin Remodeling and Polymerization Forces Control Dendritic Spine Morphology

    CERN Document Server

    Miermans, Karsten; Storm, Cornelis; Hoogenraad, Casper

    2015-01-01

    Dendritic spines are small membranous structures that protrude from the neuronal dendrite. Each spine contains a synaptic contact site that may connect its parent dendrite to the axons of neighboring neurons. Dendritic spines are markedly distinct in shape and size, and certain types of stimulation prompt spines to evolve, in fairly predictable fashion, from thin nascent morphologies to the mushroom-like shapes associated with mature spines. This striking progression is coincident with the (re)configuration of the neuronal network during early development, learning and memory formation, and has been conjectured to be part of the machinery that encodes these processes at the scale of individual neuronal connections. It is well established that the structural plasticity of spines is strongly dependent upon the actin cytoskeleton inside the spine. A general framework that details the precise role of actin in directing the transitions between the various spine shapes is lacking. We address this issue, and present...

  16. Wdr1-Dependent Actin Reorganization in Platelet Activation.

    Science.gov (United States)

    Dasgupta, Swapan K; Le, Anhquyen; Da, Qi; Cruz, Miguel; Rumbaut, Rolando E; Thiagarajan, Perumal

    2016-01-01

    In resting platelets, the integrin αIIbβ3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbβ3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbβ3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbβ3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbβ3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbβ3 during platelet activation. PMID:27627652

  17. Internal Motility in Stiffening Actin-Myosin Networks

    OpenAIRE

    Uhde, Joerg; Keller, Manfred; Sackmann, Erich; Parmeggiani, Andrea; Frey, Erwin

    2003-01-01

    We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and sug...

  18. IFT88 influences chondrocyte actin organization and biomechanics

    OpenAIRE

    Z. Wang; Wann, A.K.T.; Thompson, C L; Hassen, A.; Wang, W; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantif...

  19. Topical therapies for skin cancer and actinic keratosis.

    OpenAIRE

    Haque, T.; Rahman, K. M.; Thurston, D E; Hadgraft, J; Lane, M. E.

    2015-01-01

    The global incidence of skin cancer and actinic keratosis (AK) has increased dramatically in recent years. Although many tumours are treated with surgery or radiotherapy topical therapy has a place in the management of certain superficial skin neoplasms and AK. This review considers skin physiology, non-melanoma skin cancer (NMSC), the relationship between AK and skin cancer and drugs administered topically for these conditions. The dermal preparations for management of NMSC and AK are discus...

  20. Altered cross-bridge properties in skeletal muscle dystrophies

    Directory of Open Access Journals (Sweden)

    Aziz eGuellich

    2014-10-01

    Full Text Available Force and motion generated by skeletal muscle ultimately depends on the cyclical interaction of actin with myosin. This mechanical process is regulated by intracellular Ca2+ through the thin filament-associated regulatory proteins i.e.; troponins and tropomyosin. Muscular dystrophies are a group of heterogeneous genetic affections characterized by progressive degeneration and weakness of the skeletal muscle as a consequence of loss of muscle tissue which directly reduces the number of potential myosin cross-bridges involved in force production. Mutations in genes responsible for skeletal muscle dystrophies have been shown to modify the function of contractile proteins and cross-bridge interactions. Altered gene expression or RNA splicing or post-translational modifications of contractile proteins such as those related to oxidative stress, may affect cross-bridge function by modifying key proteins of the excitation-contraction coupling. Micro-architectural change in myofilament is another mechanism of altered cross-bridge performance. In this review, we provide an overview about changes in cross-bridge performance in skeletal muscle dystrophies and discuss their ultimate impacts on striated muscle function.

  1. Proteasomes are tightly associated to myofibrils in mature skeletal muscle.

    Science.gov (United States)

    Bassaglia, Yann; Cebrian, José; Covan, Silvia; Garcia, Monica; Foucrier, Jean

    2005-01-15

    Proteasomes are the major actors of nonlysosomal cytoplasmic protein degradation. In particular, these large protein complexes (about 2500 kDa) are considered to be responsible for muscular degradation during skeletal muscle atrophy. Despite their unusual and important size, they are widely described as soluble and mobile in the cytoplasm. In mature skeletal muscle, we have previously observed a sarcomeric distribution of proteasomes, as revealed by the distribution of alpha1/p27K, a subunit of the 20S core-particle (prosome) of proteasome. Here, we extend these observations at the electron microscopic level in vivo. We also show that this sarcomeric pattern is dependent of the extension of the sarcomere. Using isolated myofibrils, we demonstrate that proteasomes are still attached to the myofibrils after the isolation procedure, and reproduce the observations made in vivo. In addition, the extraction of actin by gelsolin largely removes proteasomes from isolated myofibrils, but some of them are held in place after this extraction, showing a sarcomeric disposition in the absence of any detectable actin, and suggesting the existence of another molecular partner for these interactions. From these results, we conclude that most of detectable 20S proteasomes in skeletal muscle cells is tightly attached to the myofibrils. PMID:15561103

  2. Dynamic Actin Controls Polarity Induction de novo in Protoplasts

    Institute of Scientific and Technical Information of China (English)

    Beatrix Zaban; Jan Maisch; Peter Nick

    2013-01-01

    Cell polarity and axes are central for plant morphogenesis.To study how polarity and axes are induced de novo,we investigated protoplasts of tobacco Nicotiana tabacum cv.BY-2 expressing fluorescentlytagged cytoskeletal markers.We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages.The synthesis of a new cell wall marks the transition to the first stage of regeneration,and proceeds after a long preparatory phase within a few minutes.During this preparatory phase,the nucleus migrates actively,and cytoplasmic strands remodel vigorously.We probed this system for the effect of anti-cytoskeletal compounds,inducible bundling of actin,RGD-peptides,and temperature.Suppression of actin dynamics at an early stage leads to aberrant tripolar cells,whereas suppression of microtubule dynamics produces aberrant sausagelike cells with asymmetric cell walls.We integrated these data into a model,where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis.Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments,and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  3. Dynamic actin controls polarity induction de novo in protoplasts.

    Science.gov (United States)

    Zaban, Beatrix; Maisch, Jan; Nick, Peter

    2013-02-01

    Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  4. Comparative studies on troponin, a Ca²⁺-dependent regulator of muscle contraction, in striated and smooth muscles of protochordates.

    Science.gov (United States)

    Obinata, Takashi; Sato, Naruki

    2012-01-01

    Troponin is well known as a Ca(2+)-dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin-myosin interaction at low Ca(2+) concentrations, and Ca(2+) at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca(2+)-dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca(2+). Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals.

  5. An autopsy case of neuroleptic malignant syndrome (NMS) and its immunohistochemical findings of muscle-associated proteins and mitochondria.

    Science.gov (United States)

    Kubo, S; Orihara, Y; Kitamura, O; Ikematsu, K; Tsuda, R; Nakasono, I

    2001-01-01

    Neuroleptic malignant syndrome (NMS) is a rare but potentially fatal disorder. In forensic cases, post-mortem diagnosis of NMS is sometimes difficult if ante-mortem information, such as neuroleptic ingestion or signs and symptoms, cannot be obtained. A 39-year-old Japanese male on a neuroleptic treatment regimen suddenly became agitated and died. Autopsy revealed muscle rigidity and hyperthermia. Post-mortem examination of blood revealed elevation of creatine phosphokinase-MM (CK-MM) and lactate dehydrogenase-4 and dehydrogenase-5 (LDH-4 and LDH-5). In renal glomeruli and tubules, myoglobin was stained immunohistochemically. From these findings, the cause of death was considered to be NMS. To support the diagnosis of NMS, both skeletal and cardiac muscles were stained with actin, myoglobin, desmin and mitochondria antibodies immunohistochemically. Actin, myoglobin, desmin, and mitochondria had been lost from skeletal, but not from the cardiac muscle, which suggested that only the skeletal muscle was damaged. Moreover, because mitochondria had disappeared only from the skeletal muscle, it was considered that skeletal muscle degeneration was caused by mitochondrial damage. Therefore, it is suggested that immunostaining of skeletal muscle by antibodies for muscle-associated proteins and mitochondria is useful to corroborate a diagnosis of NMS.

  6. LATS1 tumor suppressor is a novel actin-binding protein and negative regulator of actin polymerization

    Institute of Scientific and Technical Information of China (English)

    Stacy Visser-Grieve; Zhonghua Zhou; Yi-Min She; He Huang; Terry D Cyr; Tian Xu; Xiaolong Yang

    2011-01-01

    Dear Editor,The LATS tumor suppressor,conserved from Drosophila (dlats) to humans (LATS1,LATS2),plays a vital role in maintaining cellular homeostasis in humans since loss of either LATS1 or LATS2 leads to the development of numerous cancer types such as breast cancer and leukemia [1].Apart from its roles as a Ser/Thr kinase within the emerging Hippo pathway regulating cell proliferation and apoptosis,ultimately leading to the control of organ size and tumorigenesis [2],LATS is also implicated in a broad range of functions including regulation of genetic stability,transcription,and protein stability [1 ].Recently,tumor suppressors have also been shown to affect the later stages of tumorigenesis,including metastasis.Among this group of metastasis regulators are genes that can directly affect actin dynamics by binding to F-actin,such as the tumor suppressors p53 [3],NF2 [4] and APC [5].

  7. Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels.

    Science.gov (United States)

    Schreiber, Joerg; Végh, Marlene J; Dawitz, Julia; Kroon, Tim; Loos, Maarten; Labonté, Dorthe; Li, Ka Wan; Van Nierop, Pim; Van Diepen, Michiel T; De Zeeuw, Chris I; Kneussel, Matthias; Meredith, Rhiannon M; Smit, August B; Van Kesteren, Ronald E

    2015-11-01

    Synaptic plasticity requires remodeling of the actin cytoskeleton. Although two actin isoforms, β- and γ-actin, are expressed in dendritic spines, the specific contribution of γ-actin in the expression of synaptic plasticity is unknown. We show that synaptic γ-actin levels are regulated by the E3 ubiquitin ligase TRIM3. TRIM3 protein and Actg1 transcript are colocalized in messenger ribonucleoprotein granules responsible for the dendritic targeting of messenger RNAs. TRIM3 polyubiquitylates γ-actin, most likely cotranslationally at synaptic sites. Trim3(-/-) mice consequently have increased levels of γ-actin at hippocampal synapses, resulting in higher spine densities, increased long-term potentiation, and enhanced short-term contextual fear memory consolidation. Interestingly, hippocampal deletion of Actg1 caused an increase in long-term fear memory. Collectively, our findings suggest that temporal control of γ-actin levels by TRIM3 is required to regulate the timing of hippocampal plasticity. We propose a model in which TRIM3 regulates synaptic γ-actin turnover and actin filament stability and thus forms a transient inhibitory constraint on the expression of hippocampal synaptic plasticity. PMID:26527743

  8. Regulation of actin catch-slip bonds with a RhoA-formin module

    Science.gov (United States)

    Lee, Cho-Yin; Lou, Jizhong; Wen, Kuo-Kuang; McKane, Melissa; Eskin, Suzanne G.; Rubenstein, Peter A.; Chien, Shu; Ono, Shoichiro; Zhu, Cheng; McIntire, Larry V.

    2016-10-01

    The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated. Using AFM force-clamp experiments, we show that formin controlled by RhoA switches the actin catch-slip bonds to slip-only bonds. SMD simulations reveal that the force does not induce the K113:E195 interaction when formin binds to actin K118 and E117 residues located at the helical segment extending to K113. Actin catch-slip bonds are suppressed by single residue replacements K113E and E195K that interrupt the force-induced K113:E195 interaction; and this suppression is rescued by a K113E/E195K double mutant (E/K) restoring the interaction in the opposite orientation. These results support the biological significance of actin catch bonds, as they corroborate reported observations that RhoA and formin switch force-induced actin cytoskeleton alignment and that either K113E or E195K induces yeast cell growth defects rescued by E/K. Our study demonstrates how the mechano-regulation of actin dynamics is modulated by biochemical signaling molecules, and suggests that actin catch bonds may be important in cell functions.

  9. 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.

  10. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers.

    Science.gov (United States)

    van der Honing, Hannie S; de Ruijter, Norbert C A; Emons, Anne Mie C; Ketelaar, Tijs

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

  11. Characterization of engineered actin binding proteins that control filament assembly and structure.

    Directory of Open Access Journals (Sweden)

    Crista M Brawley

    Full Text Available BACKGROUND: Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate. METHODOLOGY/PRINCIPAL FINDINGS: We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs, are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel "reduced genetic code" phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively. CONCLUSIONS/SIGNIFICANCE: From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.

  12. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.

    Science.gov (United States)

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K

    2015-07-01

    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes.

  13. Expression of Chlamydomonas actin-gfp fusion gene in to-bacco suspension cell and polymerization of the actin-gfp protein in vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The fusion gene of actin (cDNA of Chlamydo- monas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed in E. coli and tobacco suspension cells BY2. The correct expression was observed in E. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was dis-tributed around nucleus and cell plate, while the fusion pro-tein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin in Chlamydomonas was similar with those of animals and higher plants.

  14. Rac1 signaling is required for insulin-stimulated glucose uptake and is dysregulated in insulin-resistant murine and human skeletal muscle

    DEFF Research Database (Denmark)

    Sylow, Lykke; Jensen, Thomas Elbenhardt; Kleinert, Maximilian;

    2013-01-01

    investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle, and are dysregulated in insulin resistant states.Muscle specific inducible Rac1 knockout (KO) mice and pharmacological......The actin-cytoskeleton-regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin sensitive and insulin resistant mature skeletal muscle has not previously been...

  15. Muscle strain injuries.

    Science.gov (United States)

    Garrett, W E

    1996-01-01

    One of the most common injuries seen in the office of the practicing physician is the muscle strain. Until recently, little data were available on the basic science and clinical application of this basic science for the treatment and prevention of muscle strains. Studies in the last 10 years represent action taken on the direction of investigation into muscle strain injuries from the laboratory and clinical fronts. Findings from the laboratory indicate that certain muscles are susceptible to strain injury (muscles that cross multiple joints or have complex architecture). These muscles have a strain threshold for both passive and active injury. Strain injury is not the result of muscle contraction alone, rather, strains are the result of excessive stretch or stretch while the muscle is being activated. When the muscle tears, the damage is localized very near the muscle-tendon junction. After injury, the muscle is weaker and at risk for further injury. The force output of the muscle returns over the following days as the muscle undertakes a predictable progression toward tissue healing. Current imaging studies have been used clinically to document the site of injury to the muscle-tendon junction. The commonly injured muscles have been described and include the hamstring, the rectus femoris, gastrocnemius, and adductor longus muscles. Injuries inconsistent with involvement of a single muscle-tendon junction proved to be at tendinous origins rather than within the muscle belly. Important information has also been provided regarding injuries with poor prognosis, which are potentially repairable surgically, including injuries to the rectus femoris muscle, the hamstring origin, and the abdominal wall. Data important to the management of common muscle injuries have been published. The risks of reinjury have been documented. The early efficacy and potential for long-term risks of nonsteroidal antiinflammatory agents have been shown. New data can also be applied to the field

  16. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    Science.gov (United States)

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    Summary The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its ‘scaffolding’ function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  17. Regulation of T-cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm.

    Science.gov (United States)

    Beemiller, Peter; Krummel, Matthew F

    2013-11-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering signalosome assembly and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its 'scaffolding' function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  18. 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....

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

    Science.gov (United States)

    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. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    Science.gov (United States)

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048

  1. Actin based processes that could determine the cytoplasmic architecture of plant cells.

    Science.gov (United States)

    van der Honing, Hannie S; Emons, Anne Mie C; Ketelaar, Tijs

    2007-05-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells that is likely to depend on actin-based force generation is the organisation of the cytoplasm. We compare the function of actin binding proteins of three well-studied mammalian models that depend on actin-based force generation with the function of their homologues in plants. We predict the possible role of these proteins, and thus the role of actin-based force generation, in the production of cytoplasmic organisation in plant cells.

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

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles Edward;

    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...

  3. Effect of cytochalasins on F-actin and morphology of Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Mills, J W; Falsig Pedersen, S; Walmod, P S;

    2000-01-01

    that, in intact cells, different cytochalasins can have varying effects on cell morphology and F-actin content and organization. To examine this problem in more detail, we analyzed the effects of cytochalasins on the cell morphology of and F-actin content and organization in Ehrlich ascites tumor (EAT......Cytochalasins have been used extensively to probe the role of F-actin in different aspects of cellular function. Most of the data obtained are interpreted on the basis of the well-established depolymerizing effects of cytochalasins on F-actin preparations in vitro. However, some evidence indicates......) cells. After a 3-min exposure to 0.5 microM cytochalasin D, B, or E, F-actin content was equally reduced in all cases and this correlated with a reduction in the amount of cortical F-actin associated with the EAT cell membrane. However, only with CE was cell morphology markedly altered...

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

    Directory of Open Access Journals (Sweden)

    Roman ePleskot

    2014-01-01

    Full Text Available The actin cytoskeleton plays a key role in the plant morphogenesis and is involved in polar cell growth, movement of subcellular organelles, cell division, and plant defense. Organization of actin cytoskeleton undergoes dynamic remodeling in response to internal developmental cues and diverse environmental signals. This dynamic behavior is regulated by numerous actin-binding proteins that integrate various signaling pathways. Production of the signaling lipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid affects the activity and subcellular distribution of several actin-binding proteins, and typically correlates with increased actin polymerization. Here we review current knowledge of the inter-regulatory dynamics between signaling phospholipids and the actin cytoskeleton in plant cells.

  5. Muscles of the Trunk

    Science.gov (United States)

    ... four muscle pairs, arranged in layers, and the fascia that envelops them. The abdominal wall muscles are ... formed by two muscular sheets and their associated fascia. « Previous (Head and Neck) Next (Upper Extremity) » Contact ...

  6. Healthy Muscles Matter

    Science.gov (United States)

    ... body? These muscles help you move, lift things, pump blood through your body, and even help you breathe. ... is a specialized type of involuntary muscle. It pumps blood through your body, changing its speed to keep ...

  7. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    Science.gov (United States)

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  8. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    Science.gov (United States)

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

  9. Oxidative metabolism in muscle.

    OpenAIRE

    Ferrari, M; Binzoni, T.; Quaresima, V.

    1997-01-01

    Oxidative metabolism is the dominant source of energy for skeletal muscle. Near-infrared spectroscopy allows the non-invasive measurement of local oxygenation, blood flow and oxygen consumption. Although several muscle studies have been made using various near-infrared optical techniques, it is still difficult to interpret the local muscle metabolism properly. The main findings of near-infrared spectroscopy muscle studies in human physiology and clinical medicine are summarized. The advantage...

  10. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    OpenAIRE

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has ...

  11. Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

    OpenAIRE

    Emmanuel Derivery; Emmanuèle Helfer; Véronique Henriot; Alexis Gautreau

    2012-01-01

    Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel coloca...

  12. Hypogelsolinemia, a disorder of the extracellular actin scavenger system, in patients with multiple sclerosis

    OpenAIRE

    Janmey Paul A; Szmitkowski Maciej; Drozdowski Wiesław; Mroczko Barbara; Wen Qi; Ciccarelli Nicholas J; Kułakowska Alina; Bucki Robert

    2010-01-01

    Abstract Background Extracellular gelsolin (GSN) and GC-globulin/Vitamin D-binding protein (DBP) appear to play an important role in clearing the actin from extracellular fluids and in modulating cellular responses to anionic bioactive lipids. In this study we hypothesized that cellular actin release and/or increase in bioactive lipids associated with multiple sclerosis (MS) development will translate into alteration of the actin scavenger system protein concentrations in blood and cerebrospi...

  13. Fullerenol Nanoparticles with Structural Activity Induce Variable Intracellular Actin Filament Morphologies.

    Science.gov (United States)

    Jin, Junjiang; Dong, Ying; Wang, Ying; Xia, Lin; Gu, Weihong; Bai, Xue; Chang, Yanan; Zhang, Mingyi; Chen, Kui; Li, Juan; Zhao, Lina; Xing, Gengmei

    2016-06-01

    Fullerenol nanoparticles are promising for various biological applications; many studies have shown that they induce variable and diverse biological effects including side effects. Separation and purification of two fractions of fullerenols has demonstrated that they have varied chemical structures on the surfaces of their carbon cages. Actin is an important structural protein that is able to transform functional structures under varied physiological conditions. We assessed the abilities of the two fractions of fullerenols to attach to actin and induce variable morphological features in actin filament structures. Specifically the fullerenol fraction with a surface electric charge of -1.913 ± 0.008q (x10(-6) C) has percentages of C-OH and C=O on the carbon cage of 16.14 ± 0.60 and 17.55 ± 0.69. These features allow it to form intermolecular hydrogen bonds with actin at a stoichiometric ratio of four fullerenols per actin subunit. Molecular simulations revealed these specific binding sites and binding modes in atomic details in the interaction between the active fullerenol and actin filament. Conversely, these interactions were not possible for the other fraction of fullerenol with that percentages of C-OH and C=O on the carbon cage were 15.59 ± 0.01 and 1.94 ± 0.11. Neither sample induced appreciable cytotoxicity or acute cell death. After entering cells, active fullerenol binding to actin induces variable morphological features and may transform ATP-actin to ADP-actin. These changes facilitate the binding of ADF/cofilin, allowing cofilin to sever actin filaments to form cofilin/actin/fullerenol rods. Our findings suggest that fullerenol with structural activity binding disturbs actin filament structure, which may inhibit locomotion of cell or induce chronic side effects in to cells. PMID:27319217

  14. Opposing Roles for Actin in Cdc42p PolarizationD⃞

    OpenAIRE

    Irazoqui, Javier E.; Howell, Audrey S.; Theesfeld, Chandra L.; Lew, Daniel J.

    2005-01-01

    In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399–416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization si...

  15. 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.

  16. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    OpenAIRE

    Nazila Amini; Mohadeseh Naghi Vishteh; Omid Zarei; Reza Hadavi; Negah Ahmadvand; Hodjattallah Rabbani; Mahmood Jeddi-Tehrani

    2014-01-01

    Objective(s):Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protei...

  17. Actin and nuclear myosin Ⅰ are associated with RNAP Ⅱ and function in gene transcription

    Institute of Scientific and Technical Information of China (English)

    ZHU XiaoJuan; HUANG BaiQu; WANG XingZhi; HAO Shui; ZENG XianLu

    2007-01-01

    The presence of actin in the nucleus as well as its functions in various nuclear processes has been made clear in the past few years. Actin is known to be a part of chromatin-remodeling complexes BAF,which are required for maximal ATPase activity of the Brg1 component of the BAF complex. Moreover,the essential roles of acfin in transcription mediated by RNA polymerases Ⅰ, Ⅱ and Ⅲ have been demonstrated recently. On the other hand, a myosin Ⅰ isoform, which contains a unique NH2-terminal extension for nucleus localization, has been specifically localized in nucleus. As is well known, myosin Ⅰis an actin-binding protein and plays an important role in various cellular activities. Though actin and nuclear myosin Ⅰ (NM Ⅰ) have been implicated to play distinct roles in gene expression, there has been no evidence for the actin-myosin interaction that might be involved in gene transcription mediated by RNA polymerase Ⅱ (RNAP Ⅱ). Here we show evidence that both actin and NM Ⅰ are associated with RNAP Ⅱ in nucleus by using co-localization and co-IP assays, and they may act together on gene transcription.The antibodies against β-actin or NM Ⅰ can block RNA synthesis in a eukaryotic in vitro transcription system with template DNA comprising the promoter and the coding region of human autocrine motility factor receptor (hAMFR) gene; the antibodies pre-adsorbed with purified actin and NM Ⅰ have no effect in transcriptional inhibition, indicating that the inhibition of transcription by anti-actin and anti-NM Ⅰ is specific. These results suggest a direct involvement of actin-myosin complexes in regulating transcription. It also implicates that actin and NM Ⅰ may co-exist in a same complex with RNAP Ⅱ and the interaction of RNAP Ⅱ with actin and NM Ⅰ functions in the RNAP Ⅱ-mediated transcription.

  18. Mammalian Fat1 cadherin regulates actin dynamics and cell–cell contact

    OpenAIRE

    Tanoue, Takuji; Takeichi, Masatoshi

    2004-01-01

    Fat cadherins form a distinct subfamily of the cadherin gene superfamily, and are featured by their unusually large extracellular domain. In this work, we investigated the function of a mammalian Fat cadherin. Fat1 was localized at filopodial tips, lamellipodial edges, and cell–cell boundaries, overlapping with dynamic actin structures. RNA interference–mediated knockdown of Fat1 resulted in disorganization of cell junction–associated F-actin and other actin fibers/cables, disturbance of cell...

  19. Muscle Session Summary

    Science.gov (United States)

    Baldwin, Kenneth; Feeback, Daniel

    1999-01-01

    Presentations from the assembled group of investigators involved in specific research projeects related to skeletal muscle in space flight can categorized in thematic subtopics: regulation of contractile protein phenotypes, muscle growth and atrophy, muscle structure: injury, recovery,and regeneration, metabolism and fatigue, and motor control and loading factors.

  20. Transportation of nanoscale cargoes by myosin propelled actin filaments.

    Directory of Open Access Journals (Sweden)

    Malin Persson

    Full Text Available 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. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached" or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  1. Microrheology and micromechanics of actin-coated membranes

    Science.gov (United States)

    Bourdieu, Laurent

    2002-03-01

    To study the interaction between cytoskeletal filaments and the plasma membrane, we designed composite membranes obtained by self-assembly of actin filaments on the outer leaflet of giant unilamellar fluid vesicles. Their rich dynamics is studied by micromanipulation with optical tweezers and by single particle tracking experiments. We first show that microrheology study can be carried out on such an individual microscopic object by measuring the thermally excited position fluctuations of a probed bead bound biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin filaments network first induces a finite 2D shear modulus of the order of 1 microN/m. Moreover, these membranes exhibit a clear viscoelastic behavior at high frequency: above a few tens of Hz, both the shear and the bending moduli exhibit the same frequency dependence, a power law of exponent 0.75. These results are consistent in the framework of our model with previous measurements on actin solutions. We show moreover that these complexes exhibit typical mechanical features of a solid shell. For example, a buckling instability is observed when a localized force of the order of 0.5 picoNewton is applied perpendicular to the membrane plane. Although predicted for polymerized vesicles, this is the first evidence of such an instability. This instability is a striking example of the coupling between in-plane stretch and shear and out-of-plane bending, which takes place for curves shells when it becomes more favorable energetically to concentrate the in-plane stress due to the bending within a narrow ring, centered on the force application point.

  2. Actin, RhoA, and Rab11 Participation during Encystment in Entamoeba invadens

    Directory of Open Access Journals (Sweden)

    M. Herrera-Martínez

    2013-01-01

    Full Text Available In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

  3. Cooperative and non-cooperative conformational changes of F-actin induced by cofilin

    International Nuclear Information System (INIS)

    Highlights: •Mobility of MTSL attached to C374 in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to C374 with cofilin-binding was cooperative. •Mobility of MTSL attached to V43C in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to V43C with cofilin-binding was linear. -- Abstract: Cofilin is an actin-binding protein that promotes F-actin depolymerization. It is well-known that cofilin-coated F-actin is more twisted than naked F-actin, and that the protomer is more tilted. However, the means by which the local changes induced by the binding of individual cofilin proteins proceed to the global conformational changes of the whole F-actin molecule remain unknown. Here we investigated the cofilin-induced changes in several parts of F-actin, through site-directed spin-label electron paramagnetic resonance spectroscopy analyses of recombinant actins containing single reactive cysteines. We found that the global, cooperative conformational changes induced by cofilin-binding, which were detected by the spin-label attached to the Cys374 residue, occurred without the detachment of the D-loop in subdomain 2 from the neighboring protomer. The two processes of local and global changes do not necessarily proceed in sequence

  4. Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

    Science.gov (United States)

    Herrera-Martínez, M; Hernández-Ramírez, V I; Lagunes-Guillén, A E; Chávez-Munguía, B; Talamás-Rohana, P

    2013-01-01

    In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

  5. Rapid non-equilibrium turnover fluidizes entangled F-actin solutions

    Science.gov (United States)

    McCall, Patrick M.; Kovar, David R.; Gardel, Margaret L.

    The actin cytoskeleton of living cells is a semiflexible polymer network which regulates cell division, motility, and morphogenesis by controlling cell shape. These complex shape-changing processes require both mechanical deformation and remodeling of the actin cytoskeleton. Molecular motors generate internal forces to drive deformation, while cytoskeletal remodeling is regulated by non-equilibrium polymer turnover. Although the mechanical properties of equilibrium actin filament (F-actin) networks are well-described by theories of semiflexible polymers, these theories do not incorporate the effects of non-equilibrium turnover. To address this experimentally, we developed a model system in which both the turnover rate and the length distribution of purified F-actin can be tuned independently at steady-state through the combined action of actin regulatory proteins. Specifically we tune the concentrations of cofilin, profilin, and formin to regulate F-actin severing, recycling, and nucleation, respectively. We find that the actin turnover rate can be tuned by cofilin up to 25-fold (31 +/- 2 subunits/sec/filament). Surprisingly, changes in turnover rate have no effect on the steady-state F-actin length distribution, which is instead set by formin concentration. Passive microrheology measurements show that increased turnover leads to striking fluidization in both entangled and crosslinked networks. Non-equilibrium turnover thus enables modulation of network mechanics, which impacts force transmission and material deformation.

  6. Cell-cycle regulation of formin-mediated actin cable assembly.

    Science.gov (United States)

    Miao, Yansong; Wong, Catherine C L; Mennella, Vito; Michelot, Alphée; Agard, David A; Holt, Liam J; Yates, John R; Drubin, David G

    2013-11-19

    Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.

  7. Fertilization in Torenia fournieri: actin organization and nuclear behavior in the central cell and primary endosperm

    Institute of Scientific and Technical Information of China (English)

    YUAN; Ming(袁明); FU; Ying(傅缨); WANG; Feng(王凤); HUANG; Bingquan(黄炳权); Sze-Yong; Zee(徐是雄); Peter; K.Hepler

    2002-01-01

    Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.

  8. Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly.

    Science.gov (United States)

    Roth-Johnson, Elizabeth A; Vizcarra, Christina L; Bois, Justin S; Quinlan, Margot E

    2014-02-14

    Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

  9. Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites.

    Science.gov (United States)

    Ortiz, D; del Carmen Dominguez-Robles, M; Villegas-Sepúlveda, N; Meza, I

    2000-10-01

    Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

  10. TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.

    Science.gov (United States)

    Zhu, Jinsheng; Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Di Donato, Martin; Ge, Pei; Oehri, Jacqueline; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H; Pollmann, Stephan; Azzarello, Elisa; Mancuso, Stefano; Ferro, Noel; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland; Friml, Jiří; Thomas, Clément; Geisler, Markus

    2016-04-01

    Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

  11. Protein dynamics in skeletal muscle after trauma: local and systemic effects.

    Science.gov (United States)

    Downey, R S; Monafo, W W; Karl, I E; Matthews, D E; Bier, D M

    1986-03-01

    Injury is attended by accelerated skeletal muscle proteolysis. Accurate definition of this hypercatabolic response and its mediation is requisite for specific therapy. We measured protein dynamics in the incubated and intact epitrochlearis and soleus muscles excised from both forelimbs and both hindlimbs of rats 4 days after injury by either a single hind limb scald (90 degrees C water for 3 seconds; metabolic rate (MR) + 15%, urinary urea nitrogen (UUN) + 10%) or a 5% excision (dorsal skin removed to fascia; MR + 40%, UUN + 90%). Protein synthesis (3H phenylalanine incorporation) increased only in the injured soleus from the scalded hind limb (+100%). Actin and myosin breakdown (3-methylhistidine release) increased in all muscles tested and was consistently larger in epitrochlearis than in soleus muscles. Breakdown of the mixed protein pool (tyrosine release) increased but less so than 3-methylhistidine and did not reach significance in the uninjured soleus muscle of scalded rats. With respect to fiber type, white fiber epitrochlearis muscle demonstrated a more pronounced elevation of both measures of breakdown but at a lower metabolic rate than did red fiber soleus muscle. Increasing MR was associated with a linear increase in soleus proteolysis but no further change in epitrochlearis breakdown. We conclude that protein breakdown is increased in skeletal muscle distant from injury; however, even when metabolic stress is severe, synthesis is unchanged. Muscles of different fiber composition are not equally labile. Furthermore, myofibrillar protein is more labile than the mixed protein pool.

  12. Intra-axonal myosin and actin in nerve regeneration.

    Science.gov (United States)

    McQuarrie, Irvine G; Lund, Linda M

    2009-10-01

    A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin. PMID:19927086

  13. Colocalization of synapsin and actin during synaptic vesicle recycling

    DEFF Research Database (Denmark)

    Bloom, Ona; Evergren, Emma; Tomilin, Nikolay;

    2003-01-01

    activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin......-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known...

  14. Plasma Gelsolin and Circulating Actin Correlate with Hemodialysis Mortality

    OpenAIRE

    Lee, Po-Shun; Sampath, Kartik; Karumanchi, S. Ananth; Tamez, Hector; Bhan, Ishir; Isakova, Tamara; Gutierrez, Orlando M.; Wolf, Myles; Chang, Yuchiao; Stossel, Thomas P.; Thadhani, Ravi

    2009-01-01

    Plasma gelsolin (pGSN) binds actin and bioactive mediators to localize inflammation. Low pGSN correlates with adverse outcomes in acute injury, whereas administration of recombinant pGSN reduces mortality in experimental sepsis. We found that mean pGSN levels of 150 patients randomly selected from 10,044 starting chronic hemodialysis were 140 ± 42 mg/L, 30 to 50% lower than levels reported for healthy individuals. In a larger sample, we performed a case-control analysis to evaluate the relati...

  15. Internal Motility in Stiffening Actin-Myosin Networks

    CERN Document Server

    Uhde, J; Sackmann, E; Parmeggiani, A; Frey, E; Uhde, Joerg; Keller, Manfred; Sackmann, Erich; Parmeggiani, Andrea; Frey, Erwin

    2003-01-01

    We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and suggest a "zipping" mechanism to explain the filament motion in the vicinity of the sol-gel transition.

  16. Muscle contraction and the elasticity-mediated crosstalk effect

    CERN Document Server

    Dharan, Nadiv

    2013-01-01

    Cooperative action of molecular motors is essential for many cellular processes. One possible regulator of motor coordination is the elasticity-mediated crosstalk (EMC) coupling between myosin II motors whose origin is the tensile stress that they collectively generate in actin filaments. Here, we use a statistical mechanical analysis to investigate the influence of the EMC effect on the sarcomere - the basic contractile unit of skeletal muscles. We demonstrate that the EMC effect leads to an increase in the attachment probability of motors located near the end of the sarcomere while, simultaneously, decreasing the attachment probability of the motors in the central part. Such a polarized attachment probability would impair the motors ability to cooperate efficiently. Interestingly, this undesired phenomenon becomes significant only when the system size exceeds that of the sarcomere in skeletal muscles, which provides an explanation for the remarkable lack of sarcomere variability in vertebrates. Another phen...

  17. Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle.

    Science.gov (United States)

    Rattan, Satish; Ali, Mehboob

    2015-04-01

    Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22.

  18. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients

    KAUST Repository

    Conti, Antonio

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS\\'s pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V.

  19. To the problem of cross-bridge tension in steady muscle shortening and lengthening

    CERN Document Server

    Kokshenev, Valery B

    2009-01-01

    Despite the great success of the Huxley sliding filament model proposed half a century ago for actin-myosin linkages (cross-bridges), it fails to explain the force-velocity behavior of stretching skeletal muscles. Huxley's two-state kinetic equation for cross-bridge proportions is therefore reconsidered and a new solution to the problem of steady muscle eccentric and concentric contractions is reported. When the second law of statistical thermodynamics is applied to cross-bridge proportions, the weakly bound states appear to be correlated to the strongly bound states via structural and kinetic intrinsic muscle characteristics. The explicit force-velocity curve fits the empirical tension-velocity data on frog muscle shortening using only one adjustable parameter, while the Huxley model employed four parameters.

  20. Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo

    DEFF Research Database (Denmark)

    Christensen, Britt; Lundby, Carsten; Jessen, Niels;

    2012-01-01

    myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased. Conclusions/Significance: Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or......Background: Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle. Methodology/Principal Findings: The protocols...... involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1-10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as...

  1. Housekeeping proteins: How useful are they in skeletal muscle diabetes studies and muscle hypertrophy models?

    Science.gov (United States)

    Fortes, Marco Aurélio Salomão; Marzuca-Nassr, Gabriel Nasri; Vitzel, Kaio Fernando; da Justa Pinheiro, Carlos Hermano; Newsholme, Philip; Curi, Rui

    2016-07-01

    The use of Western blot analysis is of great importance in research, and the measurement of housekeeping proteins is commonly used for loading controls. However, Ponceau S staining has been shown to be an alternative to analysis of housekeeping protein levels as loading controls in some conditions. In the current study, housekeeping protein levels were measured in skeletal muscle hypertrophy and streptozotocin-induced diabetes experimental models. The following housekeeping proteins were investigated: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, α-tubulin, γ-tubulin, and α-actinin. Evidence is presented that Ponceau S is more reliable than housekeeping protein levels for specific protein quantifications in Western blot analysis. PMID:27060530

  2. A Multimodular Tensegrity Model of an Actin Stress Fiber

    Science.gov (United States)

    Luo, Yaozhi; Xu, Xian; Lele, Tanmay; Kumar, Sanjay; Ingber, Donald E.

    2008-01-01

    Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model also can explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors, and represent a new handle on multi-scale modeling of living materials. PMID:18632107

  3. Chronic actinic dermatitis - A study of clinical features

    Directory of Open Access Journals (Sweden)

    Somani Vijay

    2005-01-01

    Full Text Available Background: Chronic actinic dermatitis (CAD, one of the immune mediated photo-dermatoses, comprises a spectrum of conditions including persistent light reactivity, photosensitive eczema and actinic reticuloid. Diagnostic criteria were laid down about 20 years back, but clinical features are the mainstay in diagnosis. In addition to extreme sensitivity to UVB, UVA and/or visible light, about three quarters of patients exhibit contact sensitivity to several allergens, which may contribute to the etiopathogenesis of CAD. This study was undertaken to examine the clinical features of CAD in India and to evaluate the relevance of patch testing and photo-aggravation testing in the diagnosis of CAD. Methods: The clinical data of nine patients with CAD were analyzed. Histopathology, patch testing and photo-aggravation testing were also performed. Results: All the patients were males. The average age of onset was 57 years. The first episode was usually noticed in the beginning of summer. Later the disease gradually tended to be perennial, without any seasonal variations. The areas affected were mainly the photo-exposed areas in all patients, and the back in three patients. Erythroderma was the presenting feature in two patients. The palms and soles were involved in five patients. Patch testing was positive in seven of nine patients. Conclusions: The diagnosis of CAD mainly depended upon the history and clinical features. The incidence of erythroderma and palmoplantar eczema was high in our series. Occupation seems to play a role in the etiopathogenesis of CAD.

  4. 5DFRXXL region of long myosin light chain kinase causes F-actin bundle formation

    Institute of Scientific and Technical Information of China (English)

    YANG Chunxiang; WEI Dongmei; CHEN Chen; YU Weiping; ZHU Minsheng

    2005-01-01

    Long myosin light chain kinase (L-MLCK) contains five DFRXXL motifs with ability to bind F-actin. Binding stoichiometry data indicated that each DFRXXL motif might bind each G-actin, but its biological significance remained unknown. We hypothesized that L-MLCK might act as an F-actin bundle peptides by its multiple binding sites of 5DFRXXL motifs to actin. In order to characterize F-actin-bundle formation properties of 5DFRXXL region of long myosin light chain kinase, we expressed and purified 5DFRXXL peptides tagged with HA in vitro. The properties of 5DFRXXL peptides binding to myofilaments or F-actin were analyzed by binding stoichiometries assays. The results indicated that 5DFRXXL peptides bound to myofilaments or F-actin with high affinity. KD values of 5DFRXXL binding to myofilaments and F-actin were 0.45 and 0.41 μmol/L, re- spectively. Cross-linking assay demonstrated that 5DFRXXL peptides could bundle F-actin efficiently. Typical F-actin bundles were observed morphologically through determina- tion of confocal and electron microscopy after adding 5DFRXXL peptides. After transfection of pEGFP-5DFRXXL plasmid into eukaryocyte, spike structure was observed around cell membrane edge. We guess that such structure formation may be attributable to F-actin over-bundle forma- tion caused by 5DFRXXL peptides. Therefore, we suppose that L-MLCK may be a new bundling protein and somehow play a certain role in organization of cell skeleton besides mediating cell contraction by it kinase activity.

  5. Stability of actin-lysozyme complexes formed in cystic fibrosis disease.

    Science.gov (United States)

    Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

    2016-08-21

    Finding the conditions for destabilizing actin-lysozyme complexes is of biomedical importance in preventing infections in cystic fibrosis. In this manuscript, the effects of different charge-mutants of lysozyme and salt concentration on the stability of actin-lysozyme complexes are studied using Langevin dynamics simulation. A coarse-grained model of F-actin is used in which both its twist and bending rigidities are considered. We observe that the attraction between F-actins is stronger in the presence of wild-type lysozymes relative to the mutated lysozymes of lower charges. By calculating the potential of mean force between F-actins, we conclude that the stability of actin-lysozyme complexes is decreased by reducing the charge of lysozyme mutants. The distributions of different lysozyme charge-mutants show that wild-type (+9e) lysozymes are mostly accumulated in the center of triangles formed by three adjacent F-actins, while lysozyme mutants of charges +7e and +5e occupy the bridging regions between F-actins. Low-charge mutants of lysozyme (+3e) distribute uniformly around F-actins. A rough estimate of the electrostatic energy for these different distributions proves that the distribution in which lysozymes reside in the center of triangles leads to more stable complexes. Also our results in the presence of a salt suggest that at physiological salt concentration of airway, F-actin complexes are not formed by charge-reduced mutants of lysozyme. The findings are interesting because if we can design charge-reduced lysozyme mutants with considerable antibacterial activity, they are not sequestered inside F-actin aggregates and can play their role as antibacterial agents against airway infection. PMID:27436705

  6. A muscle stem cell for every muscle: variability of satellite cell biology among different muscle groups

    Directory of Open Access Journals (Sweden)

    Matthew Emerson Randolph

    2015-10-01

    Full Text Available The human body contains approximately 640 individual skeletal muscles. Despite the fact that all of these muscles are composed of striated muscle tissue, the biology of these muscles and their associated muscle stem cell populations are quite diverse. Skeletal muscles are affected differentially by various muscular dystrophies, such that certain genetic mutations specifically alter muscle function in only a subset of muscles. Additionally, defective muscle stem cells have been implicated in the pathology of some muscular dystrophies. The biology of muscle stem cells varies depending on their embryologic origins and the muscles with which they are associated. Here we review the biology of skeletal muscle stem cell populations of eight different muscle groups. Understanding the biological variation of skeletal muscles and their resident stem cells could provide valuable insight into mechanisms underlying the susceptibility of certain muscles to myopathic disease.

  7. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

    Directory of Open Access Journals (Sweden)

    Alexander Belyy

    Full Text Available Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

  8. F-actin distribution and function during sexual differentiation in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Petersen, J; Nielsen, O; Egel, R;

    1998-01-01

    Sexual differentiation in Schizosaccharomyces pombe is induced from the G1 phase of the cell cycle by nitrogen starvation and the presence of mating pheromones. We describe the distribution of F-actin during sexual differentiation. Cortical F-actin dots have previously been shown to be restricted...

  9. Actin based processes that could determine the cytoplasmic architecture of plant cells

    NARCIS (Netherlands)

    Honing, van der H.S.; Emons, A.M.C.; Ketelaar, M.J.

    2007-01-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells t

  10. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

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    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  11. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

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    Kathleen A Estes

    2011-09-01

    Full Text Available The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.

  12. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

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    Yuji Henmi

    Full Text Available A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  13. Aspects of plant cell growth and the actin cytoskeleton: lessons from root hairs

    NARCIS (Netherlands)

    Ruijter, de N.C.A.

    1999-01-01

    The main topic the thesis addresses is the role of the actin cytoskeleton in the growth process of plant cells. Plant growth implies a combination of cell division and cell expansion. The cytoskeleton, which exists of microtubules and actin filaments, plays a major role in both processes. Before cel

  14. β-Actin protein expression differs in the submandibular glands of male and female mice.

    Science.gov (United States)

    Chen, Gang; Zou, Ye; Zhang, Xuan; Xu, Lingfei; Hu, Qiaoyun; Li, Ting; Yao, Chenjuan; Yu, Shali; Wang, Xiaoke; Wang, Chun

    2016-07-01

    β-actin, a cytoskeletal protein, is the most widely used housekeeping gene. Although housekeeping genes are expressed in all tissues, the β-actin gene is expressed in certain cell types because of differential binding of transcriptional factors to the regulatory elements of the gene. The expression and localization of β-actin protein in the submandibular glands (SMG) of mice were investigated in this study, using Western blot analysis and immunohistochemistry. In ICR and C57BL/6J mice, the levels of β-actin protein in the SMG of females are significantly higher than those in the SMG of males. β-actin protein is majorly distributed in acinar cells of SMG. There is no significant difference in the expression level of β-actin protein between females and castrated males. After castrated male ICR mice are treated with 10 mg/kg/day testosterone propionate (TP) for 3 weeks, the levels of β-actin protein in SMG decrease. The numbers of duct per unit area increase, whereas the numbers of acinus per unit area decrease after TP administration. These data suggest that β-actin protein is mainly distributed in acinar cells of SMG and results in a marked sexual dimorphism in mice. PMID:27079296

  15. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse.

    Science.gov (United States)

    Hartzell, Catherine A; Jankowska, Katarzyna I; Burkhardt, Janis K; Lewis, Richard S

    2016-01-01

    T cell receptor (TCR) engagement opens Ca(2+) release-activated Ca(2+) (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca(2+) influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca(2+)-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca(2+) as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca(2+) influx may modulate TCR signaling. PMID:27440222

  16. A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

    Science.gov (United States)

    Palmer, Sarah E; Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W; Ayscough, Kathryn R

    2015-03-30

    Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

  17. Opposing Roles for Actin in Cdc42p PolarizationD⃞

    Science.gov (United States)

    Irazoqui, Javier E.; Howell, Audrey S.; Theesfeld, Chandra L.; Lew, Daniel J.

    2005-01-01

    In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399–416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site. PMID:15616194

  18. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bjørnar Sporsheim

    2015-12-01

    Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

  19. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse

    Science.gov (United States)

    Hartzell, Catherine A; Jankowska, Katarzyna I; Burkhardt, Janis K; Lewis, Richard S

    2016-01-01

    T cell receptor (TCR) engagement opens Ca2+ release-activated Ca2+ (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca2+ influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca2+-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca2+ as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca2+ influx may modulate TCR signaling. DOI: http://dx.doi.org/10.7554/eLife.14850.001 PMID:27440222

  20. Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling.

    Science.gov (United States)

    Groen, Christopher M; Spracklen, Andrew J; Fagan, Tiffany N; Tootle, Tina L

    2012-12-01

    Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.

  1. Proneural proteins Achaete and Scute associate with nuclear actin to promote formation of external sensory organs.

    Science.gov (United States)

    Hsiao, Yun-Ling; Chen, Yu-Ju; Chang, Yi-Jie; Yeh, Hsiao-Fong; Huang, Yi-Chun; Pi, Haiwei

    2014-01-01

    Basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis through transcriptional regulation. Although much is known about the tissue-specific regulation of proneural gene expression, how proneural proteins interact with transcriptional machinery to activate downstream target genes is less clear. Drosophila proneural proteins Achaete (Ac) and Scute (Sc) induce external sensory organ formation by activating neural precursor gene expression. Through co-immunoprecipitation and mass spectrometric analyses, we found that nuclear but not cytoplasmic actin associated with the Ac and Sc proteins in Drosophila S2 cells. Daughterless (Da), the common heterodimeric partner of Drosophila bHLH proteins, was observed to associate with nuclear actin through proneural proteins. A yeast two-hybrid assay revealed that the binding specificity between actin and Ac or Sc was conserved in yeast nuclei without the presence of additional Drosophila factors. We further show that actin is required in external sensory organ formation. Reduction in actin gene activity impaired proneural-protein-dependent expression of the neural precursor genes, as well as formation of neural precursors. Furthermore, increased nuclear actin levels, obtained by expression of nucleus-localized actin, elevated Ac-Da-dependent gene transcription as well as Ac-mediated external sensory organ formation. Taken together, our in vivo and in vitro observations suggest a novel link for actin in proneural-protein-mediated transcriptional activation and neural precursor differentiation.

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

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    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.

  3. Kindlin-2 directly binds actin and regulates integrin outside-in signaling.

    Science.gov (United States)

    Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta; Qin, Jun; Plow, Edward F

    2016-04-11

    Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2(+/-)mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK(47)/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK(47)/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK(47)/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

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

    Science.gov (United States)

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

  5. Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

    Science.gov (United States)

    Cao, Yang; White, Howard D; Li, Xiang-Dong

    2014-01-21

    The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

  6. Establishment of artery smooth muscle cell proliferation model after subarachnoid hemorrhage in rats

    Directory of Open Access Journals (Sweden)

    Yu-jie CHEN

    2011-12-01

    Full Text Available Objective The current paper aims to simulate the effects of hemolytic products on intracranial vascular smooth muscle cell after subarachnoid hemorrhage(SAH,and probe into the molecular mechanism and strategy for the prevention and cure of vascular proliferation after SAH.Methods Thirty Sprague-Dawley rats were randomly divided into three groups,including sham-operated,24 h after SAH,and 72 h after SAH groups.The artificial hemorrhage model around the common carotid artery was established for the latter two groups.The animals were put to death after 24 h and 72 h to take the common carotid artery,and to measure the expression level of PCNA,SM-α-actin protein,and mRNA in the smooth muscle cell.Results The PCNA mRNA expression was significantly up-regulated in the 24-h group(P < 0.01.The expression in the 72-h group was lower than that of the 24-h group(P < 0.01,whereas it was still remarkably higher than that of the sham group(P < 0.01.The SM-α-actin mRNA expression in the smooth muscle cell in the 24-h and 72-h groups decreased compared with that of the Sham group(P < 0.05,whereas the 72-h group was significantly lower than that of the 24-h group(P < 0.05.The protein expression of PCNA and SM-α-actin showed a similar trend.Conclusion The current experiment simulates better effects of the hemolytic products on vascular smooth muscle cell after SAH.It also shows that artificial hemorrhage around the common carotid artery could stimulate vascular smooth muscle cell to change from contractile phenotype into synthetic phenotype,and improve it to proliferate.

  7. Double localization of F-actin in chemoattractant-stimulated polymorphonuclear leucocytes.

    Science.gov (United States)

    Lepidi, H; Benoliel, A M; Mege, J L; Bongrand, P; Capo, C

    1992-09-01

    Uniform concentrations of chemoattractants such as formylpeptides induced a morphological polarization of human polymorphonuclear leucocytes (PMNs) and a concentration of F-actin at the cell front. They also induced a transient increase in filamentous actin (F-actin) which preceded the cell shape change. We combined fluorescence microscopy and image analysis to study the localization of F-actin, as revealed by a specific probe (bodipyTM phallacidin) in suspended PMNs stimulated by chemoattractants. F-actin exhibited remarkable concentration in focal points after a 30 s exposure to 10(-8) M formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), although no shape change of PMNs was detectable. A 10-min incubation with formylpeptide (10(-6) to 10(-9) M) induced the morphological polarization of PMNs and the appearance of a principal focus of F-actin in the cell head region and a secondary focus in the cell posterior end. The distribution of F-actin-associated fluorescence in 2D images of polarized PMNs might be due to an actual concentration of F-actin in privileged areas, to a local concentration of plasma membrane drawing filamentous actin or to variations in the cell volume. Then, we studied the distribution of a cytoplasmic marker, fluorescein diacetate and a membrane probe, TMA-DPH, in unstimulated rounded PMNs and in spherical and morphologically polarized PMNs stimulated by formylpeptide. The distribution of neither of these probes was correlated with F-actin distribution, especially in rounded PMNs stimulated 30 s with 10(-8) M fMet-Leu-Phe, suggesting that F-actin was concentrated in two foci located in the cell head region and in the cell posterior end. In addition, zymosan-activated serum induced the morphological polarization of PMNs and the appearance of two foci of filamentous actin, demonstrating that binding of formylpeptide to its specific receptor was not required for F-actin reorganization. We conclude that the accumulation of F-actin probably

  8. The proline-rich domain of tau plays a role in interactions with actin

    Directory of Open Access Journals (Sweden)

    Wang Dong

    2009-11-01

    Full Text Available Abstract Background The microtubule-associated protein tau is able to interact with actin and serves as a cross-linker between the microtubule and actin networks. The microtubule-binding domain of tau is known to be involved in its interaction with actin. Here, we address the question of whether the other domains of tau also interact with actin. Results Several tau truncation and deletion mutants were constructed, namely N-terminal region (tauN, proline-rich domain (tauPRD, microtubule binding domain (tauMTBD and C-terminal region (tauC truncation mutants, and microtubule binding domain (tauΔMTBD and proline-rich domain/microtubule binding domain (tauΔPRD&MTBD deletion mutants. The proline-rich domain truncation mutant (tauPRD and the microtubule binding domain deletion mutant (tauΔMTBD promoted the formation of actin filaments. However, actin assembly was not observed in the presence of the N-terminal and C-terminal truncation mutants. These results indicate that the proline-rich domain is involved in the association of tau with G-actin. Furthermore, results from co-sedimentation, solid phase assays and electron microscopy showed that the proline-rich domain is also capable of binding to F-actin and inducing F-actin bundles. Using solid phase assays to analyze apparent dissociation constants for the binding of tau and its mutants to F-actin resulted in a sequence of affinity for F-actin: tau >> microtubule binding domain > proline-rich domain. Moreover, we observed that the proline-rich domain was able to associate with and bundle F-actin at physiological ionic strength. Conclusion The proline-rich domain is a functional structure playing a role in the association of tau with actin. This suggests that the proline-rich domain and the microtubule-binding domain of tau are both involved in binding to and bundling F-actin.

  9. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Yi Jinsoo; Schmidt, Jacob; Chien Aichi; Montemagno, Carlo D [Department of Bioengineering, University of California Los Angeles, 420 Westwood Plaza, 7523 Boelter Hall, Los Angeles, CA 90095-1600 (United States)], E-mail: montemcd@ucmail.uc.edu

    2009-02-25

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  10. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

    International Nuclear Information System (INIS)

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  11. Covalent immobilization of myosin for in-vitro motility of actin

    Indian Academy of Sciences (India)

    Ellis Bagga; Sunita Kumari; Rajesh Kumar; Rakesh Kumar; R P Bajpai; Lalit M Bharadwaj

    2005-11-01

    The present study reports the covalent immobilization of myosin on glass surface and in-vitro motility of actin-myosin biomolecular motor. Myosin was immobilized on poly-L-lysine coated glass using heterobifunctional cross linker EDC and characterized by AFM. The in-vitro motility of actin was carried out on the immobilized myosin. It was observed that velocity of actin over myosin increases with increasing actin concentration (0.4-1.0 mg/ml) and was found in the range of 0.40-3.25 m/s. The motility of actin-myosin motor on artificial surfaces is of immense importance for developing nanodevices for healthcare and engineering applications.

  12. PIP2: choreographer of actin-adaptor proteins in the HIV-1 dance

    Science.gov (United States)

    Rocha-Perugini, Vera; Gordon-Alonso, Mónica; Sánchez-Madrid, Francisco

    2014-01-01

    The actin cytoskeleton plays a key role during the replication cycle of human immunodeficiency virus-1 (HIV-1). HIV-1 infection is affected by cellular proteins that influence the clustering of viral receptors or the subcortical actin cytoskeleton. Several of these actin-adaptor proteins are controlled by the second messenger phosphatidylinositol 4,5-biphosphate (PIP2), an important regulator of actin organization. PIP2 production is induced by HIV-1 attachment and facilitates viral infection. However, the importance of PIP2 in regulating cytoskeletal proteins and thus HIV-1 infection has been overlooked. This review examines recent reports describing the roles played by actin-adaptor proteins during HIV-1 infection of CD4+ T cells, highlighting the influence of the signaling lipid PIP2 in this process. PMID:24768560

  13. Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

    Science.gov (United States)

    Cortez-Herrera, E; Yamamoto, R R; Rodrigues, J J; Farias, S E; Ferreira, H B; Zaha, A

    2001-04-01

    We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

  14. Muscle glycogenolysis during exercise

    DEFF Research Database (Denmark)

    Richter, Erik; Ruderman, N B; Gavras, H;

    1982-01-01

    The interaction of epinephrine and contractions on muscle metabolism was studied in the isolated perfused rat hindquarter. Subtetanic contractions (180/min) through 20 min elicited glycogenolysis and increased phosphorylase a activity. In the soleus, a slow-twitch red muscle, these effects were...... transient, but when epinephrine at a physiological concentration (2.4 X 10(-8) M) was added to the perfusate, glycogenolysis and phosphorylase activity were sustained throughout contractions. At this high frequency of contractions, the effect of epinephrine was much smaller in the fast-twitch red fibers...... and not significant in the fast-twitch white fibers of the gastrocnemius muscle. However, during less frequent contractions (30/min) epinephrine increased glycogenolysis and phosphorylase a activity in fast-twitch muscle. The data suggest that epinephrine and muscle contractions exert a dual control of muscle...

  15. Inhibition of pancreatic stellate cell activation by the vitamin A and vitamin E as a therapy for prevention fibrogenesis in experimental chronic alcoholic pancreatitis

    Directory of Open Access Journals (Sweden)

    Nichitaylo M. E.

    2012-01-01

    Full Text Available The aim of the study was to investigate the effects of Vitamin A and Vitamin E on activity of pancreatic stellate cells and fibrosis changes in pancreas after distal pancreatectomy in rats with experimental alcohol-induced chronic pancreatitis. Simultaneously Vitamin A and Vitamin E were administered after distal pancreatectomy in rats with experimental alcohol-induced chronic pancreatitis. The animals were treated withVitamin A at the dose of 33000 IU/kg body weight per day and Vitamin E at the dose of 100 mg/kg body weight per day for three weeks (21 days after operation. To estimate the efficacy of the treatment on activity and numbersof pancreatic stellate cells the immunohistochemicalinvestigation was made with alpha-smooth muscle actin, desmin, vimentin, glial fibrillary acidic protein (GFAP, matrix metalloproteinase 1 (MMP1, tissue inhibitor of metalloproteinase 2 (TIMP2 using. The treatment of rats after operation with vitamin A and vitamin E inhibited activity of pancreatic stellate cells and characterized by significant decreasing of the alpha-smooth muscle actin, Desmin, Vimentin, MMP1 and TIMP2 expression. The ratio of MMP1/TIMP2 was greater in the group with treatment then in the control group. This therapy had a trend to decrease the expression of GFAPand alleviate the fibrotic changes in pancreas.

  16. The role of Rho/Rho-kinase pathway and the neuroprotective effects of fasudil in chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Ya-yun Yan; Xiao-ming Wang; Yan Jiang; Han Chen; Jin-ting He; Jing Mang; Yan-kun Shao; Zhong-xin Xu

    2015-01-01

    The Rho/Rho-kinase signaling pathway plays an important role in cerebral ischemia/reperfusion injury. However, very few studies have examined in detail the changes in the Rho/Rho-kinase signaling pathway in chronic cerebral ischemia. In this study, rat models of chronic cerebral ischemia were established by permanent bilateral common carotid artery occlusion and intra-gastrically administered 9 mg/kg fasudil, a powerful ROCK inhibitor, for 9 weeks. Morris water maze results showed that cognitive impairment progressively worsened as the cerebral ischemia proceeded. Immunohistochemistry, semi-quantitative RT-PCR and western blot analysis showed that the expression levels of Rho-kinase, its substrate myosin-binding subunit, and its relat-ed protein alpha smooth muscle actin, significantly increased after chronic cerebral ischemia. TUNEL staining showed that chronic cerebral ischemia could lead to an increase in neuronal apoptosis, as well as the expression level of caspase-3 in the frontal cortex of rats subjected to chronic cerebral ischemia. Fasudil treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, and decreased the expression level of Rho-kinase, myosin-binding subunit and alpha smooth muscle actin. Furthermore, fasudil could regulate cerebral injury by reducing cell apoptosis and decreasing caspase-3 expression in the frontal cortex. These ifndings demonstrate that fasudil can protect against cognitive impairment induced by chronic cerebral ischemiavia the Rho/Rho-kinase signaling pathway and anti-apoptosis mechanism.

  17. Calomys callosus: an alternative model to study fibrosis in Schistosomiasis mansoni: the pathology of the acute phase

    Directory of Open Access Journals (Sweden)

    J. A. Lenzi

    1995-04-01

    Full Text Available Twenty Calomys callosus, Rengger, 1830 (Rodentia-Cricetidae were studied in the early stage of the acute schistosomal mansoni infection (42nd day. The same number of Swiss Webster mice were used as a comparative standard. Liver and intestinal sections, fixed in formalin-Millonig and embedded in paraffin, were stained with hematoxilin and eosin, PAS-Alcian Blue, pH = 1.0 and 2.5, Lennert's Giemsa, Picrosirius plus polarization microscopy, Periodic acid methanamine silver, Gomori's silver reticulin and resorcin-fuchsin. Immunohistological study (indirect immunofluorescence and peroxidase labeled extravidin-biotin methods was done with antibodies specific to pro-collagen III, fibronectin, elastin, condroitin-sulfate, tenascin, alpha smooth muscle actin, vimentin and desmin. The hepatic granulomas were small, reaching only 27 of the volume of the hepatic Swiss Webster granuloma. They were composed mainly by large immature macrophages, often filled by schistosomal pigment, characterizing an exsudative-macrophage granuloma type. The granulomas were situated in the parenchyma and in the portal space. They were often intravascular, poor of extracellular matrix components, except fibronectin and presented, sometimes alpha smooth muscle actin and vimentin positive cells. The C. callosus intestinal granulomas were similar to Swiss Webster, showing predominance of macrophages. Therefore, the C. callosus acquire very well the Schistosoma mansoni infection, without developing strong hepatic acute granulomatous reaction, suggesting lack of histopathological signs of hypersensitivity.

  18. Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

    International Nuclear Information System (INIS)

    Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D2O and H2O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydrated either with D2O or H2O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (DT) of the hydration water in the first layer were found to be 1.2 × 10−5 cm2/s and 1.7 × 10−5 cm2/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10−5 cm2/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The DT values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the recent observation on intracellular water that shows bulk-like behavior

  19. Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes.

    Science.gov (United States)

    Fabian, Lacramioara; Forer, Arthur

    2007-01-01

    We tested whether the mechanisms of chromosome movement during anaphase in locust (Locusta migratoria L.) spermatocytes might be similar to those described for crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes, as indicated by the effects of inhibitors and by the localisations of actin and myosin in spindles. In this study, we tested whether locust spermatocyte spindles also utilise actin and myosin, and whether actin is involved in microtubule flux. Living locust spermatocytes were treated with inhibitors of actin (latrunculin B and cytochalasin D), myosin (BDM), or myosin phosphorylation (Y-27632 and ML-7). We added drugs (individually) during anaphase. Actin inhibitors alter anaphase: chromosomes either completely stop moving, slow, or sometimes accelerate. The myosin inhibitor, BDM, also alters anaphase: in most cases, the chromosomes drastically slow or stop. ML-7, an inhibitor of MLCK, causes chromosomes to stop, slow, or sometimes accelerate, similar to actin inhibitors. Y-27632, an inhibitor of Rho-kinase, drastically slows or stops anaphase chromosome movements. The effects of the drugs on anaphase movement are reversible: most of the half-bivalents resumed movement at normal speed after these drugs were washed out. Actin and myosin were present in the spindles in locations consistent with their possible involvement in force production. Microtubule flux along kinetochore fibres is an actin-dependent process, since LatB completely removes or drastically reduces the gap in microtubule acetylation at the kinetochore. These results suggest that actin and myosin are involved in anaphase chromosome movements in locust spermatocytes. PMID:17922265

  20. Right sternalis muscle

    Directory of Open Access Journals (Sweden)

    Jetti R

    2009-03-01

    Full Text Available Knowledge regarding the muscular variations of the chest and their identification for the proper dissection planes through radiological examination is important. Sternalis is an occasional muscle, which lies along the side of the sternum. It may be confused as a tumor. The existence of sternalis muscle, its location, orientation and early identification are necessary in breast surgeries. Presence of sternalis muscle adjacent to the breast is of clinical importance.

  1. Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator.

    Science.gov (United States)

    Fabian, Lacramioara; Xia, Xuequin; Venkitaramani, Deepa V; Johansen, Kristen M; Johansen, Jørgen; Andrew, Deborah J; Forer, Arthur

    2007-07-01

    Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which we identified by mass spectrometry analysis as being titin. The spindle matrix proteins skeletor, megator and chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein. PMID:17591688

  2. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin

  3. The Role of Actin-Capping Protein and Src signalling in tissue growth and apoptosis during Drosophila wing development

    OpenAIRE

    Jezowska, Barbara Zofia

    2012-01-01

    Dissertation presented to obtain the Ph.D degree in Developmental Biology The actin cytoskeleton controls numerous cellular processes, including cell morphology and polarity, endocytosis, intracellular trafficking, contractility and cell division. Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among them Capping Protein is a highly conserved αβ heterodimer, which binds the barbed ends of actin filaments, inhibiting addit...

  4. Vital role for the Plasmodium actin capping protein (CP) beta-subunit in motility of malaria sporozoites

    OpenAIRE

    Ganter, Markus; Schüler, Herwig; Matuschewski, Kai

    2009-01-01

    Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F-actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein...

  5. Expression of TGF-β1 and CTGF Is Associated with Fibrosis of Denervated Sternocleidomastoid Muscles in Mice.

    Science.gov (United States)

    Liu, Fei; Tang, Weifang; Chen, Donghui; Li, Meng; Gao, Yinna; Zheng, Hongliang; Chen, Shicai

    2016-01-01

    Injury to the recurrent laryngeal nerve often leads to permanent vocal cord paralysis, which has a significant negative impact on the quality of life. Long-term denervation can induce laryngeal muscle fibrosis, which obstructs the muscle recovery after laryngeal reinnervation. However, the mechanisms of fibrosis remain unclear. In this study, we aimed to analyze the changes in the expression of fibrosis-related factors, including transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) in denervated skeletal muscles using a mouse model of accessory nerve transection. Because of the small size, we used sternocleidomastoid muscles instead of laryngeal muscles for denervation experiments. Masson's trichrome staining showed that the grade of atrophy and fibrosis of muscles became more severe with time, but showed a plateau at 4 weeks after denervation, followed by a slow decrease. Quantitative assessment and immunohistochemistry showed that TGF-β1 expression peaked at 1 week after denervation (p muscle cells were detected at 1 week after denervation, peaked at 2 weeks (p muscle fibrosis. They may induce the differentiation of myoblasts into myofibroblasts, as characterized by the activation of α-SMA. These findings may provide insights on key pathological processes in denervated skeletal muscle fibrosis and develop novel therapeutic strategies.

  6. Actin filaments growing against a barrier with fluctuating shape

    CERN Document Server

    Sadhu, Raj Kumar

    2016-01-01

    We study force generation by a set of parallel actin filaments growing against a non-rigid obstacle, in presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The non-rigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affects the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time-scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculation within mean-field theory shows reasonable agreement with our simulation results.

  7. Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

    Science.gov (United States)

    Roberts, Brett J.; Pashaj, Anjeza; Johnson, Keith R.; Wahl, James K.

    2011-01-01

    Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration. PMID:21945137

  8. Actin filaments on myosin beds: The velocity distribution

    Science.gov (United States)

    Bourdieu, L.; Magnasco, M. O.; Winkelmann, D. A.; Libchaber, A.

    1995-12-01

    In vitro studies of actin filaments sliding on a myosin-coated surface are analyzed, filament by filament, at a sampling rate of 30 per second. For each filament, the mean arc length coordinate is computed and histograms of instantaneous velocities, along the arc length, are established. Two types of motion are observed, depending on the experimental conditions. The first one is characterized by a homogeneous flow, with well defined velocities. In this regime, specific defects are a constitutive part of the flow. It is observed at high temperature, at high myosin coverage, and with a particular mode of attachment of myosin to the surface. The second regime shows no clear velocity selection, but a broadband distribution. It is characterized by high friction and is observed at low temperature or low myosin density. (c) 1995 The American Physical Society

  9. Actin filaments growing against a barrier with fluctuating shape

    Science.gov (United States)

    Sadhu, Raj Kumar; Chatterjee, Sakuntala

    2016-06-01

    We study force generation by a set of parallel actin filaments growing against a nonrigid obstacle, in the presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The nonrigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one-dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affect the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculations within mean-field theory show reasonable agreement with our simulation results.

  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. Percolation mechanism drives actin gels to the critically connected state

    Science.gov (United States)

    Lee, Chiu Fan; Pruessner, Gunnar

    2016-05-01

    Cell motility and tissue morphogenesis depend crucially on the dynamic remodeling of actomyosin networks. An actomyosin network consists of an actin polymer network connected by cross-linker proteins and motor protein myosins that generate internal stresses on the network. A recent discovery shows that for a range of experimental parameters, actomyosin networks contract to clusters with a power-law size distribution [J. Alvarado, Nat. Phys. 9, 591 (2013), 10.1038/nphys2715]. Here, we argue that actomyosin networks can exhibit a robust critical signature without fine-tuning because the dynamics of the system can be mapped onto a modified version of percolation with trapping (PT), which is known to show critical behavior belonging to the static percolation universality class without the need for fine-tuning of a control parameter. We further employ our PT model to generate experimentally testable predictions.

  12. Kinetics of a single cross-bridge in familial hypertrophic cardiomyopathy heart muscle measured by reverse Kretschmann fluorescence

    Science.gov (United States)

    Mettikolla, Prasad; Calander, Nils; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian

    2010-01-01

    Familial hypertrophic cardiomyopathy (FHC) is a serious heart disease that often leads to a sudden cardiac death of young athletes. It is believed that the alteration of the kinetics of interaction between actin and myosin causes FHC by making the heart to pump blood inefficiently. We set out to check this hypothesis ex vivo. During contraction of heart muscle, a myosin cross-bridge imparts periodic force impulses to actin. The impulses are analyzed by fluorescence correlation spectroscopy (FCS) of fluorescently labeled actin. To minimize observation volume and background fluorescence, we carry out FCS measurements in surface plasmon coupled emission mode in a reverse Kretschmann configuration. Fluorescence is a result of near-field coupling of fluorophores excited in the vicinity of the metal-coated surface of a coverslip with the surface plasmons propagating in the metal. Surface plasmons decouple on opposite sides of the metal film and emit in a directional manner as far-field p-polarized radiation. We show that the rate of changes of orientation is significantly faster in contracting cardiac myofibrils of transgenic mice than wild type. These results are consistent with the fact that mutated heart muscle myosin translates actin faster in in vitro motility assays.

  13. Skeletal muscle development and regeneration.

    NARCIS (Netherlands)

    Grefte, S.; Kuijpers-Jagtman, A.M.; Torensma, R.; Hoff, J.W. Von den

    2007-01-01

    In the late stages of muscle development, a unique cell population emerges that is a key player in postnatal muscle growth and muscle regeneration. The location of these cells next to the muscle fibers triggers their designation as satellite cells. During the healing of injured muscle tissue, satell

  14. Calcium and actin in the saga of awakening oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Santella, Luigia, E-mail: santella@szn.it; Limatola, Nunzia; Chun, Jong T.

    2015-04-24

    The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggs at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm–egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent “excitable media” that quickly respond to the stimulus with the Ca{sup 2+} swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca{sup 2+} signals and in the control of monospermic fertilization. - Highlights: • Besides microtubules, microfilaments may anchor the nucleus to oocyte surface. • The cortical Ca{sup 2+} flash and wave at fertilization mirror electrical membrane change. • Artificial egg activation lacks microvilli extension in the perivitelline space. • Calcium is necessary but not sufficient for cortical granules exocytosis. • Actin cytoskeleton modulates Ca{sup 2+} release at oocyte maturation

  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 li

  16. 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

    Science.gov (United States)

    Nelson, W James; Weis, William I

    2016-07-01

    Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. PMID:27166091

  17. Comparative genome analysis of cortactin and HSI : the significance of the F-actin binding repeat domain

    NARCIS (Netherlands)

    van Rossum, AGSH; Schuuring-Scholtes, E; Seggelen, VV; Kluin, PM; Schuuring, E

    2005-01-01

    Background: In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid seque

  18. Interactions between the Yeast SM22 Homologue Scp1 and Actin Demonstrate the Importance of Actin Bundling in Endocytosis*S⃞

    Science.gov (United States)

    Gheorghe, Dana M.; Aghamohammadzadeh, Soheil; Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Winder, Steve J.; Ayscough, Kathryn R.

    2008-01-01

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process. PMID:18400761

  19. Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin.

    Science.gov (United States)

    Winkelman, Jonathan D; Bilancia, Colleen G; Peifer, Mark; Kovar, David R

    2014-03-18

    Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilator-stimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin. However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. We used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascin-bundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two- to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of ∼10 s in solution, for ∼60 s when immobilized on a surface, and for ∼110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, we found that Ena's actin-assembly properties are tunable on Fascin-bundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.

  20. Skeletal muscle myofilament adaptations to aging, disease and disuse and their effects on whole muscle performance in older adult humans

    Directory of Open Access Journals (Sweden)

    Mark Stuart Miller

    2014-09-01

    Full Text Available Skeletal muscle contractile function declines with aging, disease and disuse. In vivo muscle contractile function depends on a variety of factors, but force, contractile velocity and power generating capacity ultimately derive from the summed contribution of single muscle fibers. The contractile performance of these fibers are, in turn, dependent upon the isoform and function of myofilament proteins they express, with myosin protein expression and its mechanical and kinetic characteristics playing a predominant role. Alterations in myofilament protein biology, therefore, may contribute to the development of functional limitations and disability in these conditions. Recent studies suggest that these conditions are associated with altered single fiber performance due to decreased expression of myofilament proteins and/or changes in myosin-actin cross-bridge interactions. Furthermore, cellular and myofilament-level adaptations are related to diminished whole muscle and whole body performance. Notably, the effect of these various conditions on myofilament and single fiber function tends to be larger in older women compared to older men, which may partially contribute to their higher rates of disability. To maintain functionality and provide the most appropriate and effective countermeasures to aging, disease and disuse in both sexes, a more thorough understanding is needed of the contribution of myofilament adaptations to functional disability in older men and women and their contribution to tissue level function and mobility impairment.