Alterations of the cytoskeleton in human cells in space proved by life-cell imaging

Science.gov (United States)

Corydon, Thomas J.; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-01

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24th DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31st parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization. PMID:26818711

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics.

Science.gov (United States)

Cardoso, Rita; Soares, Helena; Hemphill, Andrew; Leitão, Alexandre

2016-07-01

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells

International Nuclear Information System (INIS)

Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

1989-01-01

Epidermal growth factor receptors (EGF-R) have been shown to be associated with the detergent-insoluble cytoskeleton of A431 cells, where they retained both a functional ligand-binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF-R. The tyrosine kinase activity of the cytoskeletally associated EGF-R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF-R associated with cytoskeletons of EGF-treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent-insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF-stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF-R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF-R. Saturable binding of 125I-EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High-affinity EGF-R were preferentially retained upon detergent extraction of adherent cells, whereas both low- and high-affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF-R to that solubilized in adherent cells, indicating that EGF-R can reversibly associate with the structural elements of the cell

The cytoskeleton in cell-autonomous immunity: structural determinants of host defence

Science.gov (United States)

Mostowy, Serge; Shenoy, Avinash R.

2016-01-01

Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. PMID:26292640

Imaging Cytoskeleton Components by Electron Microscopy.

Science.gov (United States)

Svitkina, Tatyana

2016-01-01

The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell.

Mechanotransduction across the cell surface and through the cytoskeleton

Science.gov (United States)

Wang, N.; Butler, J. P.; Ingber, D. E.

1993-01-01

Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

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

System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

Science.gov (United States)

Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

2017-01-01

The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

Reorganization of the subplasmalemmal cytoskeleton in association with exocytosis in rat mast cells

DEFF Research Database (Denmark)

Nielsen, E H; Braun, K; Johansen, Torben

1989-01-01

The subplasmalemmal cytoskeleton in mast cells has been studied by scanning electron microscopy of the internal side of the plasma membrane. Rearrangement of the dense subplasmalemmal network of actin filaments took place following cell activation by compound 48/80 and secretion of histamine....... The rearrangement was a withdrawal of the subplasmalemmal cytoskeleton from the exocytotic sites and the development of bare, filament-free areas around the sites. In calcium-depleted mast cells we demonstrated a dense network that was difficult to break. Activation of the calcium-depleted cells by compound 48...

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

Science.gov (United States)

Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

2014-07-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

Cell shape can mediate the spatial organization of the bacterial cytoskeleton

Science.gov (United States)

Wang, Siyuan; Wingreen, Ned

2013-03-01

The bacterial cytoskeleton guides the synthesis of cell wall and thus regulates cell shape. Since spatial patterning of the bacterial cytoskeleton is critical to the proper control of cell shape, it is important to ask how the cytoskeleton spatially self-organizes in the first place. In this work, we develop a quantitative model to account for the various spatial patterns adopted by bacterial cytoskeletal proteins, especially the orientation and length of cytoskeletal filaments such as FtsZ and MreB in rod-shaped cells. We show that the combined mechanical energy of membrane bending, membrane pinning, and filament bending of a membrane-attached cytoskeletal filament can be sufficient to prescribe orientation, e.g. circumferential for FtsZ or helical for MreB, with the accuracy of orientation increasing with the length of the cytoskeletal filament. Moreover, the mechanical energy can compete with the chemical energy of cytoskeletal polymerization to regulate filament length. Notably, we predict a conformational transition with increasing polymer length from smoothly curved to end-bent polymers. Finally, the mechanical energy also results in a mutual attraction among polymers on the same membrane, which could facilitate tight polymer spacing or bundling. The predictions of the model can be verified through genetic, microscopic, and microfluidic approaches.

Profilin as a regulator of the membrane-actin cytoskeleton interface in plant cells

Directory of Open Access Journals (Sweden)

Tiantian eSun

2013-12-01

Full Text Available Membrane structures and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell. Recently, the molecular mechanisms operating at this interface have been progressively addressed. Many experiments have revealed that the actin cytoskeleton can interact with membranes through various discrete membrane domains. The actin-binding protein, profilin has been proven to inhibit actin polymerization and to promote F-actin elongation. This is dependent on many factors, such as the profilin/G-actin ratio and the ionic environment of the cell. Additionally, profilin has specific domains that interact with phosphoinositides and poly-L-proline rich proteins; theoretically, this gives profilin the opportunity to interact with membranes, and a large number of experiments have confirmed this possibility. In this article, we summarize recent findings in plant cells, and discuss the evidence of the connections among actin cytoskeleton, profilin and biomembranes through direct or indirect relationships.

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

Science.gov (United States)

2013-01-01

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

CecropinXJ, a silkworm antimicrobial peptide, induces cytoskeleton disruption in esophageal carcinoma cells.

Science.gov (United States)

Xia, Lijie; Wu, Yanling; Kang, Su; Ma, Ji; Yang, Jianhua; Zhang, Fuchun

2014-10-01

Antimicrobial peptides exist in the non-specific immune system of organism and participate in the innate host defense of each species. CecropinXJ, a cationic antimicrobial peptide, possesses potent anticancer activity and acts preferentially on cancer cells instead of normal cells, but the mechanism of cancer cell death induced by cecropinXJ remains largely unknown. This study was performed to investigate the cytoskeleton-disrupting effects of cecropinXJ on human esophageal carcinoma cell line Eca109 using scanning electron microscopy observation, fluorescence imaging, cell migration and invasion assays, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The electronic microscope and fluorescence imaging observation suggested that cecropinXJ could result in morphological changes and induce damage to microtubules and actin of Eca109 cells in a dose-dependent manner. The cell migration and invasion assays demonstrated that cecropinXJ could inhibit migration and invasion of tumor cells. Western blot and qRT-PCR analysis showed that there was obvious correlation between microtubule depolymerization and actin polymerization induced by cecropinXJ. Moreover, cecropinXJ might also cause decreased expression of α-actin, β-actin, γ-actin, α-tubulin, and β-tubulin genes in concentration- and time-dependent manners. In summary, this study indicates that cecropinXJ triggers cytotoxicity in Eca109 cells through inducing the cytoskeleton destruction and regulating the expression of cytoskeleton proteins. This cecropinXJ-mediated cytoskeleton-destruction effect is instrumental in our understanding of the detailed action of antimicrobial peptides in human cancer cells and cecropinXJ might be a potential therapeutic agent for the treatment of cancer in the future. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

Science.gov (United States)

Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

2014-02-01

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Progress of research on cytoskeleton and neural cell migration obstacle induced by ionizing radiation

International Nuclear Information System (INIS)

Qiu Jun; Wu Cuiping; Wang Mingming

2012-01-01

The dynamic changes of the microtubules and microfilaments provide the main force that drives the normal migration. Biological effects in tissues and cells induced by ionizing radiation are closely correlated with the changes happening to the cytoskeleton. It is that the ionizing radiation can induce the depolymeration of microfilaments and the assembly obstacles of microtubules, and make neural cell incapable of entering the model of migration or abnormally migrate. The effects of relevant changes of the cytoskeleton induced by irradiation on neural cell migration were discussed in this paper. (authors)

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

Science.gov (United States)

Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

2016-01-01

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

Nucleus and nucleus-cytoskeleton connections in 3D cell migration

Energy Technology Data Exchange (ETDEWEB)

Liu, Lingling, E-mail: liulingling2012@163.com; Luo, Qing, E-mail: qing.luo@cqu.edu.cn; Sun, Jinghui, E-mail: sunjhemail@163.com; Song, Guanbin, E-mail: song@cqu.edu.cn

2016-10-15

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at

Nucleus and nucleus-cytoskeleton connections in 3D cell migration

International Nuclear Information System (INIS)

Liu, Lingling; Luo, Qing; Sun, Jinghui; Song, Guanbin

2016-01-01

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at

A spatiotemporal characterization method for the dynamic cytoskeleton.

Science.gov (United States)

Alhussein, Ghada; Shanti, Aya; Farhat, Ilyas A H; Timraz, Sara B H; Alwahab, Noaf S A; Pearson, Yanthe E; Martin, Matthew N; Christoforou, Nicolas; Teo, Jeremy C M

2016-05-01

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to Df , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in Df and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

Science.gov (United States)

Rodríguez-García, Ruddi; López-Montero, Iván; Mell, Michael; Egea, Gustavo; Gov, Nir S.; Monroy, Francisco

2015-01-01

Erythrocytes are flexible cells specialized in the systemic transport of oxygen in vertebrates. This physiological function is connected to their outstanding ability to deform in passing through narrow capillaries. In recent years, there has been an influx of experimental evidence of enhanced cell-shape fluctuations related to metabolically driven activity of the erythroid membrane skeleton. However, no direct observation of the active cytoskeleton forces has yet been reported to our knowledge. Here, we show experimental evidence of the presence of temporally correlated forces superposed over the thermal fluctuations of the erythrocyte membrane. These forces are ATP-dependent and drive enhanced flickering motions in human erythrocytes. Theoretical analyses provide support for a direct force exerted on the membrane by the cytoskeleton nodes as pulses of well-defined average duration. In addition, such metabolically regulated active forces cause global membrane softening, a mechanical attribute related to the functional erythroid deformability. PMID:26083919

A new theoretical approach to analyze complex processes in cytoskeleton proteins.

Science.gov (United States)

Li, Xin; Kolomeisky, Anatoly B

2014-03-20

Cytoskeleton proteins are filament structures that support a large number of important biological processes. These dynamic biopolymers exist in nonequilibrium conditions stimulated by hydrolysis chemical reactions in their monomers. Current theoretical methods provide a comprehensive picture of biochemical and biophysical processes in cytoskeleton proteins. However, the description is only qualitative under biologically relevant conditions because utilized theoretical mean-field models neglect correlations. We develop a new theoretical method to describe dynamic processes in cytoskeleton proteins that takes into account spatial correlations in the chemical composition of these biopolymers. Our approach is based on analysis of probabilities of different clusters of subunits. It allows us to obtain exact analytical expressions for a variety of dynamic properties of cytoskeleton filaments. By comparing theoretical predictions with Monte Carlo computer simulations, it is shown that our method provides a fully quantitative description of complex dynamic phenomena in cytoskeleton proteins under all conditions.

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

International Nuclear Information System (INIS)

Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

2010-01-01

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca 2+ -dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

Mechanics of membrane-cytoskeleton attachment in Paramecium

Science.gov (United States)

Campillo, C.; Jerber, J.; Fisch, C.; Simoes-Betbeder, M.; Dupuis-Williams, P.; Nassoy, P.; Sykes, C.

2012-12-01

In this paper we assess the role of the protein MKS1 (Meckel syndrome type 1) in the cortical membrane mechanics of the ciliated protist Paramecium. This protein is known to be crucial in the process of cilium formation, and we investigate its putative role in membrane-cytoskeleton attachment. Therefore, we compare cells where the gene coding for MKS1 is silenced to wild-type cells. We found that scanning electron microscopy observation of the cell surface reveals a cup-like structure in wild-type cells that is lost in silenced cells. Since this structure is based on the underlying cytoskeleton, one hypothesis to explain this observation is a disruption of membrane attachment to the cytoskeleton in the absence of MKS1 that should affect plasma membrane mechanics. We test this by probing the mechanics of wild-type and silenced cells by micropipette aspiration. Strikingly, we observe that, at the same aspiration pressure, the membrane of silenced cells is easily aspirated by the micropipette whereas that of wild-type cells enters only at a moderate velocity, an effect that suggests a detachment of the membrane from the underlying cytoskeleton in silenced cells. We quantify this detachment by measuring the deformation of the cell cortex and the rate of cell membrane entry in the micropipette. This study offers a new perspective for the characterization of membrane-cytoskeleton attachment in protists and paves the way for a better understanding of the role of membrane-cortex attachment in cilium formation.

Interactions with the actin cytoskeleton are required for cell wall localization of barley stripe mosaic virus TGB proteins

Science.gov (United States)

The host cytoskeleton and membrane system are the main routes by which plant viruses move within or between cells. Barley stripe mosaic virus (BSMV) -induced actin filament thickening was visualized in the cytoskeleton of agroinfiltrated Nicotiana benthamiana epidermal cells expressing DsRed:Talin. ...

Duplication and segregation of the actin (MreB) cytoskeleton during the prokaryotic cell cycle.

Science.gov (United States)

Vats, Purva; Rothfield, Lawrence

2007-11-06

The bacterial actin homolog MreB exists as a single-copy helical cytoskeletal structure that extends between the two poles of rod-shaped bacteria. In this study, we show that equipartition of the MreB cytoskeleton into daughter cells is accomplished by division and segregation of the helical MreB array into two equivalent structures located in opposite halves of the predivisional cell. This process ensures that each daughter cell inherits one copy of the MreB cytoskeleton. The process is triggered by the membrane association of the FtsZ cell division protein. The cytoskeletal division and segregation events occur before and independently of cytokinesis and involve specialized MreB structures that appear to be intermediates in this process.

Palytoxins and cytoskeleton: An overview.

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Louzao, M Carmen; Ares, Isabel R; Cagide, Eva; Espiña, Begoña; Vilariño, Natalia; Alfonso, Amparo; Vieytes, Mercedes R; Botana, Luis M

2011-03-01

Cytoskeleton is a dynamic structure essential for a wide variety of normal cellular processes, including the maintenance of cell shape and morphology, volume regulation, membrane dynamics and signal transduction. Cytoskeleton is organized into microtubules, actin meshwork and intermediate filaments. Actin has been identified as a major target for destruction during apoptosis and is also important under pathological conditions such as cancers. Several natural compounds actively modulate actin organization by specific signaling cascades being useful tools to study cytoskeleton dynamics. Palytoxin is a large bioactive compound, first isolated from zoanthids, with a complex structure and different analogs such as ostreocin-D or ovatoxin-a. This toxin has been identified as a potent tumor promoter and cytotoxic molecule, which leads to actin filament distortion and triggers cell death or apoptosis. In this review we report the findings on the involvement of palytoxin and analogues modulating the actin cytoskeleton within different cellular models. Copyright © 2010 Elsevier Ltd. All rights reserved.

Mechanical stability of the cell nucleus: roles played by the cytoskeleton in nuclear deformation and strain recovery.

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Wang, Xian; Liu, Haijiao; Zhu, Min; Cao, Changhong; Xu, Zhensong; Tsatskis, Yonit; Lau, Kimberly; Kuok, Chikin; Filleter, Tobin; McNeill, Helen; Simmons, Craig A; Hopyan, Sevan; Sun, Yu

2018-05-18

Extracellular forces transmitted through the cytoskeleton can deform the cell nucleus. Large nuclear deformation increases the risk of disrupting the nuclear envelope's integrity and causing DNA damage. Mechanical stability of the nucleus defines its capability of maintaining nuclear shape by minimizing nuclear deformation and recovering strain when deformed. Understanding the deformation and recovery behavior of the nucleus requires characterization of nuclear viscoelastic properties. Here, we quantified the decoupled viscoelastic parameters of the cell membrane, cytoskeleton, and the nucleus. The results indicate that the cytoskeleton enhances nuclear mechanical stability by lowering the effective deformability of the nucleus while maintaining nuclear sensitivity to mechanical stimuli. Additionally, the cytoskeleton decreases the strain energy release rate of the nucleus and might thus prevent shape change-induced structural damage to chromatin. © 2018. Published by The Company of Biologists Ltd.

Mechanics of membrane–cytoskeleton attachment in Paramecium

International Nuclear Information System (INIS)

Campillo, C; Nassoy, P; Sykes, C; Jerber, J; Fisch, C; Dupuis-Williams, P; Simoes-Betbeder, M

2012-01-01

In this paper we assess the role of the protein MKS1 (Meckel syndrome type 1) in the cortical membrane mechanics of the ciliated protist Paramecium. This protein is known to be crucial in the process of cilium formation, and we investigate its putative role in membrane–cytoskeleton attachment. Therefore, we compare cells where the gene coding for MKS1 is silenced to wild-type cells. We found that scanning electron microscopy observation of the cell surface reveals a cup-like structure in wild-type cells that is lost in silenced cells. Since this structure is based on the underlying cytoskeleton, one hypothesis to explain this observation is a disruption of membrane attachment to the cytoskeleton in the absence of MKS1 that should affect plasma membrane mechanics. We test this by probing the mechanics of wild-type and silenced cells by micropipette aspiration. Strikingly, we observe that, at the same aspiration pressure, the membrane of silenced cells is easily aspirated by the micropipette whereas that of wild-type cells enters only at a moderate velocity, an effect that suggests a detachment of the membrane from the underlying cytoskeleton in silenced cells. We quantify this detachment by measuring the deformation of the cell cortex and the rate of cell membrane entry in the micropipette. This study offers a new perspective for the characterization of membrane–cytoskeleton attachment in protists and paves the way for a better understanding of the role of membrane–cortex attachment in cilium formation. (paper)

Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

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Cheng Cui

2013-11-01

Full Text Available Planar cell polarity (PCP regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin

Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

International Nuclear Information System (INIS)

Kucharzewska, Paulina; Welch, Johanna E.; Svensson, Katrin J.; Belting, Mattias

2010-01-01

The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by α-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron.

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Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

2016-01-01

Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons.

Roles of the cytoskeleton, cell adhesion and rho signalling in mechanosensing and mechanotransduction.

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Ohashi, Kazumasa; Fujiwara, Sachiko; Mizuno, Kensaku

2017-03-01

All cells sense and respond to various mechanical forces in and mechanical properties of their environment. To respond appropriately, cells must be able to sense the location, direction, strength and duration of these forces. Recent progress in mechanobiology has provided a better understanding of the mechanisms of mechanoresponses underlying many cellular and developmental processes. Various roles of mechanoresponses in development and tissue homeostasis have been elucidated, and many molecules involved in mechanotransduction have been identified. However, the whole picture of the functions and molecular mechanisms of mechanotransduction remains to be understood. Recently, novel mechanisms for sensing and transducing mechanical stresses via the cytoskeleton, cell-substrate and cell-cell adhesions and related proteins have been identified. In this review, we outline the roles of the cytoskeleton, cell-substrate and cell-cell adhesions, and related proteins in mechanosensing and mechanotransduction. We also describe the roles and regulation of Rho-family GTPases in mechanoresponses. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Understanding cytoskeleton regulators in glioblastoma multiforme for therapy design

Directory of Open Access Journals (Sweden)

Masoumi S

2016-09-01

Full Text Available Samaneh Masoumi,1,*, Aditya Harisankar,2,* Aileen Gracias,3 Fabian Bachinger,1 Temesgen Fufa,1,4 Gayathri Chandrasekar,5 Frank Gaunitz,4 Julian Walfridsson,2 Satish S Kitambi1 1Department of Microbiology Tumor and Cell Biology, 2Center for Hematology and Regenerative Medicine, Department of Medicine, 3Department of Neuroscience, Karolinska Institutet, Solna, Sweden; 4Department of Neurosurgery, University Hospital, Leipzig, Germany; 5Department of Biosciences and Nutrition, Karolinska Institutet, Solna, Sweden *These authors contributed equally to this work Abstract: The cellular cytoskeleton forms the primary basis through which a cell governs the changes in size, shape, migration, proliferation, and forms the primary means through which the cells respond to their environment. Indeed, cell and tissue morphologies are used routinely not only to grade tumors but also in various high-content screening methods with an aim to identify new small molecules with therapeutic potential. This study examines the expression of various cytoskeleton regulators in glioblastoma multiforme (GBM. GBM is a very aggressive disease with a low life expectancy even after chemo- and radiotherapy. Cancer cells of GBM are notorious for their invasiveness, ability to develop resistance to chemo- and radiotherapy, and to form secondary site tumors. This study aims to gain insight into cytoskeleton regulators in GBM cells and to understand the effect of various oncology drugs, including temozolomide, on cytoskeleton regulators. We compare the expression of various cytoskeleton regulators in GBM-derived tumor and normal tissue, CD133-postive and -negative cells from GBM and neural cells, and GBM stem-like and differentiated cells. In addition, the correlation between the expression of cytoskeleton regulators with the clinical outcome was examined to identify genes associated with longer patient survival. This was followed by a small molecule screening with US Food and Drug

Role of actin cytoskeleton at multiple levels of T cell activation

Czech Academy of Sciences Publication Activity Database

Huranová, Martina; Štěpánek, Ondřej

2016-01-01

Roč. 3, č. 4 (2016), s. 585-596 ISSN 2372-0301 R&D Projects: GA ČR GJ16-09208Y Institutional support: RVO:68378050 Keywords : T cell * actin * cytoskeleton * TCR * signal transduction * antigen recognition * antigen affinity threshold * immunological synapse Subject RIV: EB - Genetics ; Molecular Biology

The Cytoskeleton-Autophagy Connection.

Science.gov (United States)

Kast, David J; Dominguez, Roberto

2017-04-24

Actin cytoskeleton dynamics play vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also have essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Branched actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Finally, mature autophagosomes detach from the ER membrane by an as yet unknown mechanism, undergo intracellular transport and then fuse with lysosomes, endosomes and multivesicular bodies through mechanisms that involve actin- and microtubule-mediated motility, cytoskeleton-membrane scaffolds and signaling proteins. In this review, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. Published by Elsevier Ltd.

NADPH oxidase complex-derived reactive oxygen species, the actin cytoskeleton, and rho GTPases in cell migration

DEFF Research Database (Denmark)

Stanley, Alanna; Thompson, Kerry; Hynes, Ailish

2014-01-01

Abstract Significance: Rho GTPases are historically known to be central regulators of actin cytoskeleton reorganization. This affects many processes including cell migration. In addition, members of the Rac subfamily are known to be involved in reactive oxygen species (ROS) production through...... mediating cytoskeletal reorganization. Critical Issues: The role of the actin cytoskeleton in providing a scaffold for components of the Nox complex needs to be examined in the light of these new advances. During cell migration, Rho GTPases, ROS, and cytoskeletal organization appear to function as a complex...... compartments. This in conjunction with the analysis of tissues lacking specific Rho GTPases, and Nox components will facilitate a detailed examination of the interactions of these structures with the actin cytoskeleton. In combination with the analysis of ROS production, including its subcellular location...

The plant cytoskeleton controls regulatory volume increase.

Science.gov (United States)

Liu, Qiong; Qiao, Fei; Ismail, Ahmed; Chang, Xiaoli; Nick, Peter

2013-09-01

The ability to adjust cell volume is required for the adaptation to osmotic stress. Plant protoplasts can swell within seconds in response to hypoosmotic shock suggesting that membrane material is released from internal stores. Since the stability of plant membranes depends on submembraneous actin, we asked, whether this regulatory volume control depends on the cytoskeleton. As system we used two cell lines from grapevine which differ in their osmotic tolerance and observed that the cytoskeleton responded differently in these two cell lines. To quantify the ability for regulatory volume control, we used hydraulic conductivity (Lp) as readout and demonstrated a role of the cytoskeleton in protoplast swelling. Chelation of calcium, inhibition of calcium channels, or manipulation of membrane fluidity, did not significantly alter Lp, whereas direct manipulation of the cytoskeleton via specific chemical reagents, or indirectly, through the bacterial elicitor Harpin or activation of phospholipase D, was effective. By optochemical engineering of actin using a caged form of the phytohormone auxin we can break the symmetry of actin organisation resulting in a localised deformation of cell shape indicative of a locally increased Lp. We interpret our findings in terms of a model, where the submembraneous cytoskeleton controls the release of intracellular membrane stores during regulatory volume change. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression of Eag1 K+ channel and ErbBs in human pituitary adenomas: cytoskeleton arrangement patterns in cultured cells.

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del Pliego, Margarita González; Aguirre-Benítez, Elsa; Paisano-Cerón, Karina; Valdovinos-Ramírez, Irene; Rangel-Morales, Carlos; Rodríguez-Mata, Verónica; Solano-Agama, Carmen; Martín-Tapia, Dolores; de la Vega, María Teresa; Saldoval-Balanzario, Miguel; Camacho, Javier; Mendoza-Garrido, María Eugenia

2013-01-01

Pituitary adenomas can invade surrounded tissue, but the mechanism remains elusive. Ether à go-go-1 (Eag1) potassium channel and epidermal growth factor receptors (ErbB1 and ErbB2) have been associated to invasive phenotypes or poor prognosis in cancer patients. However, cells arrange their cytoskeleton in order to acquire a successful migration pattern. We have studied ErbBs and Eag1 expression, and cytoskeleton arrangements in 11 human pituitary adenomas. Eag1, ErbB1 and ErbB2 expression were studied by immunochemistry in tissue and cultured cells. The cytoskeleton arrangement was analyzed in cultured cells by immunofluorescence. Normal pituitary tissue showed ErbB2 expression and Eag1 only in few cells. However, Eag1 and ErbB2 were expressed in all the tumors analyzed. ErbB1 expression was observed variable and did not show specificity for a tumor characteristic. Cultured cells from micro- and macro-adenomas clinically functional organize their cytoskeleton suggesting a mesenchymal pattern, and a round leucocyte/amoeboid pattern from invasive clinically silent adenoma. Pituitary tumors over-express EGF receptors and the ErbB2 repeated expression suggests is a characteristic of adenomas. Eag 1 was express, in different extent, and could be a therapeutic target. The cytoskeleton arrangements observed suggest that pituitary tumor cells acquire different patterns: mesenchymal, and leucocyte/amoeboid, the last observed in the invasive adenomas. Amoeboid migration pattern has been associated with high invasion capacity.

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

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José B Gama

2014-08-01

Full Text Available Buruli ulcer (BU is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1 and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1. In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

Science.gov (United States)

Gama, José B; Ohlmeier, Steffen; Martins, Teresa G; Fraga, Alexandra G; Sampaio-Marques, Belém; Carvalho, Maria A; Proença, Fernanda; Silva, Manuel T; Pedrosa, Jorge; Ludovico, Paula

2014-08-01

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

Comparison of the cell cytoskeleton in migratory and stationary chick fibroblasts

DEFF Research Database (Denmark)

Badley, R A; Couchman, J R; Rees, D A

1980-01-01

The organization of the principal cytoskeletal components (actin, tubulin and 10 nm filament protein) have been compared by immunofluorescence microscopy in two populations of chick heart fibroblasts, previously shown to be adapted respectively for rapid, directed migration or adhesion and growth...... bundles. The variety of patients observed in the migratory cells are documented and the possible roles of the different components of the cytoskeleton in cell locomotion are discussed....

p53 regulates cytoskeleton remodeling to suppress tumor progression.

Science.gov (United States)

Araki, Keigo; Ebata, Takahiro; Guo, Alvin Kunyao; Tobiume, Kei; Wolf, Steven John; Kawauchi, Keiko

2015-11-01

Cancer cells possess unique characteristics such as invasiveness, the ability to undergo epithelial-mesenchymal transition, and an inherent stemness. Cell morphology is altered during these processes and this is highly dependent on actin cytoskeleton remodeling. Regulation of the actin cytoskeleton is, therefore, important for determination of cell fate. Mutations within the TP53 (tumor suppressor p53) gene leading to loss or gain of function (GOF) of the protein are often observed in aggressive cancer cells. Here, we highlight the roles of p53 and its GOF mutants in cancer cell invasion from the perspective of the actin cytoskeleton; in particular its reorganization and regulation by cell adhesion molecules such as integrins and cadherins. We emphasize the multiple functions of p53 in the regulation of actin cytoskeleton remodeling in response to the extracellular microenvironment, and oncogene activation. Such an approach provides a new perspective in the consideration of novel targets for anti-cancer therapy.

[The effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell in vitro].

Science.gov (United States)

Pan, Zhiguo; Shao, Yu; Geng, Yan; Chen, Jinghe; Su, Lei

2015-08-01

To study the effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell ( HUVEC ) in vitro. HUVEC was cultured in vitro in 5%CO(2) medium at 37 centigrade ( control group ) or 43 centigrade ( heat stress group ) for 1 hour. Coomassie brilliant blue R-250 staining was used to determine the effect of heat stress on the cytoskeleton. The cells in heat stress group were subsequently cultured at 37 centigradein 5%CO(2) medium after heat stress for 1 hour, and cell cycle of HUVEC was determined at 0, 6, 12, 18 and 24 hours with flow cytometry. Under light microscopy normal cytoskeleton was observed in control group, but thicker and shorter cytoskeleton was found after a rise of temperature, and stress fibers were found in heat stress group. The DNA content of HUVEC at all time points in G0/G1 stage was 38.07%-55.19% after heat stress. The DNA content in control group was 48.57%, and it was 54.06%, 55.19%, 48.23%, 38.07%, and 41.03% at 0, 6, 12, 18, 24 hours in G0/G1 stage in heat stress group. DNA content in S phase was 35.33%-48.18%. The DNA content in control group was 44.62%, and it was 35.33%, 39.50%, 42.50%, 48.18%, and 47.99% at 0, 6, 12, 18, 24 hours in S stage in heat stress group. DNA content in G2/M phase was 5.31%-13.75%. The DNA content in control group was 6.81, and it was 10.61%, 5.31%, 9.27%,13.75%, and 10.98% at 0, 6, 12, 18, 24 hours in G2/M stage in heat stress group. It was demonstrated that compared with control group, the DNA content in G0/G1 stage was significantly increased when the HUVEC were separated from heat stress within 6 hours, and it recovered at a similar level as control group at 12 hours. Heat stress can change the cytoskeleton of HUVEC, and cause stagnation at G0/G1 stage in cell cycle.

The Cytoskeleton: Mechanical, Physical, and Biological Interactions

Science.gov (United States)

1996-01-01

This workshop, entitled "The Cytoskeleton: Mechanical, Physical, and Biological Interactions," was sponsored by the Center for Advanced Studies in the Space Life Sciences at the Marine Biological Laboratory. This Center was established through a cooperative agreement between the MBL and the Life Sciences Division of the National Aeronautics and Space Administration. To achieve these goals, the Center sponsors a series of workshops on various topics in the life sciences. Elements of the cytoskeleton have been implicated in the effects of gravity on the growth of plants fungi. An intriguing finding in this regard is the report indicating that an integrin-like protein may be the gravireceptor in the internodal cells of Chara. Involvement of the cytoskeleton in cellular graviperception of the basidiomycete Flammulina velutipes has also been reported. Although the responses of mammalian cells to gravity are not well documented, it has been proposed that integrins can act as mechanochemical transducers in mammalian cells. Little is known about the integrated mechanical and physical properties of cytoplasm, this workshop would be the best place to begin developing interdisciplinary approaches to the effects of mechanical stresses on cells and their most likely responsive cytoplasmic elements- the fibrous proteins comprising the cytoskeleton.

Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells.

Science.gov (United States)

Baird, Michelle A; Billington, Neil; Wang, Aibing; Adelstein, Robert S; Sellers, James R; Fischer, Robert S; Waterman, Clare M

2017-01-15

The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A "pulses" occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells. © 2017 Baird et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells

International Nuclear Information System (INIS)

Vale, R.D.; Ignatius, M.J.; Shooter, E.M.

1985-01-01

Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125 I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin

Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton

Science.gov (United States)

Ezzell, R. M.; Goldmann, W. H.; Wang, N.; Parasharama, N.; Ingber, D. E.

1997-01-01

Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.

MCLR-induced PP2A inhibition and subsequent Rac1 inactivation and hyperphosphorylation of cytoskeleton-associated proteins are involved in cytoskeleton rearrangement in SMMC-7721 human liver cancer cell line.

Science.gov (United States)

Wang, Hao; Liu, Jinghui; Lin, Shuyan; Wang, Beilei; Xing, Mingluan; Guo, Zonglou; Xu, Lihong

2014-10-01

Cyanobacteria-derived toxin microcystin-LR (MCLR) has been widely investigated in its effects on normal cells, there is little information concerning its effects on cancer cells. In the present study, the SMMC-7721 human liver cancer cell line treated with MCLR was used to investigate the change of PP2A, cytoskeleton rearrangement, phosphorylation levels of PP2A substrates that related with cytoskeleton stability and explored underlying mechanisms. Here, we confirmed that MCLR entered into SMMC-7721 cells, bound to PP2A/C subunit and inhibited the activity of PP2A. The upregulation of phosphorylation of the PP2A/C subunit and PP2A regulation protein α4, as well as the change in the association of PP2A/C with α4, were responsible for the decrease in PP2A activity. Another novel finding is that the rearrangement of filamentous actin and microtubules led by MCLR may attribute to the increased phosphorylation of HSP27, VASP and cofilin due to PP2A inhibition. As a result of weakened interactions with PP2A and alterations in its subcellular localization, Rac1 may contribute to the cytoskeletal rearrangement induced by MCLR in SMMC-7721 cells. The current paper presents the first report demonstrating the characteristic of PP2A in MCLR exposed cancer cells, which were more susceptible to MCLR compared with the normal cell lines we previously found, which may be owing to the absence of some type of compensatory mechanisms. The hyperphosphorylation of cytoskeleton-associated proteins and Rac1 inactivation which were induced by inhibition of PP2A are shown to be involved in cytoskeleton rearrangement. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bacterial cytoskeleton and implications for new antibiotic targets.

Science.gov (United States)

Wang, Huan; Xie, Longxiang; Luo, Hongping; Xie, Jianping

2016-01-01

Traditionally eukaryotes exclusive cytoskeleton has been found in bacteria and other prokaryotes. FtsZ, MreB and CreS are bacterial counterpart of eukaryotic tubulin, actin filaments and intermediate filaments, respectively. FtsZ can assemble to a Z-ring at the cell division site, regulate bacterial cell division; MreB can form helical structure, and involve in maintaining cell shape, regulating chromosome segregation; CreS, found in Caulobacter crescentus (C. crescentus), can form curve or helical filaments in intracellular membrane. CreS is crucial for cell morphology maintenance. There are also some prokaryotic unique cytoskeleton components playing crucial roles in cell division, chromosome segregation and cell morphology. The cytoskeleton components of Mycobacterium tuberculosis (M. tuberculosis), together with their dynamics during exposure to antibiotics are summarized in this article to provide insights into the unique organization of this formidable pathogen and druggable targets for new antibiotics.

Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells in response to rigidity and shape.

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Mirjam Ochsner

2010-03-01

Full Text Available Various physical parameters, including substrate rigidity, size of adhesive islands and micro-and nano-topographies, have been shown to differentially regulate cell fate in two-dimensional (2-D cell cultures. Cells anchored in a three-dimensional (3-D microenvironment show significantly altered phenotypes, from altered cell adhesions, to cell migration and differentiation. Yet, no systematic analysis has been performed that studied how the integrated cellular responses to the physical characteristics of the environment are regulated by dimensionality (2-D versus 3-D.Arrays of 5 or 10 microm deep microwells were fabricated in polydimethylsiloxane (PDMS. The actin cytoskeleton was compared for single primary fibroblasts adhering either to microfabricated adhesive islands (2-D or trapped in microwells (3-D of controlled size, shape, and wall rigidity. On rigid substrates (Young's Modulus = 1 MPa, cytoskeleton assembly within single fibroblast cells occurred in 3-D microwells of circular, rectangular, square, and triangular shapes with 2-D projected surface areas (microwell bottom surface area and total surface areas of adhesion (microwell bottom plus wall surface area that inhibited stress fiber assembly in 2-D. In contrast, cells did not assemble a detectable actin cytoskeleton in soft 3-D microwells (20 kPa, regardless of their shapes, but did so on flat, 2-D substrates. The dependency on environmental dimensionality was also reflected by cell viability and metabolism as probed by mitochondrial activities. Both were upregulated in 3-D cultured cells versus cells on 2-D patterns when surface area of adhesion and rigidity were held constant.These data indicate that cell shape and rigidity are not orthogonal parameters directing cell fate. The sensory toolbox of cells integrates mechanical (rigidity and topographical (shape and dimensionality information differently when cell adhesions are confined to 2-D or occur in a 3-D space.

Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

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Rieder Gabriele

2011-11-01

Full Text Available Abstract Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS and the effector protein cytotoxin-associated gene A (CagA of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells.

Atomic force microscopy observation of lipopolysaccharide-induced cardiomyocyte cytoskeleton reorganization.

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Wang, Liqun; Chen, Tangting; Zhou, Xiang; Huang, Qiaobing; Jin, Chunhua

2013-08-01

We applied atomic force microscopy (AFM) to observe lipopolysaccharide (LPS)-induced intracellular cytoskeleton reorganization in primary cardiomyocytes from neonatal mouse. The nonionic detergent Triton X-100 was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized by AFM. Using three-dimensional technique of AFM, we were able to quantify the changes of cytoskeleton by the "density" and total "volume" of the cytoskeleton fibers. Compared to the control group, the density of cytoskeleton was remarkably decreased and the volume of cytoskeleton was significantly increased after LPS treatment, which suggests that LPS may induce the cytoskeleton reorganization and change the cardiomyocyte morphology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement.

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Dinicola, Simona; Fabrizi, Gianmarco; Masiello, Maria Grazia; Proietti, Sara; Palombo, Alessandro; Minini, Mirko; Harrath, Abdel Halim; Alwasel, Saleh H; Ricci, Giulia; Catizone, Angela; Cucina, Alessandra; Bizzarri, Mariano

2016-07-01

Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, β-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites.

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Gómez de León, Carmen T; Díaz Martín, Rubén Darío; Mendoza Hernández, Guillermo; González Pozos, Sirenia; Ambrosio, Javier R; Mondragón Flores, Ricardo

2014-12-05

Toxoplasma, the causative agent of toxoplasmosis in animals and humans, has a subpellicular cytoskeleton that is involved in motility, cell shape and invasion. Knowledge of components of the cytoskeleton is necessary to understand the invasion mechanisms as well as for the identification of possible therapeutic targets. To date, most cytoskeletal components of Toxoplasma remain unidentified due mainly to the lack of reproducible methods for their isolation. Based on the successful isolation of the cytoskeleton, it was possible to report for the first time, the proteomic characterization of the subpellicular cytoskeleton of Toxoplasma formed by 95 cytoskeletal proteins through proteomic analysis by tandem mass spectrometry of one dimension SDS PAGE. By bioinformatic analysis of the data, proteins were classified as: 18 conventional cytoskeletal proteins; 10 inner membrane complex proteins, including 7 with alveolin repeats; 5 new proteins with alveolin like repeats; 37 proteins associated with other organelles and 25 novel proteins of unknown function. One of the alveolin like proteins not previously described in Toxoplasma named TgArticulin was partially characterized with a specific monoclonal antibody. Presence of TgArticulin was exclusively associated with the cytoskeleton fraction with a cortical distribution. Functions for the several molecules identified are proposed. This manuscript describes, for the first time, the proteome of the subpellicular cytoskeleton of Toxoplasma gondii. The importance of this study is related to the role of the cytoskeleton in the highly invasive capability of a parasite that causes abortion, blindness, and death by encephalitis in immunocompromised patients. Proteomic characterization of the cytoskeleton of T. gondii tachyzoites was possible by the development of a successful procedure for the isolation of the subpellicular cytoskeleton. Knowledge of the composition of the cytoskeleton of Toxoplasma is fundamental for the

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

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Fernando Navarro-Garcia

2013-01-01

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

Spatial constraints and the organization of the cytoskeleton

NARCIS (Netherlands)

Ga^rlea, I.C.

2015-01-01

The shape of animal cells is in controlled by a network of filamentous polymers called the cytoskeleton. The two main components of the cytoskeleton are actin filaments and microtubules. These polymers continuously reorganize in order to performed their diverse cellular functions. For example, in

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

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Wanderley de Souza

2012-05-01

Full Text Available For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

Complete disintegration of the microtubular cytoskeleton precedes its auxin-mediated reconstruction in postmitotic maize root cells

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Baluska, F.; Barlow, P. W.; Volkmann, D.

1996-01-01

The inhibitory action of 0.1 microM auxin (IAA) on maize root growth was closely associated with a rapid and complete disintegration of the microtubular (MT) cytoskeleton, as visualized by indirect immunofluorescence of tubulin, throughout the growth region. After 30 min of this treatment, only fluorescent spots were present in root cells, accumulating either around nuclei or along cell walls. Six h later, in addition to some background fluorescence, dense but partially oriented oblique or longitudinal arrays of cortical MTs (CMTs) were found in most growing cells of the root apex. After 24 h of treatment, maize roots had adapted to the auxin, as inferred from the slowly recovering elongation rate and from the reassembly of a dense and well-ordered MT cytoskeleton which showed only slight deviations from that of the control root cells. Taxol pretreatment (100 microM, 24 h) prevented not only the rapid auxin-mediated disintegration of the MT cytoskeleton but also a reorientation of the CMT arrays, from transversal to longitudinal. The only tissue to show MTs in their cells throughout the auxin treatment was the epidermis. Significant resistance of transverse CMT arrays in these cells towards auxin was confirmed using a higher auxin concentration (100 microM, 24 h). The latter auxin dose also revealed inter-tissue-specific responses to auxin: outer cortical cell files reoriented their CMTs from the transversal to longitudinal orientation, whereas inner cortical cell files lost their MTs. This high auxin-mediated response, associated with the swelling of root apices, was abolished with the pretreatment of maize root with taxol.

Proteomic approaches to understanding the role of the cytoskeleton in host-defense mechanisms

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Radulovic, Marko; Godovac-Zimmermann, Jasminka

2014-01-01

The cytoskeleton is a cellular scaffolding system whose functions include maintenance of cellular shape, enabling cellular migration, division, intracellular transport, signaling and membrane organization. In addition, in immune cells, the cytoskeleton is essential for phagocytosis. Following the advances in proteomics technology over the past two decades, cytoskeleton proteome analysis in resting and activated immune cells has emerged as a possible powerful approach to expand our understanding of cytoskeletal composition and function. However, so far there have only been a handful of studies of the cytoskeleton proteome in immune cells. This article considers promising proteomics strategies that could augment our understanding of the role of the cytoskeleton in host-defense mechanisms. PMID:21329431

Transcription factor CREB is involved in CaSR-mediated cytoskeleton gene expression.

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Huang, Shuaishuai; Ren, Yu; Wang, Ping; Li, Yanyuan; Wang, Xue; Zhuang, Haihui; Fang, Rong; Wang, Yuduo; Liu, Ningsheng; Hehir, Michael; Zhou, Jeff X

2015-03-01

Our previous studies illustrated that a steady increase of intracellular calcium concentration ([Ca2+]i) was important for maintaining microtubules (MTs) rearrangement in apoptotic cells. However, little is known about the effect of calcium sensing receptor (CaSR)-mediated increase in [Ca2+]i on cytoskeleton gene expression. We examined the impact of taxol or CaSR agonist/antagonist on the regulation of [Ca2+]i concentration, cytoskeleton arrangement, phosphorylated CREB and cytoskeleton gene expressions in HeLa cells with dominant negative plasmid of CREB (PM). This study demonstrated that Gdcl3 (a specific CaSR agonist) evoked a rapid increase of [Ca2+]i, formed a rigid bundle of MTs which surrounded the nucleus and decreased the cytoskeleton gene expressions in HeLa cells. These effects were rescued by addition of NPS2390 (a specific CaSR antagonist). Moreover, CaSR activity affected cytoskeleton gene expression through transcription factor CREB. Histoscores of pCREB immunoreactivity in tissues of cervical adenocarcinoma, renal clear cell carcinoma, and diffuse large B-cell lymphoma were markedly increased compared with non malignant tissue. These data demonstrate, for the first time, that CaSR-mediated increase in [Ca2+]i probably modulate cytoskeleton organization and gene expression via transcription factor. © 2014 Wiley Periodicals, Inc.

Dual effect of F-actin targeted carrier combined with antimitotic drug on aggressive colorectal cancer cytoskeleton: Allying dissimilar cell cytoskeleton disrupting mechanisms.

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Taranejoo, Shahrouz; Janmaleki, Mohsen; Pachenari, Mohammad; Seyedpour, Seyed Morteza; Chandrasekaran, Ramya; Cheng, Wenlong; Hourigan, Kerry

2016-11-20

A recent approach to colon cancer therapy is to employ selective drugs with specific extra/intracellular sites of action. Alteration of cytoskeletal protein reorganization and, subsequently, to cellular biomechanical behaviour during cancer progression highly affects the cancer cell progress. Hence, cytoskeleton targeted drugs are an important class of cancer therapy agents. We have studied viscoelastic alteration of the human colon adenocarcinoma cell line, SW48, after treatment with a drug delivery system comprising chitosan as the carrier and albendazole as the microtubule-targeting agent (MTA). For the first time, we have evaluated the biomechanical characteristics of the cell line, using the micropipette aspiration (MA) method after treatment with drug delivery systems. Surprisingly, employing a chitosan-albendazole pair, in comparison with both neat materials, resulted in more significant change in the viscoelastic parameters of cells, including the elastic constants (K 1 and K 2 ) and the coefficient of viscosity (μ). This difference was more pronounced for cancer cells after 48h of the treatment. Microtubule and actin microfilament (F-actin) contents in the cell line were studied by immunofluorescent staining. Good agreement was observed between the mechanical characteristics results and microtubule/F-actin contents of the treated SW48 cell line, which declined after treatment. The results showed that chitosan affected F-actin more, while MTA was more effective for microtubules. Toxicity studies were performed against two cancer cell lines (SW48 and MCF10CA1h) and compared to normal cells, MCF10A. The results showed cancer selectiveness, safety of formulation, and enhanced anticancer efficacy of the CS/ABZ conjugate. This study suggests that employing such a suitable pair of drug-carriers with dissimilar sites of action, thus allying the different cell cytoskeleton disrupting mechanisms, may provide a more efficient cancer therapy approach. Copyright �

Protein phosphatase 2A inhibition and subsequent cytoskeleton reorganization contributes to cell migration caused by microcystin-LR in human laryngeal epithelial cells (Hep-2).

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Wang, Beilei; Liu, Jinghui; Huang, Pu; Xu, Kailun; Wang, Hanying; Wang, Xiaofeng; Guo, Zonglou; Xu, Lihong

2017-03-01

The major toxic mechanism of Microcystin-LR is inhibition of the activity of protein phosphatase 2A (PP2A), resulting in a series of cytotoxic effects. Our previous studies have demonstrated that microcystin-LR (MCLR) induced very different molecular effects in normal cells and the tumor cell line SMMC7721. To further explore the MCLR toxicity mechanism in tumor cells, human laryngeal epithelial cells (Hep-2) was examined in this study. Western blot, immunofluorescence, immunoprecipitation, and transwell migration assay were used to detect the effects of MCLR on PP2A activity, PP2A substrates, cytoskeleton, and cell migration. The results showed that the protein level of PP2A subunits and the posttranslational modification of the catalytic subunit were altered and that the binding of the AC core enzyme as well as the binding of PP2A/C and α4, was also affected. As PP2A substrates, the phosphorylation of MAPK pathway members, p38, ERK1/2, and the cytoskeleton-associated proteins, Hsp27, VASP, Tau, and Ezrin were increased. Furthermore, MCLR induced reorganization of the cytoskeleton and promoted cell migration. Taken together, direct covalent binding to PP2A/C, alteration of the protein levels and posttranslational modification, as well as the binding of subunits, are the main pattern for the effects of MCLR on PP2A in Hep-2. A dose-dependent change in p-Tau and p-Ezrin due to PP2A inhibition may contribute to the changes in the cytoskeleton and be related to the cell migration in Hep-2. Our data provide a comprehensive exposition of the MCLR mechanism on tumor cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 890-903, 2017. © 2016 Wiley Periodicals, Inc.

Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

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Hafner, Anne E.; Rieger, Heiko

2018-03-01

The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

Response of the microtubular cytoskeleton following hyperthermia as a prognostic indicator of survival of Chinese hamster ovary cells

International Nuclear Information System (INIS)

Coss, Ronald A.; Alden, Mark E.; Wachsberger, Phyllis R.; Smith, Nancy N.

1996-01-01

Purpose: The response of the microtubular (MT) cytoskeleton to hyperthermia was assessed as a prognostic indicator of cytotoxicity. Methods and Materials: Heat-induced collapse and subsequent recovery of the MT system were compared with survival for both nonthermotolerant (NT) and thermotolerant (TT) G1 populations of Chinese hamster ovary (CHO) cells. The response of the MT system was monitored using immunofluorescence staining. The G1 populations of NT and TT cells were heated by submersion in 45.0 and 43.0 deg. C waterbaths. Results: Heat-induced perinuclear collapse of the MT system did not correlate with survival for the NT and TT populations. However, recovery of the organization of the MT cytoskeleton was correlatable with survival. The regression line of survival plotted as a function of MT recovery is fit by: y = -0.43 + 1.03x, r 2 = 0.95 (p < 0.0005). Conclusion: Restoration of the organization of the MT cytoskeleton following hyperthermia may be used as a prognostic indicator of survival of CHO cells heated in G1

[Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells].

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Xiaolan, He; Guangjie, Bao; Linglu, Sun; Xue, Zhang; Shanying, Bao; Hong, Kang

2017-08-01

Objective The effect of different oxygen tensions on the cytoskeleton remodeling of goat temporomandibular joint (TMJ) disc cells were investigated. Methods Goat TMJ disc cells were cultured under normoxia (21% O₂) and hypoxia (2%, 4%, and 8% O₂). Toluidine blue, picrosirius red, and type Ⅰ collagen immunocytochemical staining were performed to observe the changes in cell phenotype under different oxygen levels. Immunofluorescent staining and real-time reverse transcription-polymerase chain reaction analysis were then performed to identify actin, tubulin, and vimentin in the cultured disc cells. Results TMJ disc cells still displayed fibroblast characteristics under different oxygen levels and their cytoskeletons had regular arrangement. The fluorescence intensities of actin and vimentin were lowest at 4% O₂(P0.05). Actin mRNA levels were considerably decreased at 2% O₂ and 4% O₂ in hypoxic conditions, while actin mRNA expression was highest in 21% O₂. Tubulin mRNA levels considerably increased at 2% O₂, while tubulin mRNA expression was lowest in 8% O₂ (Plevels among these oxygen levels (Poxygen tensions, and 2% O₂ may be the optimal oxygen level required to proliferate TMJ disc cells.

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

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Eiko Kitamura

Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

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Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

2011-01-01

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

Atomic force microscopy and confocal laser scanning microscopy on the cytoskeleton of permeabilised and embedded cells

International Nuclear Information System (INIS)

Meller, Karl; Theiss, Carsten

2006-01-01

We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 o C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton

High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization

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Andolfi, Laura; Murello, Anna; Cassese, Damiano; Ban, Jelena; Dal Zilio, Simone; Lazzarino, Marco

2017-04-01

Cell-cell and cell-matrix interactions are essential to the survival and proliferation of most cells, and are responsible for triggering a wide range of biochemical pathways. More recently, the biomechanical role of those interactions was highlighted, showing, for instance, that adhesion forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small size, high aspect ratio and anisotropic mechanical response represent a useful model to investigate the forces involved in the adhesion processes and their role in cellular development. In this work we explored and quantified, by single cell force spectroscopy (SCFS), the interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that the cell adhesion forces are comparable to those found on collagen and bare glass coverslip, analogously the membrane tether extraction forces are similar to that on collagen but stronger than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a reduced proliferation after 36 h was observed. On the contrary both cell morphology and cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was characterized by a large number of filopodia and a significant decrease of the cell mobility. The cytoskeleton organization was characterized by the absence of actin fibers, which were instead dominant on collagen and flat glass support. Such findings suggest that the mechanical properties of disordered Si NWs, and in particular their strong asymmetry, play a major role in the adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion measurements by SCFS provide out-of-plane forces values consistent with those measured on conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and migration processes.

Multiple roles for the actin cytoskeleton during regulated exocytosis

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Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

2014-01-01

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

Super-Resolution Microscopy Reveals the Native Ultrastructure of the Erythrocyte Cytoskeleton

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Leiting Pan

2018-01-01

Full Text Available The erythrocyte cytoskeleton is a textbook prototype for the submembrane cytoskeleton of metazoan cells. While early experiments suggest a triangular network of actin-based junctional complexes connected by ∼200-nm-long spectrin tetramers, later studies indicate much smaller junction-to-junction distances in the range of 25-60 nm. Through super-resolution microscopy, we resolve the native ultrastructure of the cytoskeleton of membrane-preserved erythrocytes for the N and C termini of β-spectrin, F-actin, protein 4.1, tropomodulin, and adducin. This allows us to determine an ∼80-nm junction-to-junction distance, a length consistent with relaxed spectrin tetramers and theories based on spectrin abundance. Through two-color data, we further show that the cytoskeleton meshwork often contains nanoscale voids where the cell membrane remains intact and that actin filaments and capping proteins localize to a subset of, but not all, junctional complexes. Together, our results call for a reassessment of the structure and function of the submembrane cytoskeleton.

Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells.

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Hafner, Anne E; Rieger, Heiko

2016-11-15

Intracellular transport is vital for the proper functioning and survival of a cell. Cargo (proteins, vesicles, organelles, etc) is transferred from its place of creation to its target locations via molecular motor assisted transport along cytoskeletal filaments. The transport efficiency is strongly affected by the spatial organization of the cytoskeleton, which constitutes an inhomogeneous, complex network. In cells with a centrosome microtubules grow radially from the central microtubule organizing center towards the cell periphery whereas actin filaments form a dense meshwork, the actin cortex, underneath the cell membrane with a broad range of orientations. The emerging ballistic motion along filaments is frequently interrupted due to constricting intersection nodes or cycles of detachment and reattachment processes in the crowded cytoplasm. In order to investigate the efficiency of search strategies established by the cell's specific spatial organization of the cytoskeleton we formulate a random velocity model with intermittent arrest states. With extensive computer simulations we analyze the dependence of the mean first passage times for narrow escape problems on the structural characteristics of the cytoskeleton, the motor properties and the fraction of time spent in each state. We find that an inhomogeneous architecture with a small width of the actin cortex constitutes an efficient intracellular search strategy.

Nuclear pore complex tethers to the cytoskeleton.

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Goldberg, Martin W

2017-08-01

The nuclear envelope is tethered to the cytoskeleton. The best known attachments of all elements of the cytoskeleton are via the so-called LINC complex. However, the nuclear pore complexes, which mediate the transport of soluble and membrane bound molecules, are also linked to the microtubule network, primarily via motor proteins (dynein and kinesins) which are linked, most importantly, to the cytoplasmic filament protein of the nuclear pore complex, Nup358, by the adaptor BicD2. The evidence for such linkages and possible roles in nuclear migration, cell cycle control, nuclear transport and cell architecture are discussed. Copyright © 2017. Published by Elsevier Ltd.

Physically-induced cytoskeleton remodeling of cells in three-dimensional culture.

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Sheng-Lin Lee

Full Text Available Characterizing how cells in three-dimensional (3D environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension.

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells

International Nuclear Information System (INIS)

Ohira, Koji; Homma, Koichi J.; Hirai, Hirohisa; Nakamura, Shun; Hayashi, Motoharu

2006-01-01

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton

The nesprin-cytoskeleton interface probed directly on single nuclei is a mechanically rich system.

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Balikov, Daniel A; Brady, Sonia K; Ko, Ung Hyun; Shin, Jennifer H; de Pereda, Jose M; Sonnenberg, Arnoud; Sung, Hak-Joon; Lang, Matthew J

2017-09-03

The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex. Using single-molecule on single nuclei assays, we report that the interactions between the nucleus and the cytoskeleton, thought to be nesprin-cytoskeleton interactions, are highly sensitive to force magnitude and direction depending on whether cells are historically interfaced with the matrix or with cell aggregates. Application of ∼10-30 pN forces to these nesprin linkages yielded structural transitions, with a base transition size of 5-6 nm, which are speculated to be associated with partial unfoldings of the spectrin domains of the nesprins and/or structural changes of histones within the nucleus.

The Spectrin cytoskeleton regulates the Hippo signalling pathway.

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Fletcher, Georgina C; Elbediwy, Ahmed; Khanal, Ichha; Ribeiro, Paulo S; Tapon, Nic; Thompson, Barry J

2015-04-01

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Divergent regulation of the sarcomere and the cytoskeleton.

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Schevzov, Galina; Fath, Thomas; Vrhovski, Bernadette; Vlahovich, Nicole; Rajan, Sudarsan; Hook, Jeff; Joya, Josephine E; Lemckert, Frances; Puttur, Franz; Lin, Jim J-C; Hardeman, Edna C; Wieczorek, David F; O'Neill, Geraldine M; Gunning, Peter W

2008-01-04

The existence of a feedback mechanism regulating the precise amounts of muscle structural proteins, such as actin and the actin-associated protein tropomyosin (Tm), in the sarcomeres of striated muscles is well established. However, the regulation of nonmuscle or cytoskeletal actin and Tms in nonmuscle cell structures has not been elucidated. Unlike the thin filaments of striated muscles, the actin cytoskeleton in nonmuscle cells is intrinsically dynamic. Given the differing requirements for the structural integrity of the actin thin filaments of the sarcomere compared with the requirement for dynamicity of the actin cytoskeleton in nonmuscle cells, we postulated that different regulatory mechanisms govern the expression of sarcomeric versus cytoskeletal Tms, as key regulators of the properties of the actin cytoskeleton. Comprehensive analyses of tissues from transgenic and knock-out mouse lines that overexpress the cytoskeletal Tms, Tm3 and Tm5NM1, and a comparison with sarcomeric Tms provide evidence for this. Moreover, we show that overexpression of a cytoskeletal Tm drives the amount of filamentous actin.

Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells

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Hafner, Anne E.; Rieger, Heiko

2016-12-01

Intracellular transport is vital for the proper functioning and survival of a cell. Cargo (proteins, vesicles, organelles, etc) is transferred from its place of creation to its target locations via molecular motor assisted transport along cytoskeletal filaments. The transport efficiency is strongly affected by the spatial organization of the cytoskeleton, which constitutes an inhomogeneous, complex network. In cells with a centrosome microtubules grow radially from the central microtubule organizing center towards the cell periphery whereas actin filaments form a dense meshwork, the actin cortex, underneath the cell membrane with a broad range of orientations. The emerging ballistic motion along filaments is frequently interrupted due to constricting intersection nodes or cycles of detachment and reattachment processes in the crowded cytoplasm. In order to investigate the efficiency of search strategies established by the cell’s specific spatial organization of the cytoskeleton we formulate a random velocity model with intermittent arrest states. With extensive computer simulations we analyze the dependence of the mean first passage times for narrow escape problems on the structural characteristics of the cytoskeleton, the motor properties and the fraction of time spent in each state. We find that an inhomogeneous architecture with a small width of the actin cortex constitutes an efficient intracellular search strategy.

Chronophin activation is necessary in Doxorubicin-induced actin cytoskeleton alteration.

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Lee, Su Jin; Park, Jeen Woo; Kang, Beom Sik; Lee, Dong-Seok; Lee, Hyun-Shik; Choi, Sooyoung; Kwon, Oh-Shin

2017-06-01

Although doxorubicin (Dox)-induced oxidative stress is known to be associated with cytotoxicity, the precise mechanism remains unclear. Genotoxic stress not only generates free radicals, but also affects actin cytoskeleton stability. We showed that Dox-induced RhoA signaling stimulated actin cytoskeleton alterations, resulting in central stress fiber disruption at early time points and cell periphery cortical actin formation at a later stage, in HeLa cells. Interestingly, activation of a cofilin phosphatase, chronophin (CIN), was initially evoked by Dox-induced RhoA signaling, resulting in a rapid phosphorylated cofilin turnover leading to actin cytoskeleton remodeling. In addition, a novel interaction between CIN and 14-3-3ζ was detected in the absence of Dox treatment. We demonstrated that CIN activity is quite contrary to 14-3-3ζ binding, and the interaction leads to enhanced phosphorylated cofilin levels. Therefore, initial CIN activation regulation could be critical in Dox-induced actin cytoskeleton remodeling through RhoA/cofilin signaling. [BMB Reports 2017; 50(6): 335-340].

Regional differences in endothelial cell cytoskeleton, junctional proteins and phosphorylated tyrosine labeling in the porcine vortex vein system.

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Tan, Priscilla Ern Zhi; Yu, Paula K; Yang, Hongfang; Cringle, Stephen J; Yu, Dao-Yi

2018-07-01

We previously demonstrated endothelial phenotype heterogeneity in the vortex vein system. This study is to further determine whether regional differences are present in the cytoskeleton, junctional proteins and phosphorylated tyrosine labeling within the system. The vortex vein system of twenty porcine eyes was perfused with labels for f-actin, claudin-5, VE-Cadherin, phosphorylated tyrosine and nucleic acid. The endothelial cells of eight different regions (choroidal veins, pre-ampulla, anterior ampulla, mid-ampulla, posterior ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein) were studied using confocal microscopy. There were regional differences in the endothelial cell structures. Cytoskeleton labeling was relatively even in intensity throughout Regions 1 to 6. Overall VE-Cadherin had a non-uniform distribution and thicker width endothelial cell border staining than claudin-5. Progressing downstream there was an increased variation in thickness of VE-cadherin labeling. There was an overlap in phosphorylated tyrosine and VE-Cadherin labeling in the post-ampulla, intra-scleral canal and extra-ocular vortex vein. Intramural cells were observed that were immune-positive for VE-Cadherin and phosphorylated tyrosine. There were significant differences in the number of intramural cells in different regions. Significant regional differences with endothelial cell labeling of cytoskeleton, junction proteins, and phosphorylated tyrosine were found within the vortex vein system. These findings support existing data on endothelial cell phenotype heterogeneity, and may aid in the knowledge of venous pathologies by understanding regions of vulnerability to endothelial damage within the vortex vein system. It could be valuable to further investigate and characterize the VE-cadherin and phosphotyrosine immune-positive intramural cells. Copyright © 2018. Published by Elsevier Ltd.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

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Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

International Nuclear Information System (INIS)

Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

2007-01-01

Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

Prolactin promotes breast cancer cell migration through actin cytoskeleton remodeling

Directory of Open Access Journals (Sweden)

Priscilla Ludovico da Silva

2015-12-01

Full Text Available The role of prolactin on breast cancer development and progression is debated. Breast cancer progression largely depends on cell movement and on the ability to remodel the actin cytoskeleton. In this process, actin-binding proteins are requested to achieve fibrillar actin de-polymerization and relocation at the cell membrane. Kinases such as focal adhesion kinase (FAK are later required to form actin/vinculin-enriched structures called focal adhesion complexes, which mediate firm adhesion to the extracellular matrix. These controllers are regulated by c-Src, which forms multiprotein signaling complexes with membrane receptors and is regulated by a number of hormones, including prolactin. We here show that breast cancer cells exposed to prolactin display an elevated c-Src expression and phosphorylation. In parallel, increased moesin and FAK expression and phosphorylation are found. These molecular changes are associated to relocation to the plasma membrane of cytoskeletal actin fibers and to increased horizontal cell movement. In conclusion, prolactin regulates actin remodeling and enhances breast cancer cell movement. This finding broadens the understanding of prolactin actions on breast cancer cells, highlighting new pathways that may be relevant to on breast cancer progression.

Influence of chitosan-chitin nanofiber composites on cytoskeleton structure and the proliferation of rat bone marrow stromal cells.

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Kiroshka, Victoria V; Petrova, Valentina A; Chernyakov, Daniil D; Bozhkova, Yulia O; Kiroshka, Katerina V; Baklagina, Yulia G; Romanov, Dmitry P; Kremnev, Roman V; Skorik, Yury A

2017-01-01

Chitosan scaffolds have gained much attention in various tissue engineering applications, but the effect of their microstructure on cell-material spatial interactions remains unclear. Our objective was to evaluate the effect of chitosan-based matrices doping with chitin nano-whiskers (CNW) on adhesion, spreading, cytoskeleton structure, and proliferation of rat bone marrow stromal cells (BMSCs). The behavior of BMSCs during culture on chitosan-CNW films was determined by the molecular mass, hydrophobicity, porosity, crosslinking degree, protonation degree and molecular structure of the composite chitosan-CNW films. The shape, spreading area, cytoskeleton structure, and proliferation of BMSCs on chitosan matrices with a crystalline structure and high porosity were similar to that observed for BMSCs cultured on polystyrene tissue culture plates. The amorphous polymer structure and high swelling led to a decrease in the spreading area and cell proliferation. Thus, we can control the behavior of cells in culture (adhesion, spreading, and proliferation) by changing the physico-chemical properties of the chitosan-CNW films.

SnapShot: The Bacterial Cytoskeleton.

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Fink, Gero; Szewczak-Harris, Andrzej; Löwe, Jan

2016-07-14

Most bacteria and archaea contain filamentous proteins and filament systems that are collectively known as the bacterial cytoskeleton, though not all of them are cytoskeletal, affect cell shape, or maintain intracellular organization. To view this SnapShot, open or download the PDF. Copyright © 2016. Published by Elsevier Inc.

Coupling of cytoskeleton functions for fibroblast locomotion

DEFF Research Database (Denmark)

Couchman, J R; Lenn, M; Rees, D A

1985-01-01

caused visible protrusions in projected positions at the leading edge. We conclude that fibroblast locomotion may be driven coordinately by a common set of motility mechanisms and that this coordination may be lost as a result of physical or pharmacological disturbance. Taking our evidence with results...... from other Laboratories, we propose the following cytoskeleton functions. (i) Protrusive activity, probably based on solation--gelation cycles of the actin based cytoskeleton and membrane recycling which provides cellular and membrane components for streaming through the cell body to the leading edge...

Scaffold composition affects cytoskeleton organization, cell-matrix interaction and the cellular fate of human mesenchymal stem cells upon chondrogenic differentiation.

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Li, Yuk Yin; Choy, Tze Hang; Ho, Fu Chak; Chan, Pui Barbara

2015-06-01

The stem cell niche, or microenvironment, consists of soluble, matrix, cell and mechanical factors that together determine the cellular fates and/or differentiation patterns of stem cells. Collagen and glycosaminoglycans (GAGs) are important scaffolding materials that can mimic the natural matrix niche. Here, we hypothesize that imposing changes in the scaffold composition or, more specifically, incorporating GAGs into the collagen meshwork, will affect the morphology, cytoskeletal organization and integrin expression profiles, and hence the fate of human mesenchymal stem cells (MSCs) upon the induction of differentiation. Using chondrogenesis as an example, we microencapsulated MSCs in three scaffold systems that had varying matrix compositions: collagen alone (C), aminated collagen (AC) and aminated collagen with GAGs (ACG). We then induced the MSCs to differentiate toward a chondrogenic lineage, after which, we characterized the cell viability and morphology, as well as the level of cytoskeletal organization and the integrin expression profile. We also studied the fate of the MSCs by evaluating the major chondrogenic markers at both the gene and protein level. In C, MSC chondrogenesis was successfully induced and MSCs that spread in the scaffolds had a clear actin cytoskeleton; they expressed integrin α2β1, α5 and αv; promoted sox9 nuclear localization transcription activation; and upregulated the expression of chondrogenic matrix markers. In AC, MSC chondrogenesis was completely inhibited but the scaffold still supported cell survival. The MSCs did not spread and they had no actin cytoskeleton; did not express integrin α2 or αv; they failed to differentiate into chondrogenic lineage cells even on chemical induction; and there was little colocalization or functional interaction between integrin α5 and fibronectin. In ACG, although the MSCs did not express integrin α2, they did express integrin αv and there was strong co-localization and hence functional

Stiffening of Red Blood Cells Induced by Cytoskeleton Disorders: A Joint Theory-Experiment Study.

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Lai, Lipeng; Xu, Xiaofeng; Lim, Chwee Teck; Cao, Jianshu

2015-12-01

The functions and elasticities of the cell are largely related to the structures of the cytoskeletons underlying the lipid bilayer. Among various cell types, the red blood cell (RBC) possesses a relatively simple cytoskeletal structure. Underneath the membrane, the RBC cytoskeleton takes the form of a two-dimensional triangular network, consisting of nodes of actins (and other proteins) and edges of spectrins. Recent experiments focusing on the malaria-infected RBCs (iRBCs) show that there is a correlation between the elongation of spectrins in the cytoskeletal network and the stiffening of the iRBCs. Here we rationalize the correlation between these two observations by combining the wormlike chain model for single spectrins and the effective medium theory for the network elasticity. We specifically focus on how the disorders in the cytoskeletal network affect its macroscopic elasticity. Analytical and numerical solutions from our model reveal that the stiffness of the membrane increases with increasing end-to-end distances of spectrins, but has a nonmonotonic dependence on the variance of the end-to-end distance distributions. These predictions are verified quantitatively by our atomic force microscopy and micropipette aspiration measurements of iRBCs. The model may, from a molecular level, provide guidelines for future identification of new treatment methods for RBC-related diseases, such as malaria infection. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

International Nuclear Information System (INIS)

Johnson-Lyles, Denise N.; Peifley, Kimberly; Lockett, Stephen; Neun, Barry W.; Hansen, Matthew; Clogston, Jeffrey; Stern, Stephan T.; McNeil, Scott E.

2010-01-01

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C 60 OH x ), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

DEFF Research Database (Denmark)

Walmod, P S; Skladchikova, G; Kawa, A

1999-01-01

for the VPA mediated inhibition of motility. In addition it is shown that the actomyosin cytoskeleton of VPA-treated cells was capable of contraction upon exposure to ATP, indicating that the reduced motility of VPA-treated cells was not caused by an inhibition of actomyosin contraction. On the other hand...... state. These findings indicate that VPA affects cell morphology and motility through interference with the dynamics of the actin cytoskeleton....

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

International Nuclear Information System (INIS)

Djuzenova, Cholpon S.; Fiedler, Vanessa; Memmel, Simon; Katzer, Astrid; Hartmann, Susanne; Krohne, Georg; Zimmermann, Heiko; Scholz, Claus-Jürgen; Polat, Bülent; Flentje, Michael

2015-01-01

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

Energy Technology Data Exchange (ETDEWEB)

Djuzenova, Cholpon S., E-mail: djuzenova_t@ukw.de [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Fiedler, Vanessa [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Memmel, Simon [Lehrstuhl für Biotechnologie und Biophysik, Universität Würzburg, Biozentrum Am Hubland, 97070 Würzburg (Germany); Katzer, Astrid; Hartmann, Susanne [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Krohne, Georg [Elektronenmikroskopie, Biozentrum, Universität Würzburg, Am Hubland, 97070 Würzburg (Germany); Zimmermann, Heiko [Hauptabteilung Biophysik and Kryotechnologie, Fraunhofer-Institut für Biomedizinische Technik, Lehrstuhl für Molekulare und Zelluläre Biotechnologie/Nanotechnologie, Universität des Saarlandes, Ensheimer Strasse 48, 66386 St. Ingbert (Germany); Scholz, Claus-Jürgen [Interdisciplinary Center for Clinical Research, University Hospital, Versbacher Strasse 7, 97078 Würzburg (Germany); Polat, Bülent; Flentje, Michael [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); and others

2015-01-15

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed.

The transmembrane adaptor protein NTAL signals to mast cell cytoskeleton via the small GTPase Rho

Czech Academy of Sciences Publication Activity Database

Tůmová, Magda; Koffer, Anna; Šimíček, Michal; Dráberová, Lubica; Dráber, Petr

2010-01-01

Roč. 40, č. 11 (2010), s. 3235-3245 ISSN 0014-2980 R&D Projects: GA MŠk 1M0506; GA MŠk LC545; GA ČR(CZ) GD204/05/H023; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA AV ČR KAN200520701 Institutional research plan: CEZ:AV0Z50520514 Keywords : cell activation * cytoskeleton * mast cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.942, year: 2010

Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

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Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

2010-01-01

The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

Membrane Microdomains and Cytoskeleton Organization Shape and Regulate the IL-7 Receptor Signalosome in Human CD4 T-cells*

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Tamarit, Blanche; Bugault, Florence; Pillet, Anne-Hélène; Lavergne, Vincent; Bochet, Pascal; Garin, Nathalie; Schwarz, Ulf; Thèze, Jacques; Rose, Thierry

2013-01-01

Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center. PMID:23329834

Ethanol exposure disrupts extraembryonic microtubule cytoskeleton and embryonic blastomere cell adhesion, producing epiboly and gastrulation defects

Directory of Open Access Journals (Sweden)

Swapnalee Sarmah

2013-08-01

Fetal alcohol spectrum disorder (FASD occurs when pregnant mothers consume alcohol, causing embryonic ethanol exposure and characteristic birth defects that include craniofacial, neural and cardiac defects. Gastrulation is a particularly sensitive developmental stage for teratogen exposure, and zebrafish is an outstanding model to study gastrulation and FASD. Epiboly (spreading blastomere cells over the yolk cell, prechordal plate migration and convergence/extension cell movements are sensitive to early ethanol exposure. Here, experiments are presented that characterize mechanisms of ethanol toxicity on epiboly and gastrulation. Epiboly mechanisms include blastomere radial intercalation cell movements and yolk cell microtubule cytoskeleton pulling the embryo to the vegetal pole. Both of these processes were disrupted by ethanol exposure. Ethanol effects on cell migration also indicated that cell adhesion was affected, which was confirmed by cell aggregation assays. E-cadherin cell adhesion molecule expression was not affected by ethanol exposure, but E-cadherin distribution, which controls epiboly and gastrulation, was changed. E-cadherin was redistributed into cytoplasmic aggregates in blastomeres and dramatically redistributed in the extraembryonic yolk cell. Gene expression microarray analysis was used to identify potential causative factors for early development defects, and expression of the cell adhesion molecule protocadherin-18a (pcdh18a, which controls epiboly, was significantly reduced in ethanol exposed embryos. Injecting pcdh18a synthetic mRNA in ethanol treated embryos partially rescued epiboly cell movements, including enveloping layer cell shape changes. Together, data show that epiboly and gastrulation defects induced by ethanol are multifactorial, and include yolk cell (extraembryonic tissue microtubule cytoskeleton disruption and blastomere adhesion defects, in part caused by reduced pcdh18a expression.

Astrocyte-neuron interaction in diphenyl ditelluride toxicity directed to the cytoskeleton

International Nuclear Information System (INIS)

Heimfarth, Luana; Silva Ferreira, Fernanda da; Pierozan, Paula; Mingori, Moara Rodrigues; Moreira, José Cláudio Fonseca; Batista Teixeira da Rocha, João; Pessoa-Pureur, Regina

2017-01-01

Highlights: • Diphenyl ditelluride is toxic to the cytoskeleton of neural cells in vitro. • Hypophosphorylation disrupts cytoskeletal homeostasis and causes cell dysfunction. • Calcium signaling underlies hypophosphorylation of intermediate filaments. • Actin disorganization causes altered astrocyte morphology. • Astrocyte cytoskeleton is more susceptible than neuronal cytoskeleton. - Abstract: Diphenylditelluride (PhTe) 2 is a neurotoxin that disrupts cytoskeletal homeostasis. We are showing that different concentrations of (PhTe) 2 caused hypophosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits (NFL, NFM and NFH) and altered actin organization in co-cultured astrocytes and neurons from cerebral cortex of rats. These mechanisms were mediated by N-methyl-D-aspartate (NMDA) receptors without participation of either L-type voltage-dependent calcium channels (L-VDCC) or metabotropic glutamate receptors. Upregulated Ca 2+ influx downstream of NMDA receptors activated Ca 2+ -dependent protein phosphatase 2B (PP2B) causing hypophosphorylation of astrocyte and neuron IFs. Immunocytochemistry showed that hypophosphorylated intermediate filaments (IF) failed to disrupt their organization into the cytoskeleton. However, phalloidin-actin-FITC stained cytoskeleton evidenced misregulation of actin distribution, cell spreading and increased stress fibers in astrocytes. βIII tubulin staining showed that neurite meshworks are not altered by (PhTe) 2 , suggesting greater susceptibility of astrocytes than neurons to (PheTe) 2 toxicity. These findings indicate that signals leading to IF hypophosphorylation fail to disrupt the cytoskeletal IF meshwork of interacting astrocytes and neurons in vitro however astrocyte actin network seems more susceptible. Our findings support that intracellular Ca 2+ is one of the crucial signals that modulate the action of (PhTe) 2 in co-cultured astrocytes and neurons and highlights the cytoskeleton

The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

Energy Technology Data Exchange (ETDEWEB)

Blom, Magdalena; Reis, Katarina [Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Heldin, Johan [Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala SE-751 22 Uppsala (Sweden); Kreuger, Johan [Department of Medical Cell Biology, Science for Life Laboratory, Uppsala University, SE-751 23 Uppsala (Sweden); Aspenström, Pontus, E-mail: pontus.aspenstrom@ki.se [Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden)

2017-03-15

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.

The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

International Nuclear Information System (INIS)

Blom, Magdalena; Reis, Katarina; Heldin, Johan; Kreuger, Johan; Aspenström, Pontus

2017-01-01

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

Science.gov (United States)

Kyozuka, Keiichiro; Chun, Jong T; Puppo, Agostina; Gragnaniello, Gianni; Garante, Ezio; Santella, Luigia

2008-08-15

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis.

Effect of 60Co γ-ray irradiation on cytoskeleton of human peripheral blood monocytes with whole mount cell electron microscopy in vitro

International Nuclear Information System (INIS)

Chen Xiaomei; Guo Yuhua; Yin Zhiwei

1992-01-01

Whole mount cell electron microscopy was used in combination with selective extraction to prepare cytoskeletal framework. Cytoskeleton prepared by Triton X-100 treatment of human peripheral blood monocytes appeared on electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. By three-dimensional visualization of Triton X-100 resistant cytoskeletons it was demonstrated that different doses of 60 Co γ-rays caused distinctive and reproducible alterations of the cytoskeleton of intact human peripheral blood monocytes in vitro. The alterations were similar to those caused by cytochalasin B and by colchicine. From these observations and other workers'studies, it is presumed that 60 Co γ-ray irradiation may inhibit cytoplasmic microtubule and microfilament assembling

Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

Science.gov (United States)

Kloc, Malgorzata; Bilinski, Szczepan; Kubiak, Jacek Z

2016-01-01

The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes.

Characterization of mRNA-Cytoskeleton Interactions In Situ Using FMTRIP and Proximity Ligation

Science.gov (United States)

Jung, Jeenah; Lifland, Aaron W.; Alonas, Eric J.; Zurla, Chiara; Santangelo, Philip J.

2013-01-01

Many studies have demonstrated an association between the cytoskeleton and mRNA, as well as the asymmetric distribution of mRNA granules within the cell in response to various signaling events. It is likely that the extensive cytoskeletal network directs mRNA transport and localization, with different cytoskeletal elements having their own specific roles. In order to understand the spatiotemporal changes in the interactions between the mRNA and the cytoskeleton as a response to a stimulus, a technique that can visualize and quantify these changes across a population of cells while capturing cell-to-cell variations is required. Here, we demonstrate a method for imaging and quantifying mRNA-cytoskeleton interactions on a per cell basis with single-interaction sensitivity. Using a proximity ligation assay with flag-tagged multiply-labeled tetravalent RNA imaging probes (FMTRIP), we quantified interactions between mRNAs and β-tubulin, vimentin, or filamentous actin (F-actin) for two different mRNAs, poly(A) + and β-actin mRNA, in two different cell types, A549 cells and human dermal fibroblasts (HDF). We found that the mRNAs interacted predominantly with F-actin (>50% in HDF, >20% in A549 cells), compared to β-tubulin (cytoskeleton itself and mRNA localization. Both perturbations led to a decrease in poly(A) + mRNA interactions with F-actin and an increase in the interactions with microtubules, in a time dependent manner. PMID:24040294

Extending the molecular clutch beyond actin-based cell motility

International Nuclear Information System (INIS)

Havrylenko, Svitlana; Mezanges, Xavier; Batchelder, Ellen; Plastino, Julie

2014-01-01

Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the ‘molecular clutch’ description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of major sperm protein, which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton. (paper)

Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

Science.gov (United States)

Mattila, Pieta K.; Batista, Facundo D.

2016-01-01

Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

The roles of membranes and associated cytoskeleton in plant virus replication and cell-to-cell movement.

Science.gov (United States)

Pitzalis, Nicolas; Heinlein, Manfred

2017-12-18

The infection of plants by viruses depends on cellular mechanisms that support the replication of the viral genomes, and the cell-to-cell and systemic movement of the virus via plasmodesmata (PD) and the connected phloem. While the propagation of some viruses requires the conventional endoplasmic reticulum (ER)-Golgi pathway, others replicate and spread between cells in association with the ER and are independent of this pathway. Using selected viruses as examples, this review re-examines the involvement of membranes and the cytoskeleton during virus infection and proposes potential roles of class VIII myosins and membrane-tethering proteins in controlling viral functions at specific ER subdomains, such as cortical microtubule-associated ER sites, ER-plasma membrane contact sites, and PD. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.

Science.gov (United States)

Urbancíková, M; Vozárová, G; Lesko, J; Golais, F

1999-10-01

Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.

Organization and dynamics of the actin cytoskeleton during dendritic spine morphological remodeling.

Science.gov (United States)

Chazeau, Anaël; Giannone, Grégory

2016-08-01

In the central nervous system, most excitatory post-synapses are small subcellular structures called dendritic spines. Their structure and morphological remodeling are tightly coupled to changes in synaptic transmission. The F-actin cytoskeleton is the main driving force of dendritic spine remodeling and sustains synaptic plasticity. It is therefore essential to understand how changes in synaptic transmission can regulate the organization and dynamics of actin binding proteins (ABPs). In this review, we will provide a detailed description of the organization and dynamics of F-actin and ABPs in dendritic spines and will discuss the current models explaining how the actin cytoskeleton sustains both structural and functional synaptic plasticity.

Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration.

Science.gov (United States)

Goswami, Ishan; Perry, Justin B; Allen, Mitchell E; Brown, David A; von Spakovsky, Michael R; Verbridge, Scott S

2018-06-19

Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used

A Role for the Cytoskeleton in Heart Looping

Directory of Open Access Journals (Sweden)

Kersti K. Linask

2007-01-01

Full Text Available Over the past 10 years, key genes involved in specification of left-right laterality pathways in the embryo have been defined. The read-out for misexpression of laterality genes is usually the direction of heart looping. The question of how dextral looping direction occurred mechanistically and how the heart tube bends remains unknown. It is becoming clear from our experiments and those of others that left-right differences in cell proliferation in the second heart field (anterior heart field drives the dextral direction. Evidence is accumulating that the cytoskeleton is at the center of laterality, and the bending and rotational forces associated with heart looping. If laterality pathways are modulated upstream, the cytoskeleton, including nonmuscle myosin II (NMHC-II, is altered downstream within the cardiomyocytes, leading to looping abnormalities. The cytoskeleton is associated with important mechanosensing and signaling pathways in cell biology and development. The initiation of blood flow during the looping period and the inherent stresses associated with increasing volumes of blood flowing into the heart may help to potentiate the process. In recent years, the steps involved in this central and complex process of heart development that is the basis of numerous congenital heart defects are being unraveled.

Identification of dynamic changes in proteins associated with the cellular cytoskeleton after exposure to okadaic acid

DEFF Research Database (Denmark)

Opsahl, Jill A; Ljostveit, Sonja; Solstad, Therese

2013-01-01

be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton...... of the cortical actin cytoskeleton and cell detachment....

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes.

Science.gov (United States)

Kuga, Takahisa; Sasaki, Mitsuho; Mikami, Toshinari; Miake, Yasuo; Adachi, Jun; Shimizu, Maiko; Saito, Youhei; Koura, Minako; Takeda, Yasunori; Matsuda, Junichiro; Tomonaga, Takeshi; Nakayama, Yuji

2016-05-25

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.

The axonal cytoskeleton : from organization to function

NARCIS (Netherlands)

Kevenaar, Josta T; Hoogenraad, Casper C

The axon is the single long fiber that extends from the neuron and transmits electrical signals away from the cell body. The neuronal cytoskeleton, composed of microtubules (MTs), actin filaments and neurofilaments, is not only required for axon formation and axonal transport but also provides the

Estrogen and Resveratrol Regulate Rac and Cdc42 Signaling to the Actin Cytoskeleton of Metastatic Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Nicolas G. Azios

2007-02-01

Full Text Available Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 µM, an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 µM, resveratrol at 5 µM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 µM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 µM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominantnegative Rac retain filopodia response to 50 µM resveratrol. Lamellipodia response to 5 µM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 µM signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.

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

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

2016-02-01

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

Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton.

Science.gov (United States)

Martinière, Alexandre; Gayral, Philippe; Hawes, Chris; Runions, John

2011-04-01

Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C-terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline-rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell-wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin-dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin-remodelling mechanisms. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Coupled elasticity–diffusion model for the effects of cytoskeleton deformation on cellular uptake of cylindrical nanoparticles

Science.gov (United States)

Wang, Jizeng; Li, Long

2015-01-01

Molecular dynamic simulations and experiments have recently demonstrated how cylindrical nanoparticles (CNPs) with large aspect ratios penetrate animal cells and inevitably deform cytoskeletons. Thus, a coupled elasticity–diffusion model was adopted to elucidate this interesting biological phenomenon by considering the effects of elastic deformations of cytoskeleton and membrane, ligand–receptor binding and receptor diffusion. The mechanism by which the binding energy drives the CNPs with different orientations to enter host cells was explored. This mechanism involved overcoming the resistance caused by cytoskeleton and membrane deformations and the change in configurational entropy of the ligand–receptor bonds and free receptors. Results showed that deformation of the cytoskeleton significantly influenced the engulfing process by effectively slowing down and even hindering the entry of the CNPs. Additionally, the engulfing depth was determined quantitatively. CNPs preferred or tended to vertically attack target cells until they were stuck in the cytoskeleton as implied by the speed of vertically oriented CNPs that showed much faster initial engulfing speeds than horizontally oriented CNPs. These results elucidated the most recent molecular dynamics simulations and experimental observations on the cellular uptake of carbon nanotubes and phagocytosis of filamentous Escherichia coli bacteria. The most efficient engulfment showed the stiffness-dependent optimal radius of the CNPs. Cytoskeleton stiffness exhibited more significant influence on the optimal sizes of the vertical uptake than the horizontal uptake. PMID:25411410

Engineering amount of cell-cell contact demonstrates biphasic proliferative regulation through RhoA and the actin cytoskeleton

International Nuclear Information System (INIS)

Gray, Darren S.; Liu, Wendy F.; Shen, Colette J.; Bhadriraju, Kiran; Nelson, Celeste M.; Chen, Christopher S.

2008-01-01

Endothelial cell-cell contact via VE-cadherin plays an important role in regulating numerous cell functions, including proliferation. However, using different experimental approaches to manipulate cell-cell contact, investigators have observed both inhibition and stimulation of proliferation depending on the adhesive context. In this study, we used micropatterned wells combined with active positioning of cells by dielectrophoresis in order to investigate whether the number of contacting neighbors affected the proliferative response. Varying cell-cell contact resulted in a biphasic effect on proliferation; one contacting neighbor increased proliferation, while two or more neighboring cells partially inhibited this increase. We also observed that cell-cell contact increased the formation of actin stress fibers, and that expression of dominant negative RhoA (RhoN19) blocked the contact-mediated increase in stress fibers and proliferation. Furthermore, examination of heterotypic pairs of untreated cells in contact with RhoN19-expressing cells revealed that intracellular, but not intercellular, tension is required for the contact-mediated stimulation of proliferation. Moreover, engagement of VE-cadherin with cadherin-coated beads was sufficient to stimulate proliferation in the absence of actual cell-cell contact. In all, these results demonstrate that cell-cell contact signals through VE-cadherin, RhoA, and intracellular tension in the actin cytoskeleton to regulate proliferation

Temperature response of the neuronal cytoskeleton mapped via atomic force and fluorescence microscopy

International Nuclear Information System (INIS)

Spedden, Elise; Staii, Cristian; Kaplan, David L

2013-01-01

Neuronal cells change their growth properties in response to external physical stimuli such as variations in external temperature, stiffness of the growth substrate, or topographical guidance cues. Detailed knowledge of the mechanisms that control these biomechanical responses is necessary for understanding the basic principles that underlie neuronal growth and regeneration. Here, we present elasticity maps of living cortical neurons (embryonic rat) as a function of temperature, and correlate these maps to the locations of internal structural components of the cytoskeleton. Neurons display a significant increase in the average elastic modulus upon a decrease in ambient temperature from 37 to 25 °C. We demonstrate that the dominant mechanism by which the elasticity of the neurons changes in response to temperature is the stiffening of the actin components of the cytoskeleton induced by myosin II. We also report a reversible shift in the location and composition of the high-stiffness areas of the neuron cytoskeleton with temperature. At 37 °C the areas of the cell displaying high elastic modulus overlap with the tubulin-dense regions, while at 25 °C these high-stiffness areas correspond to the actin-dense regions of the cytoskeleton. These results demonstrate the importance of considering temperature effects when investigating cytoskeletal dynamics in cells. (paper)

Characterization of mRNA-cytoskeleton interactions in situ using FMTRIP and proximity ligation.

Directory of Open Access Journals (Sweden)

Jeenah Jung

Full Text Available Many studies have demonstrated an association between the cytoskeleton and mRNA, as well as the asymmetric distribution of mRNA granules within the cell in response to various signaling events. It is likely that the extensive cytoskeletal network directs mRNA transport and localization, with different cytoskeletal elements having their own specific roles. In order to understand the spatiotemporal changes in the interactions between the mRNA and the cytoskeleton as a response to a stimulus, a technique that can visualize and quantify these changes across a population of cells while capturing cell-to-cell variations is required. Here, we demonstrate a method for imaging and quantifying mRNA-cytoskeleton interactions on a per cell basis with single-interaction sensitivity. Using a proximity ligation assay with flag-tagged multiply-labeled tetravalent RNA imaging probes (FMTRIP, we quantified interactions between mRNAs and β-tubulin, vimentin, or filamentous actin (F-actin for two different mRNAs, poly(A + and β-actin mRNA, in two different cell types, A549 cells and human dermal fibroblasts (HDF. We found that the mRNAs interacted predominantly with F-actin (>50% in HDF, >20% in A549 cells, compared to β-tubulin (<5% and vimentin (11-13%. This likely reflects differences in mRNA management by the two cell types. We then quantified changes in these interactions in response to two perturbations, F-actin depolymerization and arsenite-induced oxidative stress, both of which alter either the cytoskeleton itself and mRNA localization. Both perturbations led to a decrease in poly(A + mRNA interactions with F-actin and an increase in the interactions with microtubules, in a time dependent manner.

Chirality of the cytoskeleton in the origins of cellular asymmetry

Science.gov (United States)

2016-01-01

Self-assembly of two important components of the cytoskeleton of eukaryotic cells, actin microfilaments and microtubules (MTs) results in polar filaments of one chirality. As is true for bacterial flagella, in actin microfilaments, screw direction is important for assembly processes and motility. For MTs, polar orientation within the cell is paramount. The alignment of these elements in the cell cytoplasm gives rise to emergent properties, including the potential for cell differentiation and specialization. Complex MTs with a characteristic chirality are found in basal bodies and centrioles; this chirality is preserved in cilia. In motile cilia, it is reflected in the direction of the effective stroke. The positioning of the basal body or cilia on the cell surface depends on polarity proteins. In evolution, survival depends on global polarity information relayed to the cell in part by orientation of the MT and actin filament cytoskeletons and the chirality of the basal body to determine left and right coordinates within a defined anterior–posterior cell and tissue axis. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821520

Disruption of Spectrin-Like Cytoskeleton in Differentiating Keratinocytes by PKCδ Activation Is Associated with Phosphorylated Adducin

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Zhao, Kong-Nan; Masci, Paul P.; Lavin, Martin F.

2011-01-01

Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex. PMID:22163289

Coupled elasticity-diffusion model for the effects of cytoskeleton deformation on cellular uptake of cylindrical nanoparticles.

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Wang, Jizeng; Li, Long

2015-01-06

Molecular dynamic simulations and experiments have recently demonstrated how cylindrical nanoparticles (CNPs) with large aspect ratios penetrate animal cells and inevitably deform cytoskeletons. Thus, a coupled elasticity-diffusion model was adopted to elucidate this interesting biological phenomenon by considering the effects of elastic deformations of cytoskeleton and membrane, ligand-receptor binding and receptor diffusion. The mechanism by which the binding energy drives the CNPs with different orientations to enter host cells was explored. This mechanism involved overcoming the resistance caused by cytoskeleton and membrane deformations and the change in configurational entropy of the ligand-receptor bonds and free receptors. Results showed that deformation of the cytoskeleton significantly influenced the engulfing process by effectively slowing down and even hindering the entry of the CNPs. Additionally, the engulfing depth was determined quantitatively. CNPs preferred or tended to vertically attack target cells until they were stuck in the cytoskeleton as implied by the speed of vertically oriented CNPs that showed much faster initial engulfing speeds than horizontally oriented CNPs. These results elucidated the most recent molecular dynamics simulations and experimental observations on the cellular uptake of carbon nanotubes and phagocytosis of filamentous Escherichia coli bacteria. The most efficient engulfment showed the stiffness-dependent optimal radius of the CNPs. Cytoskeleton stiffness exhibited more significant influence on the optimal sizes of the vertical uptake than the horizontal uptake. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Subversion of the cytoskeleton by intracellular bacteria: lessons from Listeria, Salmonella, and Vibrio

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de Souza Santos, Marcela; Orth, Kim

2018-01-01

Summary Entry into host cells and intracellular persistence by invasive bacteria are tightly coupled to the ability of the bacterium to disrupt the eukaryotic cytoskeletal machinery. Herein we review the main strategies used by three intracellular pathogens to harness key modulators of the cytoskeleton. Two of these bacteria, namely Listeria monocytogenes and Salmonella enterica serovar Typhimurium, exhibit quite distinct intracellular lifestyles, and therefore, provide a comprehensive panel for the understanding of the intricate bacteria-cytoskeleton interplay during infections. The emerging intracellular pathogen Vibrio parahaemolyticus is depicted as a developing model for the uncovering of novel mechanisms used to hijack the cytoskeleton. PMID:25440316

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

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Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

2009-08-11

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

Morphed and moving: TNFα-driven motility promotes cell dissemination through MAP4K4-induced cytoskeleton remodeling

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Min Ma

2014-04-01

Full Text Available Cell dissemination from an initial site of growth is a highly coordinated and controlled process that depends on cell motility. The mechanistic principles that orchestrate cell motility, namely cell shape control, traction and force generation, are highly conserved between cells of different origins. Correspondingly, the molecular mechanisms that regulate these critical aspects of migrating cells are likely functionally conserved too. Thus, cell motility deregulation of unrelated pathogenesis could be caused and maintained by similar mechanistic principles. One such motility deregulation disorder is the leukoproliferative cattle disease Tropical Theileriosis, which is caused by the intracellular, protozoan parasite Theileria annulata. T. annulata transforms its host cell and promotes the dissemination of parasite-infected cells throughout the body of the host. An analogous condition with a fundamentally different pathogenesis is metastatic cancer, where oncogenically transformed cells disseminate from the primary tumor to form distant metastases. Common to both diseases is the dissemination of motile cells from the original site. However, unlike metastatic cancer, host cell transformation by Theileria parasites can be reverted by drug treatment and cell signaling be analyzed under transformed and non-transformed conditions. We have used this reversible transformation model and investigated parasite control of host cell motile properties in the context of inflammatory signaling in Ma M. et al. [PLoS Pathog (2014 10: e1004003]. We found that parasite infection promotes the production of the inflammatory cytokine TNFα in the host macrophage. We demonstrated that increased TNFα triggers motile and invasive properties by enhancing actin cytoskeleton remodeling and cell motility through the ser/thr kinase MAP4K4. We concluded that inflammatory conditions resulting in increased TNFα could facilitate cell dissemination by activating the actin

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

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Gadelha, Ana Paula Rocha; Benchimol, Marlene; de Souza, Wanderley

2015-06-01

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites. Copyright © 2015 Elsevier Inc. All rights reserved.

The cytoskeleton and gravitropism in higher plants

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Blancaflor, Elison B.

2002-01-01

The cellular and molecular mechanisms underlying the gravitropic response of plants have continued to elude plant biologists despite more than a century of research. Lately there has been increased attention on the role of the cytoskeleton in plant gravitropism, but several controversies and major gaps in our understanding of cytoskeletal involvement in gravitropism remain. A major question in the study of plant gravitropism is how the cytoskeleton mediates early sensing and signal transduction events in plants. Much has been made of the actin cytoskeleton as the cellular structure that sedimenting amyloplasts impinge upon to trigger the downstream signaling events leading to the bending response. There is also strong molecular and biochemical evidence that the transport of auxin, an important player in gravitropism, is regulated by actin. Organizational changes in microtubules during the growth response phase of gravitropism have also been well documented, but the significance of such reorientations in controlling differential cellular growth is unclear. Studies employing pharmacological approaches to dissect cytoskeletal involvement in gravitropism have led to conflicting results and therefore need to be interpreted with caution. Despite the current controversies, the revolutionary advances in molecular, biochemical, and cell biological techniques have opened up several possibilities for further research into this difficult area. The myriad proteins associated with the plant cytoskeleton that are being rapidly characterized provide a rich assortment of candidate regulators that could be targets of the gravity signal transduction chain. Cytoskeletal and ion imaging in real time combined with mutant analysis promises to provide a fresh start into this controversial area of research.

Infection and Transport of Herpes Simplex Virus Type 1 in Neurons: Role of the Cytoskeleton

Science.gov (United States)

2018-01-01

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that has the ability to infect and replicate within epithelial cells and neurons and establish a life-long latent infection in sensory neurons. HSV-1 depends on the host cellular cytoskeleton for entry, replication, and exit. Therefore, HSV-1 has adapted mechanisms to promote its survival by exploiting the microtubule and actin cytoskeletons to direct its active transport, infection, and spread between neurons and epithelial cells during primary and recurrent infections. This review will focus on the currently known mechanisms utilized by HSV-1 to harness the neuronal cytoskeleton, molecular motors, and the secretory and exocytic pathways for efficient virus entry, axonal transport, replication, assembly, and exit from the distinct functional compartments (cell body and axon) of the highly polarized sensory neurons. PMID:29473915

The role of cytoskeleton and adhesion proteins in the resistance to photodynamic therapy. Possible therapeutic interventions.

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Di Venosa, Gabriela; Perotti, Christian; Batlle, Alcira; Casas, Adriana

2015-08-01

It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed.

HAb18G/CD147 regulates vinculin-mediated focal adhesion and cytoskeleton organization in cultured human hepatocellular carcinoma cells.

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Qiang Liang

Full Text Available Focal adhesions (FAs, integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147 is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001, length/width ratios (P<0.0001, and mean intensities (P<0.0001. CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t1/2 recovery times (22.7±3.3 s of vinculin-mediated focal adhesions (P<0.0001. In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2. Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.

Taking control: reorganization of the host cytoskeleton by Chlamydia [version 1; referees: 5 approved

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Jordan Wesolowski

2017-11-01

Full Text Available Both actin and microtubules are major cytoskeletal elements in eukaryotic cells that participate in many cellular processes, including cell division and motility, vesicle and organelle movement, and the maintenance of cell shape. Inside its host cell, the human pathogen Chlamydia trachomatis manipulates the cytoskeleton to promote its survival and enhance its pathogenicity. In particular, Chlamydia induces the drastic rearrangement of both actin and microtubules, which is vital for its entry, inclusion structure and development, and host cell exit. As significant progress in Chlamydia genetics has greatly enhanced our understanding of how this pathogen co-opts the host cytoskeleton, we will discuss the machinery used by Chlamydia to coordinate the reorganization of actin and microtubules.

Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages

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Cui, Yizhi; Zhou, Yanlong; Yin, Xingfeng; Guo, Jiahui; Zhang, Gong; Wang, Tong; He, Qing-Yu

2016-01-01

The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit. PMID:27602764

Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages.

Science.gov (United States)

Chen, Zhipeng; Yang, Lijuan; Cui, Yizhi; Zhou, Yanlong; Yin, Xingfeng; Guo, Jiahui; Zhang, Gong; Wang, Tong; He, Qing-Yu

2016-10-11

The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit.

Morphodynamics of the Actin-Rich Cytoskeleton in Entamoeba histolytica

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Maria Manich

2018-05-01

Full Text Available Entamoeba histolytica is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in Entamoeba histolytica means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, E. histolytica has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in E. histolytica. We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by E. histolytica trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live E. histolytica cells; we observed the cortical cytoskeleton, stress fibers, “dot-like” structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.

Coronin-1A links cytoskeleton dynamics to TCR alpha beta-induced cell signaling.

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Bénédicte Mugnier

Full Text Available Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR-induced immunological synapse (IS formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of alpha beta T cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-kappaB (I kappa B. Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts alpha beta T cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

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Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live

Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture.

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Akisaka, Toshitaka; Yoshida, Hisaho; Suzuki, Reiko; Takama, Keiko

2008-03-01

The organization of the cytoskeleton in the podosomes of osteoclasts was studied by use of cell shearing, rotary replication, and fluorescence cytochemical techniques. After shearing, clathrin plaques and particles associated with the cytoskeleton were left behind on the exposed cytoplasmic side of the membrane. The cytoskeleton of the podosomes was characterized by two types of actin filaments: relatively long filaments in the portion surrounding the podosome core, and highly branched short filaments in the core. Individual actin filaments radiating from the podosomes interacted with several membrane particles along the length of the filaments. Many lateral contacts with the membrane surface by the particles were made along the length of individual actin filaments. The polarity of actin filaments in podosomes became oriented such that their barbed ends were directed toward the core of podosomes. The actin cytoskeletons terminated or branched at the podosomes, where the membrane tightly adhered to the substratum. Microtubules were not usually present in the podosome structures; however, certain microtubules appeared to be morphologically in direct contact with the podosome core. Most of the larger clathrin plaques consisted of flat sheets of clathrin lattices that interconnected neighboring clathrin lattices to form an extensive clathrin area. However, the small deeply invaginated clathrin plaques and the podosomal cytoskeleton were located close together. Thus, the clathrin plaques on the ventral membrane of osteoclasts might be involved in both cell adhesion and the formation of receptor-ligand complexes, i.e., endocytosis.

High level of reactive oxygen species impaired mesenchymal stem cell migration via overpolymerization of F-actin cytoskeleton in systemic lupus erythematosus.

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Shi, D; Li, X; Chen, H; Che, N; Zhou, S; Lu, Z; Shi, S; Sun, L

2014-12-01

Some lines of evidence have demonstrated abnormalities of bone marrow mesenchymal stem cells (MSCs) in systemic lupus erythematosus (SLE) patients, characterized by defective phenotype of MSCs and slower growth with enhanced apoptosis and senescence. However, whether SLE MSCs demonstrate aberrant migration capacity or abnormalities in cytoskeleton are issues that remain poorly understood. In this study, we found that MSCs from SLE patients did show impairment in migration capacity as well as abnormalities in F-actin cytoskeleton, accompanied by a high level of intracellular reactive oxygen species (ROS). When normal MSCs were treated in vitro with H2O2, which increases intracellular ROS level as an oxidant, both reorganization of F-actin cytoskeleton and impairment of migration capability were observed. On the other hand, treatment with N-acetylcysteine (NAC), as an exogenous antioxidant, made F-actin more orderly and increased migration ratio in SLE MSCs. In addition, oral administration of NAC markedly reduced serum autoantibody levels and ameliorated lupus nephritis (LN) in MRL/lpr mice, partially reversing the abnormalities of MSCs. These results indicate that overpolymerization of F-actin cytoskeleton, which may be associated with high levels of ROS, causes impairment in the migration capacity of SLE MSCs and that oral administration of NAC may have potential therapeutic effects on MRL/lpr mice. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton

DEFF Research Database (Denmark)

Oberli, Alexander; Zurbrügg, Laura; Rusch, Sebastian

2016-01-01

is anchored to the cytoskeleton, and the Plasmodium helical interspersed subtelomeric (PHIST) gene family plays a role in many host cell modifications including binding the intracellular domain of PfEMP1. Here, we show that conditional reduction of the PHIST protein PFE1605w strongly reduces adhesion...... interacts with both the intracellular segment of PfEMP1 and with cytoskeletal components. This is the first report of a PHIST protein interacting with key molecules of the cytoadherence complex and the host cytoskeleton, and this functional role seems to play an essential role in the pathology of P...

Thematic Minireview Series: The State of the Cytoskeleton in 2015.

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Fischer, Robert S; Fowler, Velia M

2015-07-10

The study of cytoskeletal polymers has been an active area of research for more than 70 years. However, despite decades of pioneering work by some of the brightest scientists in biochemistry, cell biology, and physiology, many central questions regarding the polymers themselves are only now starting to be answered. For example, although it has long been appreciated that the actin cytoskeleton provides contractility and couples biochemical responses with mechanical stresses in cells, only recently have we begun to understand how the actin polymer itself responds to mechanical loads. Likewise, although it has long been appreciated that the microtubule cytoskeleton can be post-translationally modified, only recently have the enzymes responsible for these modifications been characterized, so that we can now begin to understand how these modifications alter the polymerization and regulation of microtubule structures. Even the septins in eukaryotes and the cytoskeletal polymers of prokaryotes have yielded new insights due to recent advances in microscopy techniques. In this thematic series of minireviews, these topics are covered by some of the very same scientists who generated these recent insights, thereby providing us with an overview of the State of the Cytoskeleton in 2015. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell polarity, cell adhesion, and spermatogenesis: role of cytoskeletons [version 1; referees: 2 approved

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Linxi Li

2017-08-01

Full Text Available In the rat testis, studies have shown that cell polarity, in particular spermatid polarity, to support spermatogenesis is conferred by the coordinated efforts of the Par-, Crumbs-, and Scribble-based polarity complexes in the seminiferous epithelium. Furthermore, planar cell polarity (PCP is conferred by PCP proteins such as Van Gogh-like 2 (Vangl2 in the testis. On the other hand, cell junctions at the Sertoli cell–spermatid (steps 8–19 interface are exclusively supported by adhesion protein complexes (for example, α6β1-integrin-laminin-α3,β3,γ3 and nectin-3-afadin at the actin-rich apical ectoplasmic specialization (ES since the apical ES is the only anchoring device in step 8–19 spermatids. For cell junctions at the Sertoli cell–cell interface, they are supported by adhesion complexes at the actin-based basal ES (for example, N-cadherin-β-catenin and nectin-2-afadin, tight junction (occludin-ZO-1 and claudin 11-ZO-1, and gap junction (connexin 43-plakophilin-2 and also intermediate filament-based desmosome (for example, desmoglein-2-desmocollin-2. In short, the testis-specific actin-rich anchoring device known as ES is crucial to support spermatid and Sertoli cell adhesion. Accumulating evidence has shown that the Par-, Crumbs-, and Scribble-based polarity complexes and the PCP Vangl2 are working in concert with actin- or microtubule-based cytoskeletons (or both and these polarity (or PCP protein complexes exert their effects through changes in the organization of the cytoskeletal elements across the seminiferous epithelium of adult rat testes. As such, there is an intimate relationship between cell polarity, cell adhesion, and cytoskeletal function in the testis. Herein, we critically evaluate these recent findings based on studies on different animal models. We also suggest some crucial future studies to be performed.

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

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Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

2014-01-01

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. PMID:25106608

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

International Nuclear Information System (INIS)

Latonen, Leena; Jaervinen, Paeivi M.; Laiho, Marikki

2008-01-01

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1 correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions

Rho proteins − the key regulators of cytoskeleton in the progression of mitosis and cytokinesis

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Anna Klimaszewska

2011-11-01

Full Text Available The Rho proteins are members of the Ras superfamily of small GTPases. They are thought to be crucial regulators of multiple signal transduction pathways that influence a wide range of cellular functions, including migration, membrane trafficking, adhesion, polarity and cell shape changes. Thanks to their ability to control the assembly and organization of the actin and microtubule cytoskeletons, Rho GTPases are known to regulate mitosis and cytokinesis progression. These proteins are required for formation and rigidity of the cortex during mitotic cell rounding, mitotic spindle formation and attachment of the spindle microtubules to the kinetochore. In addition, during cytokinesis, they are involved in promoting division plane determination, contractile ring and cleavage furrow formation and abscission. They are also known as regulators of cell cycle progression at the G1/S and G2/M transition. Thus, the signal transduction pathways in which Rho proteins participate, appear to connect dynamics of actin and microtubule cytoskeletons to cell cycle progression. We review the current state of knowledge concerning the molecular mechanisms by which Rho GTPase signaling regulates remodeling of actin and microtubule cytoskeletons in order to control cell division progression.

A novel spiroindoline targets cell cycle and migration via modulation of microtubule cytoskeleton.

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Kumar, Naveen; Hati, Santanu; Munshi, Parthapratim; Sen, Subhabrata; Sehrawat, Seema; Singh, Shailja

2017-05-01

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.

Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells

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Weng Yuan-Yuan

2008-10-01

Full Text Available Abstract Background CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs. Tumor cells adhesion to extracellular matrix (ECM proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA against CD147 (si-CD147 on hepatocellular carcinoma cells' (SMMC-7721 architecture and functions. Methods Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK, vinculiln and mitogen-activated protein kinase (MAPK expression in SMMC-7721 cells. Results Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression. Conclusion CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

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Sikder, K. U.; Stone, K. A.; Kumar, P. B. S.

2014-01-01

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that mic......We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find...... that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. (C) 2014 AIP Publishing LLC....

Self-assembling enzymes and the origins of the cytoskeleton

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Barry, Rachael; Gitai, Zemer

2011-01-01

The bacterial cytoskeleton is composed of a complex and diverse group of proteins that self-assemble into linear filaments. These filaments support and organize cellular architecture and provide a dynamic network controlling transport and localization within the cell. Here, we review recent discoveries related to a newly appreciated class of self-assembling proteins that expand our view of the bacterial cytoskeleton and provide potential explanations for its evolutionary origins. Specifically, several types of metabolic enzymes can form structures similar to established cytoskeletal filaments and, in some cases, these structures have been repurposed for structural uses independent of their normal role. The behaviors of these enzymes suggest that some modern cytoskeletal proteins may have evolved from dual-role proteins with catalytic and structural functions. PMID:22014508

The sarcomeric cytoskeleton: from molecules to motion.

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Gautel, Mathias; Djinović-Carugo, Kristina

2016-01-01

Highly ordered organisation of striated muscle is the prerequisite for the fast and unidirectional development of force and motion during heart and skeletal muscle contraction. A group of proteins, summarised as the sarcomeric cytoskeleton, is essential for the ordered assembly of actin and myosin filaments into sarcomeres, by combining architectural, mechanical and signalling functions. This review discusses recent cell biological, biophysical and structural insight into the regulated assembly of sarcomeric cytoskeleton proteins and their roles in dissipating mechanical forces in order to maintain sarcomere integrity during passive extension and active contraction. α-Actinin crosslinks in the Z-disk show a pivot-and-rod structure that anchors both titin and actin filaments. In contrast, the myosin crosslinks formed by myomesin in the M-band are of a ball-and-spring type and may be crucial in providing stable yet elastic connections during active contractions, especially eccentric exercise. © 2016. Published by The Company of Biologists Ltd.

[At the plant side of formins--organizers of the actin cytoskeleton].

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Maruniewicz, Michalina; Kasprowicz, Anna; Wojtaszek, Przemysław

2009-01-01

Rearrangements of actin cytoskeleton enable proper functioning of the cells under normal conditions, and also cellular adaptations to changes in the direct surroundings. Formins are actin binding proteins, responsible for actin nucleation and further elongation of microfilaments. The distinguishing feature of formins is the presence of conserved FH2 (formin homology domain 2) domain, as well as other domains typical for distinct formin classes. In animal cells formins are involved in cytokinesis and determination and maintenance of the cell shape and polarity, but also in the formation of filopodia, endocytosis and many other processes. The presence of proteins from the formin family in plant cells, and their involvement in the tip growth and cytokinesis, has been determined only recently. As the functional organization of plant and animal cells is different, one can assume that the range of putative functions of plant formins might also be diverse. One of such proposed functions for formins in plants is the role of linker protein within WMC continuum (cell wall-plasma membrane-cytoskeleton). Unfortunately, for that moment the state of knowledge about plant formins in comparison with animal or fungal ones is much poorer.

The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

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Basseres, Eugene; Coppotelli, Giuseppe; Pfirrmann, Thorsten

2010-01-01

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocyto......Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria...... of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S...

Mechanotransduction through Cytoskeleton

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Ingber, Donald

2002-01-01

The goal of this project was to characterize the molecular mechanism by which cells recognize and respond to physical forces in their local environment. The project was based on the working hypothesis that cells sense mechanical stresses, such as those due to gravity, through their cell surface adhesion receptors (e.g., integrins) and that they respond as a result of structural arrangements with their internal cytoskeleton (CSK) which are orchestrated through use of tensegrity architecture. In this project, we carried out studies to define the architectural and molecular basis of cellular mechanotransduction. Our major goal was to define the molecular pathway that mediates mechanical force transfer between integrins and the CSK and to determine how mechanical deformation of integrin-CSK linkages is transduced into a biochemical response. Elucidation of the mechanism by which cells sense mechanical stresses through integrins and translate them into a biochemical response should help us to understand the molecular basis of the cellular response to gravity as well as many other forms of mechanosensation and tissue regulation. The specific aims of this proposal were: 1. To define the molecular basis of mechanical coupling between integrins, vinculin, and the actin CSK; 2. To develop a computer simulation of how mechanical stresses alter CSK structure and test this model in living cells; 3. To determine how mechanical deformation of integrin-CSK linkages is transduced into a biochemical response.

Tubulin cytoskeleton during microsporogenesis in the male-sterile genotype of Allium sativum and fertile Allium ampeloprasum L.

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Tchórzewska, Dorota; Deryło, Kamil; Błaszczyk, Lidia; Winiarczyk, Krystyna

2015-12-01

Microsporogenesis in garlic. The male-sterile Allium sativum (garlic) reproduces exclusively in the vegetative mode, and anthropogenic factors seem to be the cause of the loss of sexual reproduction capability. There are many different hypotheses concerning the causes of male sterility in A.sativum; however, the mechanisms underlying this phenomenon have not been comprehensively elucidated.Numerous attempts have been undertaken to understand the causes of male sterility, but the tubulin cytoskeleton in meiotically dividing cells during microsporogenesis has never been investigated in this species. Using sterile A.sativum genotype L13 and its fertile close relative A. ampeloprasum (leek), we have analysed the distribution of the tubulin cytoskeleton during microsporogenesis. We observed that during karyokinesis and cytokinesis, in both meiotic divisions I and II, the microtubular cytoskeleton in garlic L13 formed configurations that resembled tubulin arrangement typical of monocots. However, the tubulin cytoskeleton in garlic was distinctly poorer (composed of a few MT filaments) compared with that found in meiotically dividing cells in A. ampeloprasum. These differences did not affect the course of karyogenesis, chondriokinesis, and cytokinesis, which contributed to completion of microsporogenesis, but there was no further development of the male gametophyte. At the very beginning of the successive stage of development of fertile pollen grains, i.e. gametogenesis, there were disorders involving the absence of a normal cortical cytoskeleton and dramatically progressive degeneration of the cytoplasm in garlic. Therefore,we suggest that, due to disturbances in cortical cytoskeleton formation at the very beginning of gametogenesis, the intracellular transport governed by the cytoskeleton might be perturbed, leading to microspore decay in the male-sterile garlic genotype.

Nuclear Import of β-Dystroglycan Is Facilitated by Ezrin-Mediated Cytoskeleton Reorganization

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Vásquez-Limeta, Alejandra; Wagstaff, Kylie M.; Ortega, Arturo; Crouch, Dorothy H.; Jans, David A.; Cisneros, Bulmaro

2014-01-01

The β-dystroglycan (β-DG) protein has the ability to target to multiple sites in eukaryotic cells, being a member of diverse protein assemblies including the transmembranal dystrophin-associated complex, and a nuclear envelope-localised complex that contains emerin and lamins A/C and B1. We noted that the importin α2/β1-recognised nuclear localization signal (NLS) of β-DG is also a binding site for the cytoskeletal-interacting protein ezrin, and set out to determine whether ezrin binding might modulate β-DG nuclear translocation for the first time. Unexpectedly, we found that ezrin enhances rather than inhibits β-DG nuclear translocation in C2C12 myoblasts. Both overexpression of a phosphomimetic activated ezrin variant (Ez-T567D) and activation of endogenous ezrin through stimulation of the Rho pathway resulted in both formation of actin-rich surface protrusions and significantly increased nuclear translocation of β-DG as shown by quantitative microscopy and subcellular fractionation/Western analysis. In contrast, overexpression of a nonphosphorylatable inactive ezrin variant (Ez-T567A) or inhibition of Rho signaling, decreased nuclear translocation of β-DG concomitant with a lack of cell surface protrusions. Further, a role for the actin cytoskeleton in ezrin enhancement of β-DG nuclear translocation was implicated by the observation that an ezrin variant lacking its actin-binding domain failed to enhance nuclear translocation of β-DG, while disruption of the actin cytoskeleton led to a reduction in β-DG nuclear localization. Finally, we show that ezrin-mediated cytoskeletal reorganization enhances nuclear translocation of the cytoplasmic but not the transmembranal fraction of β-DG. This is the first study showing that cytoskeleton reorganization can modulate nuclear translocation of β-DG, with the implication that β-DG can respond to cytoskeleton-driven changes in cell morphology by translocating from the cytoplasm to the nucleus to orchestrate

Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

International Nuclear Information System (INIS)

Li Guoping; Yang Lusheng; Huang Qunce; Qin Guangyong

2008-01-01

Pollen grains of Pinus thunbergii Parl. (Japanese black pine) were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.

Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

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Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

2011-01-01

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

The nano-architecture of the axonal cytoskeleton.

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Leterrier, Christophe; Dubey, Pankaj; Roy, Subhojit

2017-12-01

The corporeal beauty of the neuronal cytoskeleton has captured the imagination of generations of scientists. One of the easiest cellular structures to visualize by light microscopy, its existence has been known for well over 100 years, yet we have only recently begun to fully appreciate its intricacy and diversity. Recent studies combining new probes with super-resolution microscopy and live imaging have revealed surprising details about the axonal cytoskeleton and, in particular, have discovered previously unknown actin-based structures. Along with traditional electron microscopy, these newer techniques offer a nanoscale view of the axonal cytoskeleton, which is important for our understanding of neuronal form and function, and lay the foundation for future studies. In this Review, we summarize existing concepts in the field and highlight contemporary discoveries that have fundamentally altered our perception of the axonal cytoskeleton.

The prehistory of the cytoskeleton concept.

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Zampieri, Fabio; Coen, Matteo; Gabbiani, Giulio

2014-08-01

Here we discuss how the concept and the name of cytoskeleton were generated and started to evolve over the last two centuries into what is presently a basic topic of modern biology. We also attempt to describe some facets of the emergence of cytoskeleton component characterization in which our laboratory was in part involved. © 2014 Wiley Periodicals, Inc.

Cytoskeleton dynamics: Fluctuations within the network

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Bursac, Predrag; Fabry, Ben; Trepat, Xavier; Lenormand, Guillaume; Butler, James P.; Wang, Ning; Fredberg, Jeffrey J.; An, Steven S.

2007-01-01

Out-of-equilibrium systems, such as the dynamics of a living cytoskeleton (CSK), are inherently noisy with fluctuations arising from the stochastic nature of the underlying biochemical and molecular events. Recently, such fluctuations within the cell were characterized by observing spontaneous nano-scale motions of an RGD-coated microbead bound to the cell surface [Bursac et al., Nat. Mater. 4 (2005) 557-561]. While these reported anomalous bead motions represent a molecular level reorganization (remodeling) of microstructures in contact with the bead, a precise nature of these cytoskeletal constituents and forces that drive their remodeling dynamics are largely unclear. Here, we focused upon spontaneous motions of an RGD-coated bead and, in particular, assessed to what extent these motions are attributable to (i) bulk cell movement (cell crawling), (ii) dynamics of focal adhesions, (iii) dynamics of lipid membrane, and/or (iv) dynamics of the underlying actin CSK driven by myosin motors

Differential nuclear shape dynamics of invasive andnon-invasive breast cancer cells are associated with actin cytoskeleton organization and stability.

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Chiotaki, Rena; Polioudaki, Hara; Theodoropoulos, Panayiotis A

2014-08-01

Cancer cells often exhibit characteristic aberrations in their nuclear architecture, which are indicative of their malignant potential. In this study, we have examined the nuclear and cytoskeletal composition, attachment configuration dynamics, and osmotic or drug treatment response of invasive (Hs578T and MDA-MB-231) and non-invasive (MCF-10A and MCF-7) breast cancer cell lines. Unlike MCF-10A and MCF-7, Hs578T and MDA-MB-231 cells showed extensive nuclear elasticity and deformability and displayed distinct kinetic profiles during substrate attachment. The nuclear shape of MCF-10A and MCF-7 cells remained almost unaffected upon detachment, hyperosmotic shock, or cytoskeleton depolymerization, while Hs578T and MDA-MB-231 revealed dramatic nuclear contour malformations following actin reorganization.

Cyclooxygenase and cAMP-dependent protein kinase reorganize the actin cytoskeleton for motility in HeLa cells.

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Glenn, Honor L; Jacobson, Bruce S

2003-08-01

The adhesion of a cell to its surrounding matrix is a key determinant in many aspects of cell behavior. Adhesion consists of distinct stages : attachment, cell spreading, motility, and/or immobilization. Interrelated signaling pathways regulate these stages, and many adhesion-related signals control the architecture of the cytoskeleton. The various cytoskeletal organizations then give rise to the specific stages of adhesion. It has been shown that arachidonic acid acts at a signaling branch point during cell attachment. Arachidonic acid is metabolized via lipoxygenase to activate actin polymerization and cell spreading. It is also metabolized by cyclooxygenase to generate small actin bundles. We have used confocal microscopy and indirect immunofluorescence to investigate the structure of these cyclooxygenase dependent actin bundles in HeLa cells. We have also employed cell migration assays and pharmacological modulation of cyclooxygenase and downstream signals. The results indicate that cyclooxygenase and PKA stimulate the formation of actin bundles that contain myosin II and associate with small focal adhesions. In addition, we demonstrate that this cytoskeletal organization correlates with increased cell motility. Copyright 2003 Wiley-Liss, Inc.

A comparative study of the effect of oxidative stress on the cytoskeleton in human cortical neurons

International Nuclear Information System (INIS)

Allani, Pramod K.; Sum, Tak; Bhansali, Suraj G.; Mukherjee, Suman K.; Sonee, Manisha

2004-01-01

Cytoskeleton disruption is a process by which oxidative stress disrupts cellular function. This study compares and contrasts the effect of oxidative stress on the three major cytoskeleton filaments, microfilaments (MFs), microtubule (MT), and vimentin in human cortical neuronal cell line (HCN2). HCN2 cells were treated with 100 μM tertiary butylhydroperoxide (t-BuOOH), a free radical generating neurotoxin for 1, 3, or 6 h. Cell viability studies demonstrated significant cell death although the morphology studies showed that there was a substantial loss in neurites of neurons treated with t-BuOOH for 6 h. Because the cytoskeleton plays a role in neurite outgrowth, the effect of oxidative stress on the cytoskeletal was studied. In neurons subjected to oxidative stress for 30 min or 1 h, there were no major changes in microfilament distribution though there was altered distribution of microtubule and vimentin filaments as compared to controls. However, loss and disruption of all the three cytoskeletal filaments was observed at later times (3 and 6 h), which was confirmed by Western Blot analysis. Further studies were done to measure the gene expression levels of actin, tubulin, and vimentin. Results indicated that the overall loss of the cytoskeletal proteins in neurons treated with free radical generating toxin might not be a direct result of the downregulation of the cytoskeletal genes. This study shows that free radical generation in human neurons leads to the disruption of the cytoskeleton, though there may be a difference in the susceptibility to oxidative stress among the individual components of the cytoskeletal filaments

Root cytoskeleton: its role in perception of and response to gravity

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Baluska, F.; Hasenstein, K. H.

1997-01-01

We have critically evaluated the possible functions of the plant cytoskeleton in root gravisensing and graviresponse and discussed the evidence that microtubules (MTs) and actin microfilaments (MFs) do not control differential cell growth during bending of roots. On the other hand, MF and MT networks are envisaged to participate in gravisensing because of the mechanical properties of the cytoskeletal structures that interconnect plant cell organelles with the plasma membrane. In restrained gravisensing, forces are suggested to be transmitted to membranes because large-scale gravity-dependent repositioning of organelles is effectively prevented due to the cytoskeleton-mediated anchorage of their envelopes at the plasma membrane. From the cytoskeletal point of view, we can also envisage an unrestrained gravity sensing when cytoskeletal tethers are not strong enough to preserve the tight control over distribution of organelles and the latter, if heavy enough, are allowed to sediment towards the physical bottom of cells. This situation obviously occurs in root cap statocytes because these uniquely organized cells are depleted of prominent actin MF bundles, endoplasmic MT arrays, and ER elements in their internal cytoplasm. Nevertheless, indirect evidence clearly indicates that sedimented root cap statoliths are enmeshed within fine but dynamic MF networks and that their behaviour is obviously under, at least partial, cytoskeletal control. The actomyosin-enriched domain among and around amyloplasts is proposed to increase the perception of gravity due to the grouping effect of sedimenting statoliths. Cytoskeletal links between myosin-rich statoliths, and cell peripheries well equipped with dense cortical MTs, membrane-associated cytoskeleton, as well as with ER elements, would allow efficient restrained gravisensing only at the statocyte cell cortex. As a consequence of cytoskeletal depletion in the internal statocyte cytoplasm and bulk sedimentation of large

The role of the cytoskeleton in cellular force generation in 2D and 3D environments

International Nuclear Information System (INIS)

Kraning-Rush, Casey M; Carey, Shawn P; Califano, Joseph P; Smith, Brooke N; Reinhart-King, Cynthia A

2011-01-01

To adhere and migrate, cells generate forces through the cytoskeleton that are transmitted to the surrounding matrix. While cellular force generation has been studied on 2D substrates, less is known about cytoskeletal-mediated traction forces of cells embedded in more in vivo-like 3D matrices. Recent studies have revealed important differences between the cytoskeletal structure, adhesion, and migration of cells in 2D and 3D. Because the cytoskeleton mediates force, we sought to directly compare the role of the cytoskeleton in modulating cell force in 2D and 3D. MDA-MB-231 cells were treated with agents that perturbed actin, microtubules, or myosin, and analyzed for changes in cytoskeletal organization and force generation in both 2D and 3D. To quantify traction stresses in 2D, traction force microscopy was used; in 3D, force was assessed based on single cell-mediated collagen fibril reorganization imaged using confocal reflectance microscopy. Interestingly, even though previous studies have observed differences in cell behaviors like migration in 2D and 3D, our data indicate that forces generated on 2D substrates correlate with forces within 3D matrices. Disruption of actin, myosin or microtubules in either 2D or 3D microenvironments disrupts cell-generated force. These data suggest that despite differences in cytoskeletal organization in 2D and 3D, actin, microtubules and myosin contribute to contractility and matrix reorganization similarly in both microenvironments

ANDROGENS REGULATE T47D CELLS MOTILITY AND INVASION THROUGH ACTIN CYTOSKELETON REMODELLING

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Maria Magdalena Montt-Guevara

2016-09-01

Full Text Available The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgens receptor (AR is expressed in approximately 70% to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple negative breast cancers. Recent studies have associated the actin-binding proteins of the Ezrin-Radixin-Moesin (ERM family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T, dihydrotestosterone (DHT and dehydroepiandrosterone (DHEA may regulate breast cancer cell motility and invasion through the control of actin remodelling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER, while the non aromatizable androgen – DHT only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer, and eventually to develop new strategies for treatment of breast cancer.

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

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Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

2006-07-26

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (Ø confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

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Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

2016-10-07

Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

The Evolving Complexity of the Podocyte Cytoskeleton.

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Schell, Christoph; Huber, Tobias B

2017-11-01

Podocytes exhibit a unique cytoskeletal architecture that is fundamentally linked to their function in maintaining the kidney filtration barrier. The cytoskeleton regulates podocyte shape, structure, stability, slit diaphragm insertion, adhesion, plasticity, and dynamic response to environmental stimuli. Genetic mutations demonstrate that even slight impairment of the podocyte cytoskeletal apparatus results in proteinuria and glomerular disease. Moreover, mechanisms underpinning all acquired glomerular pathologies converge on disruption of the cytoskeleton, suggesting that this subcellular structure could be targeted for therapeutic purposes. This review summarizes our current understanding of the function of the cytoskeleton in podocytes and the associated implications for pathophysiology. Copyright © 2017 by the American Society of Nephrology.

Sirtuin1 Maintains Actin Cytoskeleton by Deacetylation of Cortactin in Injured Podocytes

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Motonishi, Shuta; Wada, Takehiko; Ishimoto, Yu; Ohse, Takamoto; Matsusaka, Taiji; Kubota, Naoto; Shimizu, Akira; Kadowaki, Takashi; Tobe, Kazuyuki

2015-01-01

Recent studies have highlighted the renoprotective effect of sirtuin1 (SIRT1), a deacetylase that contributes to cellular regulation. However, the pathophysiologic role of SIRT1 in podocytes remains unclear. Here, we investigated the function of SIRT1 in podocytes. We first established podocyte-specific Sirt1 knockout (SIRT1pod−/−) mice. We then induced glomerular disease by nephrotoxic serum injection. The increase in urinary albumin excretion and BUN and the severity of glomerular injury were all significantly greater in SIRT1pod−/− mice than in wild-type mice. Western blot analysis and immunofluorescence showed a significant decrease in podocyte-specific proteins in SIRT1pod−/− mice, and electron microscopy showed marked exacerbation of podocyte injury, including actin cytoskeleton derangement in SIRT1pod−/− mice compared with wild-type mice. Protamine sulfate-induced podocyte injury was also exacerbated by podocyte-specific SIRT1 deficiency. In vitro, actin cytoskeleton derangement in H2O2-treated podocytes became prominent when the cells were pretreated with SIRT1 inhibitors. Conversely, this H2O2-induced derangement was ameliorated by SIRT1 activation. Furthermore, SIRT1 activation deacetylated the actin-binding and -polymerizing protein cortactin in the nucleus and facilitated deacetylated cortactin localization in the cytoplasm. Cortactin knockdown or inhibition of the nuclear export of cortactin induced actin cytoskeleton derangement and dissociation of cortactin from F-actin, suggesting the necessity of cytoplasmic cortactin for maintenance of the actin cytoskeleton. Taken together, these findings indicate that SIRT1 protects podocytes and prevents glomerular injury by deacetylating cortactin and thereby, maintaining actin cytoskeleton integrity. PMID:25424328

X-ray-enhanced cancer cell migration requires the linker of nucleoskeleton and cytoskeleton complex.

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Imaizumi, Hiromasa; Sato, Katsutoshi; Nishihara, Asuka; Minami, Kazumasa; Koizumi, Masahiko; Matsuura, Nariaki; Hieda, Miki

2018-04-01

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, including nuclear migration, mechanotransduction, chromatin tethering and DNA damage response. We recently showed that a nuclear envelope protein, Sad1 and UNC84 domain protein 1 (SUN1), a component of the LINC complex, has a critical function in cell migration. Although ionizing radiation activates cell migration and invasion in vivo and in vitro, the underlying molecular mechanism remains unknown. Here, we examined the involvement of the LINC complex in radiation-enhanced cell migration and invasion. A sublethal dose of X-ray radiation promoted human breast cancer MDA-MB-231 cell migration and invasion, whereas carbon ion beam radiation suppressed these processes in a dose-dependent manner. Depletion of SUN1 and SUN2 significantly suppressed X-ray-enhanced cell migration and invasion. Moreover, depletion or overexpression of each SUN1 splicing variant revealed that SUN1_888 containing 888 amino acids of SUN1 but not SUN1_916 was required for X-ray-enhanced migration and invasion. In addition, the results suggested that X-ray irradiation affected the expression level of SUN1 splicing variants and a SUN protein binding partner, nesprins. Taken together, our findings supported that the LINC complex contributed to photon-enhanced cell migration and invasion. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

The cytoskeleton as a novel therapeutic target for old neurodegenerative disorders.

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Eira, Jessica; Silva, Catarina Santos; Sousa, Mónica Mendes; Liz, Márcia Almeida

2016-06-01

Cytoskeleton defects, including alterations in microtubule stability, in axonal transport as well as in actin dynamics, have been characterized in several unrelated neurodegenerative conditions. These observations suggest that defects of cytoskeleton organization may be a common feature contributing to neurodegeneration. In line with this hypothesis, drugs targeting the cytoskeleton are currently being tested in animal models and in human clinical trials, showing promising effects. Drugs that modulate microtubule stability, inhibitors of posttranslational modifications of cytoskeletal components, specifically compounds affecting the levels of tubulin acetylation, and compounds targeting signaling molecules which regulate cytoskeleton dynamics, constitute the mostly addressed therapeutic interventions aiming at preventing cytoskeleton damage in neurodegenerative disorders. In this review, we will discuss in a critical perspective the current knowledge on cytoskeleton damage pathways as well as therapeutic strategies designed to revert cytoskeleton-related defects mainly focusing on the following neurodegenerative disorders: Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic Lateral Sclerosis and Charcot-Marie-Tooth Disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

2007-01-01

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 data demonstrate that

Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth.

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Fuchino, Katsuya; Bagchi, Sonchita; Cantlay, Stuart; Sandblad, Linda; Wu, Di; Bergman, Jessica; Kamali-Moghaddam, Masood; Flärdh, Klas; Ausmees, Nora

2013-05-21

Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

IGF-1 modulates gene expression of proteins involved in inflammation, cytoskeleton, and liver architecture.

Science.gov (United States)

Lara-Diaz, V J; Castilla-Cortazar, I; Martín-Estal, I; García-Magariño, M; Aguirre, G A; Puche, J E; de la Garza, R G; Morales, L A; Muñoz, U

2017-05-01

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1 +/- , and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.

Cytoskeleton-amyloplast interactions in sweet clover

Science.gov (United States)

Guikema, J. A.; Hilaire, E.; Odom, W. R.; Spooner, B. S. (Principal Investigator)

1993-01-01

The distribution of organelles within columella cells of sweet clover was examined by transmission electron microscopy following growth under static or clinorotating conditions. A developmentally conditioned polarity was observed, with a proximal location of the nucleus and a distal accumulation of the endoplasmic reticulum. This polarity was insensitive to clinorotation. In contrast, clinorotation altered the location of amyloplasts. Application of cytoskeletal poisons (colchicine, cytochalasin D, taxol, and phalloidin), especially during clinorotation, had interesting effects on the maintenance of columella cell polarity, with a profound effect on the extent, location, and structure of the endoplasmic reticulum. The site of cytoskeletal interactions with sedimenting amyloplasts is thought to be the amyloplast envelope. An envelope fraction, having over 17 polypeptides, was isolated using immobilized antibody technology, and will provide a means of assessing the role of specific peptides in cytoskeleton/amyloplast interactions.

A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts

International Nuclear Information System (INIS)

Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

2006-01-01

Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation

A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts.

Science.gov (United States)

Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

2006-09-15

Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation.

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

International Nuclear Information System (INIS)

Pradhan, D.; Schlegel, R.A.; Williamson, P.

1991-01-01

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[ 14 C] ethanolamine ([ 14 C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [ 14 C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes

The cytoskeleton of digitonin-treated rat hepatocytes.

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Fiskum, G; Craig, S W; Decker, G L; Lehninger, A L

1980-06-01

Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.

Airborne particulate matter in vitro exposure induces cytoskeleton remodeling through activation of the ROCK-MYPT1-MLC pathway in A549 epithelial lung cells.

Science.gov (United States)

Chirino, Yolanda I; García-Cuellar, Claudia María; García-García, Carlos; Soto-Reyes, Ernesto; Osornio-Vargas, Álvaro Román; Herrera, Luis A; López-Saavedra, Alejandro; Miranda, Javier; Quintana-Belmares, Raúl; Pérez, Irma Rosas; Sánchez-Pérez, Yesennia

2017-04-15

Airborne particulate matter with an aerodynamic diameter ≤10μm (PM 10 ) is considered a risk factor for the development of lung cancer. Little is known about the cellular mechanisms by which PM 10 is associated with cancer, but there is evidence that its exposure can lead to an acquired invasive phenotype, apoptosis evasion, inflammasome activation, and cytoskeleton remodeling in lung epithelial cells. Cytoskeleton remodeling occurs through actin stress fiber formation, which is partially regulated through ROCK kinase activation, we aimed to investigate if this protein was activated in response to PM 10 exposure in A549 lung epithelial cells. Results showed that 10μg/cm 2 of PM 10 had no influence on cell viability but increased actin stress fibers, cytoplasmic ROCK expression, and phosphorylation of myosin phosphatase-targeting 1 (MYPT1) and myosin light chain (MLC) proteins, which are targeted by ROCK. The inhibition of ROCK prevented actin stress fiber formation and the phosphorylation of MYPT1 and MLC, suggesting that PM 10 activated the ROCK-MYPT1-MLC pathway in lung epithelial cells. The activation of ROCK1 has been involved in the acquisition of malignant phenotypes, and its induction by PM 10 exposure could contribute to the understanding of PM 10 as a risk factor for cancer development through the mechanisms associated with invasive phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

 Oxidative stress modulates the organization of erythrocyte membrane cytoskeleton

Directory of Open Access Journals (Sweden)

Maria Olszewska

2012-07-01

Full Text Available  Background:Apart from their main role in transporting oxygen and carbon dioxide, erythrocytes play also an important role in organism antioxidative defence. Direct exposure to reactive oxygen species (ROS results in shortening of their half-life, even by 50�20The presence of glucose, being the substrate in pentose phosphate pathway (PPP cycle, is one of the factors that can have influence on the level of oxidative stress. The activity of PPP increases during oxidative stress. Glucose guarantees normal PPP functioning with the production of reductive equivalents in the amounts necessary to reproduction of glutathione – nonenzymatic free radical scavenger. In available literature there are no reports regarding the changes in protein contents of erythrocyte cytoskeleton exposed to t-butyl hydroperoxide in relation to glucose presence in incubation medium.Material/methods:Erythrocytes taken from 10 healthy subjects were used to assess the influence of generated free radicals on erythrocyte proteins and chosen parameters of oxidative stress. Erythrocytes were incubated in the solutions containing deferent concentrations of t-butyl hydroperoxide and glucose. Electrophoresis was performed on polyacrylamide gel in denaturating conditions. The contents of tryptophan in membranes was evaluated spectrofluorometrically.Results/conclusions:In vitro conditions oxidative stress leads to protein damage in erythrocyte cytoskeleton, both in proteins inside the cell as well as having contact with extracellular environment. In consequence, the amount of low-molecular proteins – mainly globin, which bind to cytoskeleton, increases. This process takes place independently of glucose presence in incubation medium. One of the element of protein cytoskeleton, tryptophan, also undergoes degradation. The decrease of its contents is higher during erythrocyte exposure to t-BOOH in environment containing glucose, what can suggest prooxidative influence of glucose in

Fungal lectin of Peltigera canina induces chemotropism of compatible Nostoc cells by constriction-relaxation pulses of cyanobiont cytoskeleton.

Science.gov (United States)

Díaz, Eva Maria; Vicente-Manzanares, Miguel; Sacristan, Mara; Vicente, Carlos; Legaz, Maria-Estrella

2011-10-01

A glycosylated arginase acting as a fungal lectin from Peltigera canina is able to produce recruitment of cyanobiont Nostoc cells and their adhesion to the hyphal surface. This implies that the cyanobiont would develop organelles to motility towards the chemoattractant. However when visualized by transmission electron microscopy, Nostoc cells recently isolated from P. canina thallus do not reveal any motile, superficial organelles, although their surface was covered by small spindles and serrated layer related to gliding. The use of S-(3,4-dichlorobenzyl)isothiourea, blebbistatin, phalloidin and latrunculin A provide circumstantial evidence that actin microfilaments rather than MreB, the actin-like protein from prokaryota, and, probably, an ATPase which develops contractile function similar to that of myosin II, are involved in cell motility. These experimental facts, the absence of superficial elements (fimbriae, pili or flagellum) related to cell movement, and the appearance of sunken cells during of after movement verified by scanning electron microscopy, support the hypothesis that the motility of lichen cyanobionts could be achieved by contraction-relaxation episodes of the cytoskeleton induced by fungal lectin act as a chemoattractant.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

Science.gov (United States)

Cerecedo, Doris; Martínez-Vieyra, Ivette; Maldonado-García, Deneb; Hernández-González, Enrique; Winder, Steve J

2015-11-01

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics. © 2015 Wiley Periodicals, Inc.

Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton

DEFF Research Database (Denmark)

Ly, Donald L.; Waheed, Faiza; Lodyga, Monika

2013-01-01

Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin-regulated coactiv......Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin......-regulated coactivator of serum response factor, is a major link between the actin skeleton and transcriptional control. We therefore investigated whether MRTF is regulated by hyperosmotic stress. Here we show that hypertonicity induces robust, rapid, and transient translocation of MRTF from the cytosol to the nucleus...... in kidney tubular cells. We found that the hyperosmolarity-triggered MRTF translocation is mediated by the RhoA/Rho kinase (ROK) pathway. Moreover, the Rho guanine nucleotide exchange factor GEF-H1 is activated by hyperosmotic stress, and it is a key contributor to the ensuing RhoA activation and MRTF...

Osmoconditioning prevents the onset of microtubular cytoskeleton and activation of cell cycle and is detrimental for germination of Jatropha curcas L. seeds.

Science.gov (United States)

de Brito, C D; Loureiro, M B; Ribeiro, P R; Vasconcelos, P C T; Fernandez, L G; de Castro, R D

2016-11-01

Jatropha curcas is an oilseed crop renowned for its tolerance to a diverse range of environmental stresses. In Brazil, this species is grown in semiarid regions where crop establishment requires a better understanding of the mechanisms underlying appropriate seed, seedling and plant behaviour under water restriction conditions. In this context, the objective of this study was to investigate the physiological and cytological profiles of J. curcas seeds in response to imbibition in water (control) and in polyethylene glycol solution (osmoticum). Seed germinability and reactivation of cell cycle events were assessed by means of different germination parameters and immunohistochemical detection of tubulin and microtubules, i.e. tubulin accumulation and microtubular cytoskeleton configurations in water imbibed seeds (control) and in seeds imbibed in the osmoticum. Immunohistochemical analysis revealed increasing accumulation of tubulin and appearance of microtubular cytoskeleton in seed embryo radicles imbibed in water from 48 h onwards. Mitotic microtubules were only visible in seeds imbibed in water, after radicle protrusion, as an indication of cell cycle reactivation and cell proliferation, with subsequent root development. Imbibition in osmoticum prevented accumulation of microtubules, i.e. activation of cell cycle, therefore germination could not be resumed. Osmoconditioned seeds were able to survive re-drying and could resume germination after re-imbibition in water, however, with lower germination performance, possibly due to acquisition of secondary dormancy. This study provides important insights into understanding of the physiological aspects of J. curcas seed germination in response to water restriction conditions. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Effect of omega-3 polyunsaturated fatty acids on the cytoskeleton: an open-label intervention study.

Science.gov (United States)

Schmidt, Simone; Willers, Janina; Riecker, Sabine; Möller, Katharina; Schuchardt, Jan Philipp; Hahn, Andreas

2015-02-14

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.

The Prediction of Key Cytoskeleton Components Involved in Glomerular Diseases Based on a Protein-Protein Interaction Network.

Science.gov (United States)

Ding, Fangrui; Tan, Aidi; Ju, Wenjun; Li, Xuejuan; Li, Shao; Ding, Jie

2016-01-01

Maintenance of the physiological morphologies of different types of cells and tissues is essential for the normal functioning of each system in the human body. Dynamic variations in cell and tissue morphologies depend on accurate adjustments of the cytoskeletal system. The cytoskeletal system in the glomerulus plays a key role in the normal process of kidney filtration. To enhance the understanding of the possible roles of the cytoskeleton in glomerular diseases, we constructed the Glomerular Cytoskeleton Network (GCNet), which shows the protein-protein interaction network in the glomerulus, and identified several possible key cytoskeletal components involved in glomerular diseases. In this study, genes/proteins annotated to the cytoskeleton were detected by Gene Ontology analysis, and glomerulus-enriched genes were selected from nine available glomerular expression datasets. Then, the GCNet was generated by combining these two sets of information. To predict the possible key cytoskeleton components in glomerular diseases, we then examined the common regulation of the genes in GCNet in the context of five glomerular diseases based on their transcriptomic data. As a result, twenty-one cytoskeleton components as potential candidate were highlighted for consistently down- or up-regulating in all five glomerular diseases. And then, these candidates were examined in relation to existing known glomerular diseases and genes to determine their possible functions and interactions. In addition, the mRNA levels of these candidates were also validated in a puromycin aminonucleoside(PAN) induced rat nephropathy model and were also matched with existing Diabetic Nephropathy (DN) transcriptomic data. As a result, there are 15 of 21 candidates in PAN induced nephropathy model were consistent with our predication and also 12 of 21 candidates were matched with differentially expressed genes in the DN transcriptomic data. By providing a novel interaction network and prediction, GCNet

Elevated phosphatase of regenerating liver 3 (PRL-3) promotes cytoskeleton reorganization, cell migration and invasion in endometrial stromal cells from endometrioma.

Science.gov (United States)

Zhan, Hong; Ma, Junyan; Ruan, Fei; Bedaiwy, Mohamed A; Peng, Bo; Wu, Ruijin; Lin, Jun

2016-04-01

Is phosphatase of regenerating liver-3 (PRL-3) associated with increased motility of endometriotic cells from endometrioma? Elevated PRL-3 promotes cytoskeleton reorganization, cell migration and invasion of endometrial stromal cells (ESCs) from endometrioma. Overexpression of PRL-3 is associated with cancer cell migration, invasion and metastatic phenotype. Primary human ESCs were isolated from eutopic endometrium of women without endometriosis (EuCo, n = 10), with histologically proven endometrioma (EuEM, n = 19) and from the cyst wall of ovarian endometriosis (OvEM, n = 26). The expression of PRL-3 in ESCs derived from EuCo, EuEM and OvEM at different phases of menstrual cycle were compared. The protein and mRNA levels of PRL-3 were examined by western blot and RT-qPCR, respectively. ESCs from OvEM were transfected with/without short hairpin RNA (shRNA) or small interfering RNA (siRNA). Additionally, a plasmid-mediated delivery system was used to achieve PRL-3 overexpression in ESCs from EuEM. The cellular distribution of F-actin and α-tubulin were examined by immunocytochemistry. Cell motility was evaluated by a transwell migration/invasion assay. The protein and mRNA levels of PRL-3 are significantly elevated in ESCs from OvEM compared with EuCo and EuEM. The expression of PRL-3 was not altered between proliferative phase and secretory phase in ESCs from all groups. Knockdown of PRL-3 significantly modified the distribution of F-actin and α-tubulin cytoskeleton, inhibited cell migration and invasion. Endogenous inhibition of PRL-3 attenuated the expression of Ras homolog gene family members A and C (RhoA, RhoC), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and matrix metalloproteinase (MMP) 9, but not MMP2 in ESCs from OvEM. Additionally, overexpression of PRL-3 in ESCs from EuEM up-regulates cell migration and invasion, and increases the expression of RhoA, RhoC, ROCK1 and MMP9. Lack of in vivo animal studies is the major limitation of our

Sonic hedgehog signaling regulates actin cytoskeleton via Tiam1-Rac1 cascade during spine formation.

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Sasaki, Nobunari; Kurisu, Junko; Kengaku, Mineko

2010-12-01

The sonic hedgehog (Shh) pathway has essential roles in several processes during development of the vertebrate central nervous system (CNS). Here, we report that Shh regulates dendritic spine formation in hippocampal pyramidal neurons via a novel pathway that directly regulates the actin cytoskeleton. Shh signaling molecules Patched (Ptc) and Smoothened (Smo) are expressed in several types of postmitotic neurons, including cerebellar Purkinje cells and hippocampal pyramidal neurons. Knockdown of Smo induces dendritic spine formation in cultured hippocampal neurons independently of Gli-mediated transcriptional activity. Smo interacts with Tiam1, a guanine nucleotide exchange factor for Rac1, via its cytoplasmic C-terminal region. Inhibition of Tiam1 or Rac1 activity suppresses spine induction by Smo knockdown. Shh induces remodeling of the actin cytoskeleton independently of transcriptional activation in mouse embryonic fibroblasts. These findings demonstrate a novel Shh pathway that regulates the actin cytoskeleton via Tiam1-Rac1 activation. Copyright © 2010 Elsevier Inc. All rights reserved.

Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation

International Nuclear Information System (INIS)

Packham, M.A.; Guccione, M.A.; Bryant, N.L.; Livne, A.

1990-01-01

Previous experiments showed that during the early stages (20-30 seconds) of aggregation induced by adenosine diphosphate (ADP, 2 microM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with [3H]palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(beta-aminoethyl ether)]-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated cytoskeletons. In both species, and with either ADP or thrombin as aggregating agent, 70-85% of the label in both intact platelets and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets. The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and palmitic acid into the cytoskeleton

Coordination of membrane and actin cytoskeleton dynamics during filopodia protrusion.

Directory of Open Access Journals (Sweden)

Changsong Yang

2009-05-01

Full Text Available Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.

Changes in Cell Adhesivity and Cytoskeleton-Related Proteins During Imatinib-Induced Apoptosis of Leukemic JURL-MK1 Cells

Czech Academy of Sciences Publication Activity Database

Kuželová, K.; Pluskalová, M.; Grebeňová, D.; Pavlásková, Kateřina; Halada, Petr; Hrkal, Z.

2010-01-01

Roč. 111, č. 6 (2010), s. 1413-1425 ISSN 0730-2312 R&D Projects: GA MŠk LC07017; GA MZd NR9243 Institutional research plan: CEZ:AV0Z50200510 Keywords : imatinib * adhesion * cytoskeleton Subject RIV: CE - Biochemistry Impact factor: 3.122, year: 2010

S-Nitrosylation of Cofilin-1 Mediates Estradiol-17β-Stimulated Endothelial Cytoskeleton Remodeling

Science.gov (United States)

Zhang, Hong-hai; Lechuga, Thomas J.; Tith, Tevy; Wang, Wen; Wing, Deborah A.

2015-01-01

Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNOCys80 of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling. PMID:25635941

Multiciliated cell basal bodies align in stereotypical patterns coordinated by the apical cytoskeleton

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Herawati, Elisa; Kanoh, Hatsuho

2016-01-01

Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating, which requires properly organized basal bodies (BBs). Airway MCCs have large numbers of BBs, which are uniformly oriented and, as we show here, align linearly. The mechanism for BB alignment is unexplored. To study this mechanism, we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein–centrin2–labeled BBs in cultured mouse tracheal MCCs. During MCC differentiation, the BB array adopted four stereotypical patterns, from a clustering “floret” pattern to the linear “alignment.” This alignment process was correlated with BB orientations, revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF). The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. We constructed a theoretical model, which indicated that the apical cytoskeleton, acting like a viscoelastic fluid, provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport. PMID:27573463

Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies.

Science.gov (United States)

Méplan, Catherine; Johnson, Ian T; Polley, Abigael C J; Cockell, Simon; Bradburn, David M; Commane, Daniel M; Arasaradnam, Ramesh P; Mulholland, Francis; Zupanic, Anze; Mathers, John C; Hesketh, John

2016-08-01

Epidemiologic studies highlight the potential role of dietary selenium (Se) in colorectal cancer prevention. Our goal was to elucidate whether expression of factors crucial for colorectal homoeostasis is affected by physiologic differences in Se status. Using transcriptomics and proteomics followed by pathway analysis, we identified pathways affected by Se status in rectal biopsies from 22 healthy adults, including 11 controls with optimal status (mean plasma Se = 1.43 μM) and 11 subjects with suboptimal status (mean plasma Se = 0.86 μM). We observed that 254 genes and 26 proteins implicated in cancer (80%), immune function and inflammatory response (40%), cell growth and proliferation (70%), cellular movement, and cell death (50%) were differentially expressed between the 2 groups. Expression of 69 genes, including selenoproteins W1 and K, which are genes involved in cytoskeleton remodelling and transcription factor NFκB signaling, correlated significantly with Se status. Integrating proteomics and transcriptomics datasets revealed reduced inflammatory and immune responses and cytoskeleton remodelling in the suboptimal Se status group. This is the first study combining omics technologies to describe the impact of differences in Se status on colorectal expression patterns, revealing that suboptimal Se status could alter inflammatory signaling and cytoskeleton in human rectal mucosa and so influence cancer risk.-Méplan, C., Johnson, I. T., Polley, A. C. J., Cockell, S., Bradburn, D. M., Commane, D. M., Arasaradnam, R. P., Mulholland, F., Zupanic, A., Mathers, J. C., Hesketh, J. Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies. © The Author(s).

Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton

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Hoyer-Fender Sigrid

2010-04-01

Full Text Available Abstract Background Pelota (PELO is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation. Results We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC, we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments. Conclusion We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

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Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

YeeV is an Escherichia coli toxin that inhibits cell division by targeting the cytoskeleton proteins, FtsZ and MreB.

Science.gov (United States)

Tan, Qian; Awano, Naoki; Inouye, Masayori

2011-01-01

Toxin-antitoxin (TA) systems of free-living bacteria have recently demonstrated that these toxins inhibit cell growth by targeting essential functions of cellular metabolism. Here we show that YeeV toxin inhibits cell division, leads to a change in morphology and lysis of Escherichia coli cells. YeeV interacts with two essential cytoskeleton proteins, FtsZ and MreB. Purified YeeV inhibits both the GTPase activity and the GTP-dependent polymerization of FtsZ. YeeV also inhibits ATP-dependent polymerization of MreB. Truncated C-terminal deletions of YeeV result in elongation of cells, and a deletion of the first 15 amino acids from the N-terminus of YeeV caused lemon-shaped cell formation. The YeeV toxin is distinct from other well-studied toxins: it directs the binding of two cytoskeletal proteins and inhibits FtsZ and MreB simultaneously. © 2010 Blackwell Publishing Ltd.

Marine toxins and the cytoskeleton: a new view of palytoxin toxicity.

Science.gov (United States)

Louzao, M Carmen; Ares, Isabel R; Cagide, Eva

2008-12-01

Palytoxin is a marine toxin first isolated from zoanthids (genus Palythoa), even though dinoflagellates of the genus Ostreopsis are the most probable origin of the toxin. Ostreopsis has a wide distribution in tropical and subtropical areas, but recently these dinoflagellates have also started to appear in the Mediterranean Sea. Two of the most remarkable properties of palytoxin are the large and complex structure (with different analogs, such as ostreocin-D or ovatoxin-a) and the extreme acute animal toxicity. The Na(+)/K(+)-ATPase has been proposed as receptor for palytoxin. The marine toxin is known to act on the Na(+) pump and elicit an increase in Na(+) permeability, which leads to depolarization and a secondary Ca(2+) influx, interfering with some functions of cells. Studies on the cellular cytoskeleton have revealed that the signaling cascade triggered by palytoxin leads to actin filament system distortion. The activity of palytoxin on the actin cytoskeleton is only partially associated with the cytosolic Ca(2+) changes; therefore, this ion represents an important factor in altering this structure, but it is not the only cause. The goal of the present minireview is to compile the findings reported to date about: (a) how palytoxin and analogs are able to modify the actin cytoskeleton within different cellular models; and (b) what signaling mechanisms could be involved in the modulation of cytoskeletal dynamics by palytoxin.

MICAL, the Flavoenzyme Participating in Cytoskeleton Dynamics

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Daniela Zucchini

2013-03-01

Full Text Available MICAL (from the Molecule Interacting with CasL indicates a family of recently discovered cytosolic, multidomain proteins, which uniquely couple an N-terminal FAD-containing monooxygenase-like domain to typical calponine homology, LIM and coiled-coil protein-interaction modules. Genetic and cell biology approaches have demonstrated an essential role of the catalytic activity of the monooxygenase-like domain in transducing the signal initiated by semaphorins interaction with their plexin receptors, which results in local actin cytoskeleton disassembly as part of fundamental processes that include differentiation, migration and cell-cell contacts in neuronal and non-neuronal cell types. This review focuses on the structure-function relations of the MICAL monooxygenase-like domain as they are emerging from the available in vitro studies on mouse, human and Drosophila MICAL forms that demonstrated a NADPH-dependent actin depolymerizing activity of MICAL. With Drosophila MICAL forms, actin depolymerization was demonstrated to be associated to conversion of Met44 to methionine sulfone through a postulated hydroxylating reaction. Arguments supporting the concept that MICAL effect on F-actin may be reversible will be discussed.

The Cytoskeleton and Force Response Mechanisms

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Allen, Philip Goodwin

2003-01-01

The long term aim of this project was to define the mechanisms by which cells sense and respond to the physical forces experienced at 1g and missing in microgravity. Identification and characterization of the elements of the cells force response mechanism could provide pathways and molecules to serve as targets for pharmacological intervention to mitigate the pathologic effects of microgravity. Mechanical forces experienced by the organism can be transmitted to cells through molecules that allow cells to bind to the extracellular matrix and through other types of molecules which bind cells to each other. These molecules are coupled in large complexes of proteins to structural elements such as the actin cytoskeleton that give the cell the ability to sense, resist and respond to force. Application of small forces to tissue culture cells causes local elevation of intracellular calcium through stretch activated ion channels, increased tyrosine phosphorylation and a restructuring of the actin cytoskeleton. Using collagen coated iron oxide beads and strong magnets, we can apply different levels of force to cells in culture. We have found that force application causes the cells to polymerize actin at the site of mechanical deformation and unexpectedly, to depolymerize actin across the rest of the cell. Observations of GFP- actin expressing cells demonstrate that actin accumulates at the site of deformation within the first five minutes of force application and is maintained for many tens of minutes after force is removed. Consistent with the reinforcement of the cytoskeletal structures underlying the integrin-bead interaction, force also alters the motion of bound magnetic beads. This effect is seen following the removal of the magnetic field, and is only partially ablated by actin disruption with cytochalsin B. While actin is polymerizing locally at the site of force application, force also stimulates a global reduction in actin filament content within the cells. We have

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

Science.gov (United States)

Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

2015-07-20

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. © 2015 Treuner-Lange et al.

Astrocyte-neuron interaction in diphenyl ditelluride toxicity directed to the cytoskeleton.

Science.gov (United States)

Heimfarth, Luana; da Silva Ferreira, Fernanda; Pierozan, Paula; Mingori, Moara Rodrigues; Moreira, José Cláudio Fonseca; da Rocha, João Batista Teixeira; Pessoa-Pureur, Regina

2017-03-15

Diphenylditelluride (PhTe) 2 is a neurotoxin that disrupts cytoskeletal homeostasis. We are showing that different concentrations of (PhTe) 2 caused hypophosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits (NFL, NFM and NFH) and altered actin organization in co-cultured astrocytes and neurons from cerebral cortex of rats. These mechanisms were mediated by N-methyl-d-aspartate (NMDA) receptors without participation of either L-type voltage-dependent calcium channels (L-VDCC) or metabotropic glutamate receptors. Upregulated Ca 2+ influx downstream of NMDA receptors activated Ca 2+ -dependent protein phosphatase 2B (PP2B) causing hypophosphorylation of astrocyte and neuron IFs. Immunocytochemistry showed that hypophosphorylated intermediate filaments (IF) failed to disrupt their organization into the cytoskeleton. However, phalloidin-actin-FITC stained cytoskeleton evidenced misregulation of actin distribution, cell spreading and increased stress fibers in astrocytes. βIII tubulin staining showed that neurite meshworks are not altered by (PhTe) 2 , suggesting greater susceptibility of astrocytes than neurons to (PheTe) 2 toxicity. These findings indicate that signals leading to IF hypophosphorylation fail to disrupt the cytoskeletal IF meshwork of interacting astrocytes and neurons in vitro however astrocyte actin network seems more susceptible. Our findings support that intracellular Ca 2+ is one of the crucial signals that modulate the action of (PhTe) 2 in co-cultured astrocytes and neurons and highlights the cytoskeleton as an end-point of the neurotoxicity of this compound. Cytoskeletal misregulation is associated with cell dysfunction, therefore, the understanding of the molecular mechanisms mediating the neurotoxicity of this compound is a matter of increasing interest since tellurium compounds are increasingly released in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Computer simulation of cytoskeleton-induced blebbing in lipid membranes

DEFF Research Database (Denmark)

Spangler, E. J.; Harvey, C. W.; Revalee, J. D.

2011-01-01

for large values of the ratio between the areas of the bilayer and the cytoskeleton. We also found that blebbing can be induced when the cytoskeleton is subject to a localized ablation or a uniform compression. The results obtained are qualitatively in agreement with the experimental evidence and the model...

Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

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L. López-Contreras

2013-01-01

Full Text Available Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

CLASP2 Links Reelin to the Cytoskeleton during Neocortical Development.

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Dillon, Gregory M; Tyler, William A; Omuro, Kerilyn C; Kambouris, John; Tyminski, Camila; Henry, Shawna; Haydar, Tarik F; Beffert, Uwe; Ho, Angela

2017-03-22

The Reelin signaling pathway plays a crucial role in regulating neocortical development. However, little is known about how Reelin controls the cytoskeleton during neuronal migration. Here, we identify CLASP2 as a key cytoskeletal effector in the Reelin signaling pathway. We demonstrate that CLASP2 has distinct roles during neocortical development regulating neuron production and controlling neuron migration, polarity, and morphogenesis. We found downregulation of CLASP2 in migrating neurons leads to mislocalized cells in deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. We discovered that Reelin regulates several phosphorylation sites within the positively charged serine/arginine-rich region that constitute consensus GSK3β phosphorylation motifs of CLASP2. Furthermore, phosphorylation of CLASP2 regulates its interaction with the Reelin adaptor Dab1 and this association is required for CLASP2 effects on neurite extension and motility. Together, our data reveal that CLASP2 is an essential Reelin effector orchestrating cytoskeleton dynamics during brain development. Copyright © 2017 Elsevier Inc. All rights reserved.

Formation of compact myelin is required for maturation of the axonal cytoskeleton

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Brady, S. T.; Witt, A. S.; Kirkpatrick, L. L.; de Waegh, S. M.; Readhead, C.; Tu, P. H.; Lee, V. M.

1999-01-01

Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.

Microfilamin - a new protein of the cytoskeleton with molecular weight 53,000

International Nuclear Information System (INIS)

Fridlyanskaya, I.I.; Galaktionov, K.I.; D'yakonova, M.Yu.; Pinaev, G.P.

1987-01-01

The microfilaments in cultured cells participate in many vitally important processes: endocytosis, movement of cells, division, etc. One of the approaches to the study of the proteins of the microfilaments is the use of the hybridoma technique. This method permits the use of cell structures as an immunogen and the investigation of their protein assortment by means of monoclonal antibodies. Using such an approach, the authors were able to identify a protein with molecular weight 53 kilodaltons, not previously described in the microfilaments. BABL/c mice were immunized with an extract of the cytoskeleton of murine cells 3T3. It was produced by treating the cells with 0.5% Triton in a buffer stabilizing the cytoskeleton (10 mM sodium phosphate buffer, pH 6.8, 0.1 M KCl, 1 mMEGTA, 0.1 mM EDTA, 5 mM MgCl 2 , and 2 M sucrose), and then with the same buffer, containing 1 micrograms/ml DNase I, 1mM PMSF, and 20 units/ml trasylol. The extract was clarified by successive centrifugation at 1000, 1200, and 10,000 g. Splenocytes of immune animals were hybridized with murine myeloma SP 2/0-Ag 14 cells and screening of the hybridomas was performed by the method of indirect immunofluorescence on murine cells of the 3T3 line stained with Coomassie blue. Data from autograph after incubation with E9-48 antibodies and 125 I-labeled rabbit anti-mouse immunoglobulins are presented as well. The authors named this new technique microfilamin since it is detected in structures containing microfilaments: stress fibrils, the active edge, surface villi, and others

Epiplasmins and epiplasm in paramecium: the building of a submembraneous cytoskeleton.

Science.gov (United States)

Aubusson-Fleury, Anne; Bricheux, Geneviève; Damaj, Raghida; Lemullois, Michel; Coffe, Gérard; Donnadieu, Florence; Koll, France; Viguès, Bernard; Bouchard, Philippe

2013-07-01

In ciliates, basal bodies and associated appendages are bound to a submembrane cytoskeleton. In Paramecium, this cytoskeleton takes the form of a thin dense layer, the epiplasm, segmented into regular territories, the units where basal bodies are inserted. Epiplasmins, the main component of the epiplasm, constitute a large family of 51 proteins distributed in 5 phylogenetic groups, each characterized by a specific molecular design. By GFP-tagging, we analyzed their differential localisation and role in epiplasm building and demonstrated that: 1) The epiplasmins display a low turnover, in agreement with the maintenance of an epiplasm layer throughout the cell cycle; 2) Regionalisation of proteins from different groups allows us to define rim, core, ring and basal body epiplasmins in the interphase cell; 3) Their dynamics allows definition of early and late epiplasmins, detected early versus late in the duplication process of the units. Epiplasmins from each group exhibit a specific combination of properties. Core and rim epiplasmins are required to build a unit; ring and basal body epiplasmins seem more dispensable, suggesting that they are not required for basal body docking. We propose a model of epiplasm unit assembly highlighting its implication in structural heredity in agreement with the evolutionary history of epiplasmins. Copyright © 2013 Elsevier GmbH. All rights reserved.

Microscale force response and morphology of tunable co-polymerized cytoskeleton networks

Science.gov (United States)

Ricketts, Shea; Yadav, Vikrant; Ross, Jennifer L.; Robertson-Anderson, Rae M.

The cytoskeleton is largely comprised of actin and microtubules that entangle and crosslink to form complex networks and structures, giving rise to nonlinear multifunctional mechanics in cells. The relative concentrations of semiflexible actin filaments and rigid microtubules tune cytoskeleton function, allowing cells to move and divide while maintaining rigidity and resilience. To elucidate this complex tunability, we create in vitro composites of co-polymerized actin and microtubules with actin:microtubule molar ratios of 0:1-1:0. We use optical tweezers and confocal microscopy to characterize the nonlinear microscale force response and morphology of the composites. We optically drag a microsphere 30 μm through varying actin-microtubule networks at 10 μm/s and 20 μm/s, and measure the force the networks exerts to resist the strain and the force relaxation following strain. We use dual-color confocal microscopy to image distinctly-labeled filaments in the networks, and characterize the integration of actin and microtubules, network connectivity, and filament rigidity. We find that increasing the fraction of microtubules in networks non-monotonically increases elasticity and stiffness, and hinders force relaxation by suppressing network mobility and fluctuations. NSF CAREER Award (DMR-1255446), Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

Fluxes of water through aquaporin 9 weaken membrane-cytoskeleton anchorage and promote formation of membrane protrusions.

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Thommie Karlsson

Full Text Available All modes of cell migration require rapid rearrangements of cell shape, allowing the cell to navigate within narrow spaces in an extracellular matrix. Thus, a highly flexible membrane and a dynamic cytoskeleton are crucial for rapid cell migration. Cytoskeleton dynamics and tension also play instrumental roles in the formation of different specialized cell membrane protrusions, viz. lamellipodia, filopodia, and membrane blebs. The flux of water through membrane-anchored water channels, known as aquaporins (AQPs has recently been implicated in the regulation of cell motility, and here we provide novel evidence for the role of AQP9 in the development of various forms of membrane protrusion. Using multiple imaging techniques and cellular models we show that: (i AQP9 induced and accumulated in filopodia, (ii AQP9-associated filopodial extensions preceded actin polymerization, which was in turn crucial for their stability and dynamics, and (iii minute, local reductions in osmolarity immediately initiated small dynamic bleb-like protrusions, the size of which correlated with the reduction in osmotic pressure. Based on this, we present a model for AQP9-induced membrane protrusion, where the interplay of water fluxes through AQP9 and actin dynamics regulate the cellular protrusive and motile activity of cells.

Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton

DEFF Research Database (Denmark)

Jensen, Morten Østergaard; Hansen, Gry Aune; Vorum, Henrik

2006-01-01

cytoskeleton and is involved in cytokinesis. Labeling of S phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin possesses a paracrine role on the cells in its vicinity and therefore may be involved...

MIIP, a cytoskeleton regulator that blocks cell migration and invasion, delays mitosis, and suppresses tumorogenesis.

Science.gov (United States)

Wang, Yingmei; Wen, Jing; Zhang, Wei

2011-02-01

The migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). Recent studies have shown that MIIP not only modulates IGFBP2 but also regulates microtubule by binding to and inhibiting HDAC6, a class 2 histone deacetylase that deacetylates α-tubulin, heat-shock protein 90 (HSP90), and cortactin. In addition, MIIP also regulates the mitosis checkpoint, another microtubule-associated process. The location of the MIIP gene in chromosomal region 1p36, a commonly deleted region in a broad spectrum of human cancers, and the observation that MIIP attenuates tumorigenesis in a mouse model suggest that it functions as a tumor-suppressor gene. This review summarizes the recent progress in characterizing this novel protein, which regulates cell migration and mitosis, two processes that rely on the highly coordinated dynamics of the microtubule and cytoskeleton systems.

New insights into the structural organization of eukaryotic and prokaryotic cytoskeletons using cryo-electron tomography

International Nuclear Information System (INIS)

Kuerner, Julia; Medalia, Ohad; Linaroudis, Alexandros A.; Baumeister, Wolfgang

2004-01-01

Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3-D) imaging at molecular resolution (<5 nm) with a close-to-life preservation of the specimen. In conjunction with pattern recognition techniques, it enables us to map the molecular landscape inside cells. The application of cryo-ET to intact cells provides novel insights into the structure and the spatial organization of the cytoskeleton in prokaryotic and eukaryotic cells

ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function

DEFF Research Database (Denmark)

Kawaguchi, Nobuko; Sundberg, Christina; Kveiborg, Marie

2003-01-01

-100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary...

Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton

Science.gov (United States)

Kostal, Vratislav; Arriaga, Edgar A.

2011-01-01

Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for non-bound ‘free’ subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count and quantify properties of individual binding events of mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zeptomole F-actin, which theoretically represents a ~ 2.5-μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections. PMID:21309532

Multiscale evaluation of cellular adhesion alteration and cytoskeleton remodeling by magnetic bead twisting.

Science.gov (United States)

Isabey, Daniel; Pelle, Gabriel; André Dias, Sofia; Bottier, Mathieu; Nguyen, Ngoc-Minh; Filoche, Marcel; Louis, Bruno

2016-08-01

Cellular adhesion forces depend on local biological conditions meaning that adhesion characterization must be performed while preserving cellular integrity. We presently postulate that magnetic bead twisting provides an appropriate stress, i.e., basically a clamp, for assessment in living cells of both cellular adhesion and mechanical properties of the cytoskeleton. A global dissociation rate obeying a Bell-type model was used to determine the natural dissociation rate ([Formula: see text]) and a reference stress ([Formula: see text]). These adhesion parameters were determined in parallel to the mechanical properties for a variety of biological conditions in which either adhesion or cytoskeleton was selectively weakened or strengthened by changing successively ligand concentration, actin polymerization level (by treating with cytochalasin D), level of exerted stress (by increasing magnetic torque), and cell environment (by using rigid and soft 3D matrices). On the whole, this multiscale evaluation of the cellular and molecular responses to a controlled stress reveals an evolution which is consistent with stochastic multiple bond theories and with literature results obtained with other molecular techniques. Present results confirm the validity of the proposed bead-twisting approach for its capability to probe cellular and molecular responses in a variety of biological conditions.

Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

NARCIS (Netherlands)

van Belzen, N.; Spaargaren, M.; Verkleij, A. J.; Boonstra, J.

1990-01-01

Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction,

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

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Huang, C-L; Ueno, M; Liu, D; Masuya, D; Nakano, J; Yokomise, H; Nakagawa, T; Miyake, M

2006-10-19

Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (PMRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (PMRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (PMRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

Cell Culturing of Cytoskeleton

Science.gov (United States)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

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Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

2008-06-01

Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

Orchestrating cytoskeleton and intracellular vesicle traffic to build functional immunological synapses.

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Soares, Helena; Lasserre, Rémi; Alcover, Andrés

2013-11-01

Immunological synapses are specialized cell-cell contacts formed between T lymphocytes and antigen-presenting cells. They are induced upon antigen recognition and are crucial for T-cell activation and effector functions. The generation and function of immunological synapses depend on an active T-cell polarization process, which results from a finely orchestrated crosstalk between the antigen receptor signal transduction machinery, the actin and microtubule cytoskeletons, and controlled vesicle traffic. Although we understand how some of these particular events are regulated, we still lack knowledge on how these multiple cellular elements are harmonized to ensure appropriate T-cell responses. We discuss here our view on how T-cell receptor signal transduction initially commands cytoskeletal and vesicle traffic polarization, which in turn sets the immunological synapse molecular design that regulates T-cell activation. We also discuss how the human immunodeficiency virus (HIV-1) hijacks some of these processes impairing immunological synapse generation and function. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity

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Méndez-Olvera, Estela T.; Bustos-Martínez, Jaime A.; López-Vidal, Yolanda; Verdugo-Rodríguez, Antonio; Martínez-Gómez, Daniel

2016-01-01

Background Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. Objectives The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. Methods Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). Results The results obtained showed that the eight strains of C. jejuni, including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. Conclusions This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA, cdtB and cdtC genes. PMID:27942359

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity.

Science.gov (United States)

Méndez-Olvera, Estela T; Bustos-Martínez, Jaime A; López-Vidal, Yolanda; Verdugo-Rodríguez, Antonio; Martínez-Gómez, Daniel

2016-10-01

Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). The results obtained showed that the eight strains of C. jejuni , including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA , cdtB and cdtC genes.

Axon Initial Segment Cytoskeleton: Architecture, Development, and Role in Neuron Polarity

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Svitkina, Tatyana M.

2016-01-01

The axon initial segment (AIS) is a specialized structure in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in neurons is ideal for its two major functions: it serves as the site of action potential firing and helps to maintain neuron polarity. It has become increasingly clear that the AIS cytoskeleton is fundamental to AIS functions. In this review, we discuss current understanding of the AIS cytoskeleton with particular interest in its unique architecture and role in maintenance of neuron polarity. The AIS cytoskeleton is divided into two parts, submembrane and cytoplasmic, based on localization, function, and molecular composition. Recent studies using electron and subdiffraction fluorescence microscopy indicate that submembrane cytoskeletal components (ankyrin G, βIV-spectrin, and actin filaments) form a sophisticated network in the AIS that is conceptually similar to the polygonal/triangular network of erythrocytes, with some important differences. Components of the AIS cytoplasmic cytoskeleton (microtubules, actin filaments, and neurofilaments) reside deeper within the AIS shaft and display structural features distinct from other neuronal domains. We discuss how the AIS submembrane and cytoplasmic cytoskeletons contribute to different aspects of AIS polarity function and highlight recent advances in understanding their AIS cytoskeletal assembly and stability. PMID:27493806

Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

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Golla, R; Philp, N; Safer, D; Chintapalli, J; Hoffman, R; Collins, L; Nachmias, V T

1997-01-01

In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

International Nuclear Information System (INIS)

Peerschke, E.I.

1991-01-01

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding

Roles of cell adhesion and cytoskeleton activity in Entamoeba histolytica pathogenesis: a delicate balance.

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Tavares, Paulo; Rigothier, Marie-Christine; Khun, Huot; Roux, Pascal; Huerre, Michel; Guillén, Nancy

2005-03-01

The protozoan parasite Entamoeba histolytica colonizes the human large bowel. Invasion of the intestinal epithelium causes amoebic colitis and opens the route for amoebic liver abscesses. The parasite relies on its dynamic actomyosin cytoskeleton and on surface adhesion molecules for dissemination in the human tissues. Here we show that the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin clusters in focal structures localized in the region of E. histolytica that contacts monolayers of enterocytes. Disruption of myosin II activity impairs the formation of these structures and renders the trophozoites avirulent for liver abscess development. Production of the cytoplasmic domain of the E. histolytica Gal/GalNAc lectin in engineered trophozoites causes reduced adhesion to enterocytes. Intraportal delivery of these parasites to the liver leads to the formation of a large number of small abscesses with disorganized morphology that are localized in the vicinity of blood vessels. The data support a model for invasion in which parasite motility is essential for establishment of infectious foci, while the adhesion to host cells modulates the distribution of trophozoites in the liver and their capacity to migrate in the hepatic tissue.

Neuronal cytoskeleton in synaptic plasticity and regeneration.

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Gordon-Weeks, Phillip R; Fournier, Alyson E

2014-04-01

During development, dynamic changes in the axonal growth cone and dendrite are necessary for exploratory movements underlying initial axo-dendritic contact and ultimately the formation of a functional synapse. In the adult central nervous system, an impressive degree of plasticity is retained through morphological and molecular rearrangements in the pre- and post-synaptic compartments that underlie the strengthening or weakening of synaptic pathways. Plasticity is regulated by the interplay of permissive and inhibitory extracellular cues, which signal through receptors at the synapse to regulate the closure of critical periods of developmental plasticity as well as by acute changes in plasticity in response to experience and activity in the adult. The molecular underpinnings of synaptic plasticity are actively studied and it is clear that the cytoskeleton is a key substrate for many cues that affect plasticity. Many of the cues that restrict synaptic plasticity exhibit residual activity in the injured adult CNS and restrict regenerative growth by targeting the cytoskeleton. Here, we review some of the latest insights into how cytoskeletal remodeling affects neuronal plasticity and discuss how the cytoskeleton is being targeted in an effort to promote plasticity and repair following traumatic injury in the central nervous system. © 2013 International Society for Neurochemistry.

Analysis of the cytoskeleton organization and its possible functions in male earthworm germ-line cysts equipped with a cytophore.

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Małota, Karol; Świątek, Piotr

2016-10-01

We studied the organization of F-actin and the microtubular cytoskeleton in male germ-line cysts in the seminal vesicles of the earthworm Dendrobaena veneta using light, fluorescent and electron microscopy along with both chemically fixed tissue and life cell imaging. Additionally, in order to follow the functioning of the cytoskeleton, we incubated the cysts in colchicine, nocodazole, cytochalasin D and latrunculin A. The male germ-line cells of D. veneta are interconnected via stable intercellular bridges (IB), and form syncytial cysts. Each germ cell has only one IB that connects it to the anuclear central cytoplasmic mass, the cytophore. During the studies, we analyzed the cytoskeleton in spermatogonial, spermatocytic and spermatid cysts. F-actin was detected in the cortical cytoplasm and forms distinct rings in the IBs. The arrangement of the microtubules changed dynamically during spermatogenesis. The microtubules are distributed evenly in whole spermatogonial and spermatocytic cysts; however, they primarily accumulate within the IBs in spermatogonia. In early spermatids, microtubules pass through the IBs and are present in whole cysts. During spermatid elongation, the microtubules form a manchette while they are absent in the cytophore and in the IBs. Use of cytoskeletal drugs did not alter the general morphology of the cysts. Detectable effects-the occurrence of nuclei in the late spermatids and manchette fragments in the cytophore-were observed only after incubation in nocodazole. Our results suggest that the microtubules are responsible for cytoplasmic/organelle transfer between the germ cells and the cytophore during spermatogenesis and for the positioning of the spermatid nuclei.

The roles of the actin cytoskeleton in fear memory formation

Directory of Open Access Journals (Sweden)

Raphael eLamprecht

2011-07-01

Full Text Available The formation and storage of fear memory is needed to adapt behavior and avoid danger during subsequent fearful events. However, fear memory may also play a significant role in stress and anxiety disorders. When fear becomes disproportionate to that necessary to cope with a given stimulus, or begins to occur in inappropriate situations, a fear or anxiety disorder exists. Thus, the study of cellular and molecular mechanisms underpinning fear memory may shed light on the formation of memory and on anxiety and stress related disorders. Evidence indicates that fear learning leads to changes in neuronal synaptic transmission and morphology in brain areas underlying fear memory formation including the amygdala and hippocampus. The actin cytoskeleton has been shown to participate in these key neuronal processes. Recent findings show that the actin cytoskeleton is needed for fear memory formation and extinction. Moreover, the actin cytoskeleton is involved in synaptic plasticity and in neuronal morphogenesis in brain areas that mediate fear memory. The actin cytoskeleton may therefore mediate between synaptic transmission during fear learning and long-term cellular alterations mandatory for fear memory formation.

Cell Motility

CERN Document Server

Lenz, Peter

2008-01-01

Cell motility is a fascinating example of cell behavior which is fundamentally important to a number of biological and pathological processes. It is based on a complex self-organized mechano-chemical machine consisting of cytoskeletal filaments and molecular motors. In general, the cytoskeleton is responsible for the movement of the entire cell and for movements within the cell. The main challenge in the field of cell motility is to develop a complete physical description on how and why cells move. For this purpose new ways of modeling the properties of biological cells have to be found. This long term goal can only be achieved if new experimental techniques are developed to extract physical information from these living systems and if theoretical models are found which bridge the gap between molecular and mesoscopic length scales. Cell Motility gives an authoritative overview of the fundamental biological facts, theoretical models, and current experimental developments in this fascinating area.

MAPK/p38 regulation of cytoskeleton rearrangement accelerates induction of macrophage activation by TLR4, but not TLR3.

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Bian, Hongjun; Li, Feifei; Wang, Wenwen; Zhao, Qi; Gao, Shanshan; Ma, Jincai; Li, Xiao; Ren, Wanhua; Qin, Chengyong; Qi, Jianni

2017-11-01

Toll-like receptor 3 (TLR3) and TLR4 utilize adaptor proteins to activate mitogen‑activated protein kinase (MAPK), resulting in the acute but transient inflammatory response aimed at the clearance of pathogens. In the present study, it was demonstrated that macrophage activation by lipopolysaccharide (LPS) or poly(I:C), leading to changes in cell morphology, differed significantly between the mouse macrophage cell line RAW264.7 and mouse primary peritoneal macrophages. Moreover, the expression of α- and β-tubulin was markedly decreased following LPS stimulation. By contrast, α- and β-tubulin expression were only mildly increased following poly(I:C) treatment. However, the expression of β-actin and GAPDH was not significantly affected. Furthermore, it was verified that vincristine pretreatment abrogated the cytoskeleton rearrangement and decreased the synthesis and secretion of proinflammatory cytokines and migration of macrophages caused by LPS. Finally, it was observed that the MAPK/p38 signaling pathway regulating cytoskeleton rearrangement may participate in LPS‑induced macrophage cytokine production and migration. Overall, the findings of the present study indicated that MAPK/p38 regulation of the cytoskeleton, particularly tubulin proteins, plays an important role in LPS-induced inflammatory responses via alleviating the synthesis and secretion of proinflammatory cytokines and inhibiting the migration of macrophages.

Plant actin cytoskeleton re-modeling by plant parasitic nematodes.

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Engler, Janice de Almeida; Rodiuc, Natalia; Smertenko, Andrei; Abad, Pierre

2010-03-01

The cytoskeleton is an important component of the plant's defense mechanism against the attack of pathogenic organisms. Plants however, are defenseless against parasitic root-knot and cyst nematodes and respond to the invasion by the development of a special feeding site that supplies the parasite with nutrients required for the completion of its life cycle. Recent studies of nematode invasion under treatment with cytoskeletal drugs and in mutant plants where normal functions of the cytoskeleton have been affected, demonstrate the importance of the cytoskeleton in the establishment of a feeding site and successful nematode reproduction. It appears that in the case of microfilaments, nematodes hijack the intracellular machinery that regulates actin dynamics and modulate the organization and properties of the actin filament network. Intervening with this process reduces the nematode infection efficiency and inhibits its life cycle. This discovery uncovers a new pathway that can be exploited for the protection of plants against nematodes.

The tumor suppressor CDX2 opposes pro-metastatic biomechanical modifications of colon cancer cells through organization of the actin cytoskeleton.

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Platet, Nadine; Hinkel, Isabelle; Richert, Ludovic; Murdamoothoo, Devadarssen; Moufok-Sadoun, Ahlam; Vanier, Marie; Lavalle, Philippe; Gaiddon, Christian; Vautier, Dominique; Freund, Jean-Noel; Gross, Isabelle

2017-02-01

The vast majority of cancer deaths are caused by the formation of metastases rather than the primary tumor itself. Despite this clinical importance, the molecular and cellular events that support the dissemination of cancer cells are not yet fully unraveled. We have previously shown that CDX2, a homeotic transcription factor essential for gut development, acts as a colon-specific tumor suppressor and opposes metastasis. Here, using a combination of biochemical, biophysical, and immunofluorescence techniques, we further investigated the mechanisms promoted by CDX2 that might antagonize tumor cell dissemination. We found that CDX2 expression regulates the transcription of RHO GEFs, thereby activating RHO signaling cascades that lead to reorganization of the actin cytoskeleton and enhanced adherent junctions. Accordingly, we observed by atomic force microscopy (AFM) that colon cancer cells expressing CDX2 are less deformable, a feature that has been shown to correlate with poor metastatic potential. Thus, this study illustrates how the loss of expression of a transcription factor during colon cancer progression modifies the biomechanical characteristics of tumor cells and hence facilitates invasion and metastasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tensegrity and mechanoregulation: from skeleton to cytoskeleton

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Chen, C. S.; Ingber, D. E.

1999-01-01

OBJECTIVE: To elucidate how mechanical stresses that are applied to the whole organism are transmitted to individual cells and transduced into a biochemical response. DESIGN: In this article, we describe fundamental design principles that are used to stabilize the musculoskeletal system at many different size scales and show that these design features are embodied in one particular form of architecture that is known as tensegrity. RESULTS: Tensegrity structures are characterized by use of continuous tension and local compression; architecture, prestress (internal stress prior to application of external force), and triangulation play the most critical roles in terms of determining their mechanical stability. In living organisms, use of a hierarchy of tensegrity networks both optimizes structural efficiency and provides a mechanism to mechanically couple the parts with the whole: mechanical stresses applied at the macroscale result in structural rearrangements at the cell and molecular level. CONCLUSION: Due to use of tensegrity architecture, mechanical stress is concentrated and focused on signal transducing molecules that physically associate with cell surface molecules that anchor cells to extracellular matrix, such as integrins, and with load-bearing elements within the internal cytoskeleton and nucleus. Mechanochemical transduction may then proceed through local stress-dependent changes in molecular mechanics, thermodynamics, and kinetics within the cell. In this manner, the entire cellular response to stress may be orchestrated and tuned by altering the prestress in the cell, just as changing muscular tone can alter mechanical stability and structural coordination throughout the whole musculoskeletal system.

TGFβ1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton

Directory of Open Access Journals (Sweden)

Mona Elsafadi

2018-01-01

Full Text Available TGFβ is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs is currently poorly understood. In the present study, we demonstrate that a single dose of TGFβ1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGFβ-mediated enhancement of lineage commitment, we compared the gene expression profiles of TGFβ1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFβl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGFβ1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFβ1 and cytochalasin D. Our study demonstrates that TGFβ1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

Remodeling of tick cytoskeleton in response to infection with Anaplasma phagocytophilum.

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Cabezas-Cruz, Alejandro; Alberdi, Pilar; Valdes, James J; Villar, Margarita; de la Fuente, Jose

2017-06-01

The obligate intracellular pathogen Anaplasma phagocytophilum infects vertebrate and tick hosts. In this study, a genome-wide search for cytoskeleton components was performed in the tick vector, Ixodes scapularis . The available transcriptomics and proteomics data was then used to characterize the mRNA and protein levels of I. scapularis cytoskeleton components in response to A. phagocytophilum infection. The results showed that cytoskeleton components described in other model organisms were present in the I. scapularis genome. One type of intermediate filaments (lamin), a family of septins that was recently implicated in the cellular response to intracellular pathogens, and several members of motor proteins (kinesins and dyneins) that could be implicated in the cytoplasmic movements of A. phagocytophilum were found. The results showed that levels of tubulin, actin, septin, actin-related proteins and motor proteins were affected by A. phagocytophilum , probably to facilitate infection in I. scapularis . Functional studies demonstrated a role for selected cytoskeleton components in pathogen infection. These results provided a more comprehensive view of the cytoskeletal components involved in the response to A. phagocytophilum infection in ticks.

Origin and evolution of the self-organizing cytoskeleton in the network of eukaryotic organelles.

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Jékely, Gáspár

2014-09-02

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing "active gel," the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Regulation from within: the cytoskeleton in transmembrane signaling

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Jaqaman, Khuloud; Grinstein, Sergio

2013-01-01

There is mounting evidence that the plasma membrane is highly dynamic and organized in a complex manner. The cortical cytoskeleton is proving to be a particularly important regulator of plasmalemmal organization, modulating the mobility of proteins and lipids in the membrane, facilitating their segregation and influencing their clustering. This organization plays a critical role in receptor-mediated signaling, especially in the case of immunoreceptors, which require lateral clustering for their activation. Based on recent developments, we discuss the structures and mechanisms whereby the cortical cytoskeleton regulates membrane dynamics and organization, and how the non-uniform distribution of immunoreceptors and their self-association may affect activation and signaling. PMID:22917551

CRMPs colocalize and interact with cytoskeleton in hippocampal neurons

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Yang, Yuhao; Zhao, Bo; Ji, Zhisheng; Zhang, Guowei; Zhang, Jifeng; Li, Sumei; Guo, Guoqing; Lin, Hongsheng

2015-01-01

CRMP family proteins (CRMPs) are widely expressed in the developing neurons, mediating a variety of fundamental functions such as growth cone guidance, neuronal polarity and axon elongation. However, whether all the CRMP proteins interact with cytoskeleton remains unknown. In this study, we found that in cultured hippocampal neurons, CRMPs mainly colocalized with tubulin and actin network in neurites. In growth cones, CRMPs colocalized with tubulinmainly in the central (C-) domain and transition zone (T-zone), less in the peripheral (P-) domain and colocalized with actin in all the C-domain, T-zone and P-domain. The correlation efficiency of CRMPs between actin was significantly higher than that between tubulin, especially in growth cones. We successfully constructed GST-CRMPs plasmids, expressed and purified the GST-CRMP proteins. By GST-pulldown assay, all the CRMP family proteins were found to beinteracted with cytoskeleton proteins. Taken together, we revealed that CRMPs were colocalized with cytoskeleton in hippocampal neurons, especially in growth cones. CRMPs can interact with both tubulin and actin, thus mediating neuronal development. PMID:26885211

Axon initial segment cytoskeleton comprises a multiprotein submembranous coat containing sparse actin filaments

Science.gov (United States)

Jones, Steven L.; Korobova, Farida

2014-01-01

The axon initial segment (AIS) of differentiated neurons regulates action potential initiation and axon–dendritic polarity. The latter function depends on actin dynamics, but actin structure and functions at the AIS remain unclear. Using platinum replica electron microscopy (PREM), we have characterized the architecture of the AIS cytoskeleton in mature and developing hippocampal neurons. The AIS cytoskeleton assembly begins with bundling of microtubules and culminates in formation of a dense, fibrillar–globular coat over microtubule bundles. Immunogold PREM revealed that the coat contains a network of known AIS proteins, including ankyrin G, spectrin βIV, neurofascin, neuronal cell adhesion molecule, voltage-gated sodium channels, and actin filaments. Contrary to existing models, we find neither polarized actin arrays, nor dense actin meshworks in the AIS. Instead, the AIS contains two populations of sparse actin filaments: short, stable filaments and slightly longer dynamic filaments. We propose that stable actin filaments play a structural role for formation of the AIS diffusion barrier, whereas dynamic actin may promote AIS coat remodeling. PMID:24711503

Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton

Science.gov (United States)

Sawyer, S. J.; Norvell, S. M.; Ponik, S. M.; Pavalko, F. M.

2001-01-01

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.

High postnatal susceptibility of hippocampal cytoskeleton in response to ethanol exposure during pregnancy and lactation.

Science.gov (United States)

Reis, Karina Pires; Heimfarth, Luana; Pierozan, Paula; Ferreira, Fernanda; Loureiro, Samanta Oliveira; Fernandes, Carolina Gonçalves; Carvalho, Rônan Vivian; Pessoa-Pureur, Regina

2015-11-01

Ethanol exposure to offspring during pregnancy and lactation leads to developmental disorders, including central nervous system dysfunction. In the present work, we have studied the effect of chronic ethanol exposure during pregnancy and lactation on the phosphorylating system associated with the astrocytic and neuronal intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), and neurofilament (NF) subunits of low, medium, and high molecular weight (NFL, NFM, and NFH, respectively) in 9- and 21-day-old pups. Female rats were fed with 20% ethanol in their drinking water during pregnancy and lactation. The homeostasis of the IF phosphorylation was not altered in the cerebral cortex, cerebellum, or hippocampus of 9-day-old pups. However, GFAP, NFL, and NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. PKA had been activated in the hippocampus, and Ser55 in the N-terminal region of NFL was hyperphosphorylated. In addition, JNK/MAPK was activated and KSP repeats in the C-terminal region of NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. Decreased NFH immunocontent but an unaltered total NFH/phosphoNFH ratio suggested altered stoichiometry of NFs in the hippocampus of ethanol-exposed 21-day-old pups. In contrast to the high susceptibility of hippocampal cytoskeleton in developing rats, the homeostasis of the cytoskeleton of ethanol-fed adult females was not altered. Disruption of the cytoskeletal homeostasis in neural cells supports the view that regions of the brain are differentially vulnerable to alcohol insult during pregnancy and lactation, suggesting that modulation of JNK/MAPK and PKA signaling cascades target the hippocampal cytoskeleton in a window of vulnerability in 21-day-old pups. Our findings are relevant, since disruption of the cytoskeleton in immature hippocampus could contribute to later hippocampal damage associated with ethanol toxicity. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

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

A growing family: the expanding universe of the bacterial cytoskeleton.

Science.gov (United States)

Ingerson-Mahar, Michael; Gitai, Zemer

2012-01-01

Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

Science.gov (United States)

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

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

Deoxypodophyllotoxin suppresses tumor vasculature in HUVECs by promoting cytoskeleton remodeling through LKB1-AMPK dependent Rho A activatio.

Science.gov (United States)

Wang, Yurong; Wang, Bin; Guerram, Mounia; Sun, Li; Shi, Wei; Tian, Chongchong; Zhu, Xiong; Jiang, Zhenzhou; Zhang, Luyong

2015-10-06

Angiogenesis plays a critical role in the growth and metastasis of tumors, which makes it an attractive target for anti-tumor drug development. Deoxypodophyllotoxin (DPT), a natural product isolated from Anthriscus sylvestris, inhibits cell proliferation and migration in various cancer cell types. Our previous studies indicate that DPT possesses both anti-angiogenic and vascular-disrupting activities. Although the RhoA/ RhoA kinase (ROCK) signaling pathway is implicated in DPT-stimulated cytoskeleton remodeling and tumor vasculature suppressing, the detailed mechanisms by which DPT mediates these effects are poorly understood. In the current study, we found that DPT promotes cytoskeleton remodeling in human umbilical vein endothelial cells (HUVECs) via stimulation of AMP-activated protein kinase (AMPK) and that this effect is abolished by either treatment with a selective AMPK inhibitor or knockdown. Moreover, the cellular levels of LKB1, a kinase upstream of AMPK, were enhanced following DPT exposure. DPT-induced activation of AMPK in tumor vasculature effect was also verified by transgenic zebrafish (VEGFR2:GFP), Matrigel plug assay, and xenograft model in nude mice. The present findings may lay the groundwork for a novel therapeutic approach in treating cancer.

RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages

DEFF Research Database (Denmark)

von Wichert, Gotz; Jiang, Guoying; Kostic, Ana

2003-01-01

Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha). RPTPalpha colocalizes with alphav...... of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPalpha is an early component in force-dependent signal transduction pathways leading...

The effect of 60Co-irradiation on platelet cytoskeleton and function

International Nuclear Information System (INIS)

Zhuang Qingqi; Li Chengzhu

1987-01-01

Changes in rat platelet cytoskeleton and platelet function by 60 Co-irradiation were investigated. When platelet-rich plasma (PRP) was stimulated with ADP, PRP aggregation rates on the 3rd, 6th and 9th day after irradiation were 135%, 96% and 0% of the controls respectively. Although control rat PRP responded to collagen stimulation well, PRP from treated rats had lost the aggregation ability induced by collagen. When same amounts of platelets from the control and the treated rats were extracted with 1% triton X-100-EGTA buffer and the cytoskeleton pellet analysed by SDS-PAGE, it was found that the amount and components of the rat platelet cytoskeleton especially actin and myosin on the 3rd day after irradiation were greatly increased. Those from rat platelets on the 6th day after irradiation showed no difference from the controls, but was significantly decreased on the 9th day after irradiation. Based on these results, the authors concluded that the platelets from rats on the 3rd day after irradiation were in an active state. Exhaustion of granule contents or impairment of collagen receptors might render platelets unable to respond to collagen. Decreased platelet function on the 9th day after irradiation was due to a failure of assembly of its cytoskeleton

A New Invasion and Metastasis Molecule, Tiam1 and its Interaction with the Cytoskeleton are Involved in Human Breast Cancer Progression

National Research Council Canada - National Science Library

Bourguignon, Lilly

2001-01-01

...) both in vivo and in vitro. Specifically, the binding of HA to the CD44v3 isoform stimulates Tiam1-specific GDP/GTP exchange for Rho-like GTPases such as Rac1, and promotes cytoskeleton-mediated breast tumor cell migration...

Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organising cytoskeleton upon platelet activation

NARCIS (Netherlands)

Asijee, G. M.; Sturk, A.; Bruin, T.; Wilkinson, J. M.; ten Cate, J. W.

1990-01-01

Vinculin, a 130-kDa protein discovered in chicken gizzard smooth-muscle cells and subsequently also described in platelets, is believed to be involved in membrane-cytoskeleton interactions. In this study we investigated vinculin distribution in human blood platelets. Two skeletal fractions and a

Cytoskeleton, L-type Ca2+ and stretch activated channels in injured skeletal muscle

Directory of Open Access Journals (Sweden)

Fabio Francini

2013-07-01

Full Text Available The extra-sarcomeric cytoskeleton (actin microfilaments and anchoring proteins is involved in maintaining the sarco-membrane stiffness and integrity and in turn the mechanical stability and function of the intra- and sub-sarcoplasmic proteins. Accordingly, it regulates Ca2+ entry through the L-type Ca2+ channels and the mechano-sensitivity of the stretch activated channels (SACs. Moreover, being intra-sarcomeric cytoskeleton bound to costameric proteins and other proteins of the sarcoplasma by intermediate filaments, as desmin, it integrates the properties of the sarcolemma with the skeletal muscle fibres contraction. The aim of this research was to compare the cytoskeleton, SACs and the ECC alterations in two different types of injured skeletal muscle fibres: by muscle denervation and mechanical overload (eccentric contraction. Experiments on denervation were made in isolated Soleus muscle of male Wistar rats; forced eccentric-contraction (EC injury was achieved in Extensor Digitorum Longus muscles of Swiss mice. The method employed conventional intracellular recording with microelectrodes inserted in a single fibre of an isolated skeletal muscle bundle. The state of cytoskeleton was evaluated by recording SAC currents and by evaluating the resting membrane potential (RMP value determined in current-clamp mode. The results demonstrated that in both injured skeletal muscle conditions the functionality of L-type Ca2+ current, ICa, was affected. In parallel, muscle fibres showed an increase of the resting membrane permeability and of the SAC current. These issues, together with a more depolarized RMP are an index of altered cytoskeleton. In conclusion, we found a symilar alteration of ICa, SAC and cytoskeleton in both injured skeletal muscle conditions.

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

Directory of Open Access Journals (Sweden)

Julie Carnesecchi

Full Text Available The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2 addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

Science.gov (United States)

Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

2015-01-01

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

Directory of Open Access Journals (Sweden)

Jennifer Pöhlmann

2014-09-01

Full Text Available Microtubules (MTs are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

Effects of X irradiation on the cytoskeleton of rat alveolar macrophages in vitro

International Nuclear Information System (INIS)

Ladyman, S.J.; Townsend, K.M.S.; Edwards, C.

1984-01-01

The three-dimensional visualization of Triton X-100 resistant cytoskeletons has been used to demonstrate that an absorbed dose of 120 Gy from X rays causes a distinctive and reproducible alteration of the cytoskeleton of intact rat alveolar macrophages in vitro. The alteration has also been shown to be rapidly and completely ''repaired'' and to be apparently similar to alterations caused by colchicine but dissimilar to those caused by cytochalasin B. From these observations and those of other workers who have studied the irradiation of extracted microtubular proteins in vitro, the authors think it likely that microtubules rather than microfilaments are the radiosensitive component of the macrophage cytoskeleton

An αII Spectrin-Based Cytoskeleton Protects Large-Diameter Myelinated Axons from Degeneration.

Science.gov (United States)

Huang, Claire Yu-Mei; Zhang, Chuansheng; Zollinger, Daniel R; Leterrier, Christophe; Rasband, Matthew N

2017-11-22

Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with βIV and βII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1 f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K + channels. We show that the density of nodal βIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1 f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier. SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in

Investigation on cytoskeleton dynamics for no-adherent cells subjected to point-like stimuli by digital holographic microscopy and holographic optical trapping

Science.gov (United States)

Miccio, Lisa; Merola, Francesco; Memmolo, Pasquale; Mugnano, Martina; Fusco, Sabato; Netti, Paolo A.; Ferraro, Pietro

2014-05-01

Guiding, controlling and studying cellular functions are challenging themes in the biomedical field, as they are fundamental prerequisites for new therapeutic strategies from tissue regeneration to controlled drug delivery. In recent years, multidisciplinary studies in nanotechnology offer new tools to investigate important biophysical phenomena in response to the local physical characteristics of the extracellular environment, some examples are the mechanisms of cell adhesion, migration, communication and differentiation. Indeed for reproducing the features of the extracellular matrix in vitro, it is essential to develop active devices that evoke as much as possible the natural cellular environment. Our investigation is in the framework of studying and clarifying the biophysical mechanisms of the interaction between cells and the microenvironment in which they exist. We implement an optical tweezers setup to investigate cell material interaction and we use Digital Holography as non-invasive imaging technique in microscopy. We exploit Holographic Optical Tweezers arrangement in order to trap and manage functionalized micrometric latex beads to induce mechanical deformation in suspended cells. A lot of papers in literature examine the dynamics of the cytoskeleton when cells adhere on substrates and nowadays well established cell models are based on such research activities. Actually, the natural cell environment is made of a complex extracellular matrix and the single cell behavior is due to intricate interactions with the environment and are strongly correlated to the cell-cell interactions. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence.

Gfi1b controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes.

Science.gov (United States)

Beauchemin, Hugues; Shooshtarizadeh, Peiman; Vadnais, Charles; Vassen, Lothar; Pastore, Yves D; Möröy, Tarik

2017-03-01

Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content. Copyright© Ferrata Storti Foundation.

Mechanism of aftered cytoskeleton organization in influenza virus infection

International Nuclear Information System (INIS)

Krizanova, O.; Ciampor, F.; Zavodska, E.; Matis, J.; Stancek, D.; Krivjanska, M.

1989-01-01

The autophosphorylation was followed of cytoskeleton (CS) isolated from control chick embryo cell membranes (CS-C) and from these membranes after influenza virus adsorption (CS-V) under conditions allowing to determine the activity of a single type proteinkinase. The Ca 2+ dependent calmodulin (CaM) kinase used different substrates from CS-V than did the c'AMP dependent proteinkinase. The catalytic subunit (c-subunit) of the c'AMP dependent proteinkinase added from outside phosphorylated the same polypeptides than the endogeneous c'AMP dependent proteinkinase, the further being more active than the latter. The purified influenza virus incorporated 32 P in the presence of the c-subunit only. Incubation of influenza virus with the c-subunit caused morphological changes visible by electron microscopy. The pleomorphy of the particles as well as their electron transmissibility were enhanced in the result of structural alterations and rarefaction of surface spikes of the haemagglutinin and neuraminidase. The contractibility of CS isolated from normal CEC and of the CS from CEC by 15 min postinfection (p.i.) was determined according to the actomyosin ATPase activity. The ATPase activity of the cytoskeleton in the presence of the Ca 2+ /CaM and that in the presence of c'AMP were used as controls. The virus as well as the Ca 2+ /CaM increased the ATPase activity. EGTA had no effect but did not interfere with virus stimulation, while c'AMP blocked the virus-induced enhancement of the ATPase activity. (author). 3 figs., 1 tab., 36 refs

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification.

Science.gov (United States)

Wang, C L; Xu, H Y; Xie, L; Lu, Y Q; Yang, X G; Lu, S S; Lu, K H

2016-06-01

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12 μg/mL CB for 30 min. The optimum concentration of CB treatment (8 μg/mL for 30 min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8 μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8 μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (P < 0.01) than those of the control groups. In conclusion, pretreatment with 8 μg/mL CB for 30 min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

The effect of ionizing radiation on the filamentous actin of vascular endothelial cell

International Nuclear Information System (INIS)

Yao Xiaowu; Chen Shisheng; Yang Lihe; Lin Juelong; Yang Haiwei

2006-01-01

Objective: To observe the ionizing radiation effect on filamentous actin of vascular endothelial cell and explore its mechanism. Methods: The vascular endothelial cells were irradiated with 0, 2, 4, 6, 8, 10 and 12 Gy 60 Co γ-rays. The cytoskeleton was observed with CLSM at 6 hs after the irradiation and the cytoskeleton protein F-actin detected with flow cytometry after 12 and 24 hs. Results: The damage to cytoskeletons increased with the radiation dose. The cytoskeleton protein F-actin was significantly decreased at 12 hs after the irradiation, and then recovered after 24 hs. Conclusion: Ionizing radiation caused vascular endothelial cell injury by damaging the cytoskeleton and depolymerizating the F-actin. (authors)

Sun-mediated mechanical LINC between nucleus and cytoskeleton regulates βcatenin nuclear access.

Science.gov (United States)

Uzer, Gunes; Bas, Guniz; Sen, Buer; Xie, Zhihui; Birks, Scott; Olcum, Melis; McGrath, Cody; Styner, Maya; Rubin, Janet

2018-06-06

βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of βcatenin in the cytoplasm has been well studied but βcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of βcatenin destruction complex element GSK3β (glycogen synthase kinase 3β) to increase nuclear βcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3β due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, βcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of βcatenin, resulting in decreased nuclear βcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in βcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of βcatenin. Copyright © 2018 Elsevier Ltd. All rights reserved.

A 23Na Multiple-Quantum-Filtered NMR Study of the Effect of the Cytoskeleton Conformation on the Anisotropic Motion of Sodium Ions in Red Blood Cells

Science.gov (United States)

Knubovets, Tatyana; Shinar, Hadassah; Eliav, Uzi; Navon, Gil

1996-01-01

Recently, it has been shown that23Na double-quantum-filtered NMR spectroscopy can be used to detect anisotropic motion of bound sodium ions in biological systems. The technique is based on the formation of the second-rank tensor when the quadrupolar interaction is not averaged to zero. Using this method, anisotropic motion of bound sodium in human and dog red blood cells was detected, and the effect was shown to depend on the integrity of the membrane cytoskeleton. In the present study, multiple-quantum-filtered techniques were applied in combination with a quadrupolar echo to measure the transverse-relaxation times,T2fandT2s. Line fitting was performed to obtain the values of the residual quadrupolar interaction, which was measured for sodium in a variety of mammalian erythrocytes of different size, shape, rheological properties, and sodium concentrations. Human unsealed white ghosts were used to study sodium bound at the anisotropic sites on the inner side of the RBC membrane. Modulations of the conformation of the cytoskeleton by the variation of either the ionic strength or pH of the suspending medium caused drastic changes in both the residual quadrupolar interaction andT2fdue to changes in the fraction of bound sodium ions as well as changes in the structure of the binding sites. By combining the two spectroscopic parameters, structural change can be followed. The changes in the structure of the sodium anisotropic binding sites deduced by this method were found to correlate with known conformational changes of the membrane cytoskeleton. Variations of the medium pH affected both the fraction of bound sodium ions and the structure of the anisotropic binding sites. Sodium and potassium were shown to bind to the anisotropic binding sites with the same affinity.

All-Round Manipulation of the Actin Cytoskeleton by HIV.

Science.gov (United States)

Ospina Stella, Alberto; Turville, Stuart

2018-02-05

While significant progress has been made in terms of human immunodeficiency virus (HIV) therapy, treatment does not represent a cure and remains inaccessible to many people living with HIV. Continued mechanistic research into the viral life cycle and its intersection with many aspects of cellular biology are not only fundamental in the continued fight against HIV, but also provide many key observations of the workings of our immune system. Decades of HIV research have testified to the integral role of the actin cytoskeleton in both establishing and spreading the infection. Here, we review how the virus uses different strategies to manipulate cellular actin networks and increase the efficiency of various stages of its life cycle. While some HIV proteins seem able to bind to actin filaments directly, subversion of the cytoskeleton occurs indirectly by exploiting the power of actin regulatory proteins, which are corrupted at multiple levels. Furthermore, this manipulation is not restricted to a discrete class of proteins, but rather extends throughout all layers of the cytoskeleton. We discuss prominent examples of actin regulators that are exploited, neutralized or hijacked by the virus, and address how their coordinated deregulation can lead to changes in cellular behavior that promote viral spreading.

Transforming growth factor β-induced superficial zone protein accumulation in the surface zone of articular cartilage is dependent on the cytoskeleton.

Science.gov (United States)

McNary, Sean M; Athanasiou, Kyriacos A; Reddi, A Hari

2014-03-01

The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. Whereas dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP, with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and transforming growth factor β (TGFβ) 1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFβ1. A decrease in TGFβ1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified using paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFβ-induced SZP accumulation. TGFβ-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1

Nanoengineered Polystyrene Surfaces with Nanopore Array Pattern Alters Cytoskeleton Organization and Enhances Induction of Neural Differentiation of Human Adipose-Derived Stem Cells.

Science.gov (United States)

Jung, Ae Ryang; Kim, Richard Y; Kim, Hyung Woo; Shrestha, Kshitiz Raj; Jeon, Seung Hwan; Cha, Kyoung Je; Park, Yong Hyun; Kim, Dong Sung; Lee, Ji Youl

2015-07-01

Human adipose-derived stem cells (hADSCs) can differentiate into various cell types depending on chemical and topographical cues. One topographical cue recently noted to be successful in inducing differentiation is the nanoengineered polystyrene surface containing nanopore array-patterned substrate (NP substrate), which is designed to mimic the nanoscale topographical features of the extracellular matrix. In this study, efficacies of NP and flat substrates in inducing neural differentiation of hADSCs were examined by comparing their substrate-cell adhesion rates, filopodia growth, nuclei elongation, and expression of neural-specific markers. The polystyrene nano Petri dishes containing NP substrates were fabricated by a nano injection molding process using a nickel electroformed nano-mold insert (Diameter: 200 nm. Depth of pore: 500 nm. Center-to-center distance: 500 nm). Cytoskeleton and filopodia structures were observed by scanning electron microscopy and F-actin staining, while cell adhesion was tested by vinculin staining after 24 and 48 h of seeding. Expression of neural specific markers was examined by real-time quantitative polymerase chain reaction and immunocytochemistry. Results showed that NP substrates lead to greater substrate-cell adhesion, filopodia growth, nuclei elongation, and expression of neural specific markers compared to flat substrates. These results not only show the advantages of NP substrates, but they also suggest that further study into cell-substrate interactions may yield great benefits for biomaterial engineering.

Fluorescence Imaging of the Cytoskeleton in Plant Roots.

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Dyachok, Julia; Paez-Garcia, Ana; Yoo, Cheol-Min; Palanichelvam, Karuppaiah; Blancaflor, Elison B

2016-01-01

During the past two decades the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis roots, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. For roots with a larger diameter such as Medicago truncatula, seeds are first germinated in moist paper, grown vertically in between plastic trays, and roots mounted on glass slides for confocal imaging. Parallel with our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use non-embedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.

Immunolocation of mitochondria-rich cells in epidermis of the common toad, Bufo bufo L

DEFF Research Database (Denmark)

Spies, Ingrid B.M.

1997-01-01

Adult toads, skin, flask-shaped cells, cytoskeleton, keratin expression, immunohistochemistry, differentiation.......Adult toads, skin, flask-shaped cells, cytoskeleton, keratin expression, immunohistochemistry, differentiation....

Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve an 89K protein

International Nuclear Information System (INIS)

Gupta, S.K.; Woda, B.

1986-01-01

Membrane immunoglobulin of B-lymphocytes is thought to play an important role in antigen recognition and cellular activation. Binding of cross-linking ligands to surface immunoglobulin (SIg) on intact cells converts it to a detergent insoluble state, and this conversion is associated with the transmission of a mitogenic signal. Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons in buffers which convert F-actin to G-actin [(Buffer 1), 0.34M sucrose, 0.5mM ATP, 0.5mM Dithiothrietol and lmM EDTA]. Immunoprecipitation of SIg from the detergent soluble fraction of 35 S-methionine labeled non ligand treated rat B-cells results in the co-isolation of an 89K protein and a 44K protein, presumably actin. The 89K protein is not associated with the fraction of endogenous detergent insoluble SIg. On treatment of rat B cells with cross-linking ligand (anti-Ig) the 89K protein becomes detergent insoluble along with most of the SIg and co-isolates with SIg on immunoprecipitation of the detergent insoluble, buffer l solubilized fraction. The migration of the SIg-associated 89K protein from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that this protein might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of a mitogenic signal

Cortical microtubule nucleation can organise the cytoskeleton of Drosophila oocytes to define the anteroposterior axis

Science.gov (United States)

Khuc Trong, Philipp; Doerflinger, Hélène; Dunkel, Jörn; St Johnston, Daniel; Goldstein, Raymond E

2015-01-01

Many cells contain non-centrosomal arrays of microtubules (MTs), but the assembly, organisation and function of these arrays are poorly understood. We present the first theoretical model for the non-centrosomal MT cytoskeleton in Drosophila oocytes, in which bicoid and oskar mRNAs become localised to establish the anterior-posterior body axis. Constrained by experimental measurements, the model shows that a simple gradient of cortical MT nucleation is sufficient to reproduce the observed MT distribution, cytoplasmic flow patterns and localisation of oskar and naive bicoid mRNAs. Our simulations exclude a major role for cytoplasmic flows in localisation and reveal an organisation of the MT cytoskeleton that is more ordered than previously thought. Furthermore, modulating cortical MT nucleation induces a bifurcation in cytoskeletal organisation that accounts for the phenotypes of polarity mutants. Thus, our three-dimensional model explains many features of the MT network and highlights the importance of differential cortical MT nucleation for axis formation. DOI: http://dx.doi.org/10.7554/eLife.06088.001 PMID:26406117

The actin cytoskeleton may control the polar distribution of an auxin transport protein

Science.gov (United States)

Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

2000-01-01

The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

The effects of 60Co γ-ray irradiation on the cytoskeleton of mouse peritoneal macrophages and human peripheral blood monocytes in vitro

International Nuclear Information System (INIS)

Chen Xiaomei; Guo Yuhua; Yin Zhiwei; Mao Zijun

1990-03-01

The whole mount cell electron microscopy in combination with selective extraction method for preparing cytoskeletal framework was applied. Cy toskeleton prepared by Triton X-100 treatment of mouse peritoneal macrophages and human peripheral blood monocytes appeared in electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. Since such cytoskeletons are open membrane-free system, individual fibrous organizations can be identified by specific antibodies. An indirect immunogold procedure using monoclonal anti-tubulin or anti-actin antibodies was applied to visualize tubulin-or actin-containing structures. The three-dimensional visualization of Triton X-100 resistant cytoskeletons had been used to demonstrate that different doses of 60 Co γ-ray caused a distinctive and reproducible alterations of the cytoskeletons of intact mouse peritoneal macrophages and human peripheral blood monocytes in vitro. The results showed that there were some similar alterations with those caused by cytochalasin B and by colchicine. From these observations and other workers' studies, it's likely that 60 Co γ-ray irradiation may inhibit cytoplasmic microtubule and microfilament assembling

The Roles of RNase-L in Antimicrobial Immunity and the Cytoskeleton-Associated Innate Response.

Science.gov (United States)

Ezelle, Heather J; Malathi, Krishnamurthy; Hassel, Bret A

2016-01-08

The interferon (IFN)-regulated endoribonuclease RNase-L is involved in multiple aspects of the antimicrobial innate immune response. It is the terminal component of an RNA cleavage pathway in which dsRNA induces the production of RNase-L-activating 2-5A by the 2'-5'-oligoadenylate synthetase. The active nuclease then cleaves ssRNAs, both cellular and viral, leading to downregulation of their expression and the generation of small RNAs capable of activating retinoic acid-inducible gene-I (RIG-I)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome. This leads to IFNβ expression and IL-1β activation respectively, in addition to broader effects on immune cell function. RNase-L is also one of a growing number of innate immune components that interact with the cell cytoskeleton. It can bind to several cytoskeletal proteins, including filamin A, an actin-binding protein that collaborates with RNase-L to maintain the cellular barrier to viral entry. This antiviral activity is independent of catalytic function, a unique mechanism for RNase-L. We also describe here the interaction of RNase-L with the E3 ubiquitin ligase and scaffolding protein, ligand of nump protein X (LNX), a regulator of tight junction proteins. In order to better understand the significance and context of these novel binding partners in the antimicrobial response, other innate immune protein interactions with the cytoskeleton are also discussed.

Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis.

Science.gov (United States)

Jeganathan, Niranjan; Predescu, Dan; Zhang, Jin; Sha, Fei; Bardita, Cristina; Patel, Monal; Wood, Stephen; Borgia, Jeffrey A; Balk, Robert A; Predescu, Sanda

2016-09-14

The mechanisms involved in lung cancer (LC) progression are poorly understood making discovery of successful therapies difficult. Adaptor proteins play a crucial role in cancer as they link cell surface receptors to specific intracellular pathways. Intersectin-1s (ITSN-1s) is an important multidomain adaptor protein implicated in the pathophysiology of numerous pulmonary diseases. To date, the role of ITSN-1s in LC has not been studied. Human LC cells, human LC tissue and A549 LC cells stable transfected with myc-ITSN-1s construct (A549 + ITSN-1s) were used in correlation with biochemical, molecular biology and morphological studies. In addition scratch assay with time lapse microscopy and in vivo xenograft tumor and mouse metastasis assays were performed. ITSN-1s, a prevalent protein of lung tissue, is significantly downregulated in human LC cells and LC tissue. Restoring ITSN-1s protein level decreases LC cell proliferation and clonogenic potential. In vivo studies indicate that immunodeficient mice injected with A549 + ITSN-1s cells develop less and smaller metastatic tumors compared to mice injected with A549 cells. Our studies also show that restoring ITSN-1s protein level increases the interaction between Cbl E3 ubiquitin ligase and Eps8 resulting in enhanced ubiquitination of the Eps8 oncoprotein. Subsequently, downstream unproductive assembly of the Eps8-mSos1 complex leads to impaired activation of the small GTPase Rac1. Impaired Rac1 activation mediated by ITSN-1s reorganizes the cytoskeleton (increased thick actin bundles and focal adhesion (FA) complexes as well as collapse of the vimentin filament network) in favor of decreased LC cell migration and metastasis. ITSN-1s induced Eps8 ubiquitination and impaired Eps8-mSos1 complex formation, leading to impaired activation of Rac1, is a novel signaling mechanism crucial for abolishing the progression and metastatic potential of LC cells.

Understanding the role of the cytoskeleton in wood formation in angiosperm trees: hybrid aspen (Populus tremula x P. tremuloides) as a model species

Energy Technology Data Exchange (ETDEWEB)

Chaffey, N.; Barlow, P. [Bristol Univ., Dept. of Agricultural Sciences, Long Ashton, (United Kingdom); Sundberg, B. [Swedish Univ. of Agricultural Sciences, Dept. of Forest Genetics and Plant Physiology, Umea (Sweden)

2002-03-01

The involvement of microfilaments (MFs) and microtubules (MTs) in the development of the radial and axial components of secondary wood in hybrid aspen (Populus tremula X P. tremuloides) was studied by indirect immunofluorescent localization techniques in order to elucidate a consensus view of the roles of the cytoskeleton during wood formation in angiosperm trees. Early and late vessel elements, axial parenchyma, normal-wood fibres and contact and isolation cells were included in addition to cambial cells. Microfilaments were found to be rare in cambial cells, but were abundant and axially arranged in their derivatives once cell elongation begun. Microtubules were randomly oriented in ray and fusiform cells of the cambial zone. Ellipses of microfilaments were associated with pit development in fiber cells and isolation ray cells. Rings of localized microtubules and microfilaments were associated with developing inter-vessel bordered pits and vessel-contact ray cell contact pits. Although only microtubules were seen in the periphery of the perforation plate of vessel elements, a prominent meshwork of microfilaments overlaid the perforation plate itself. These observations indicate that there are corresponding subcellular control points whose manipulation could lead to the development of 'designer wood'. However, such development would require a better understanding of the physiological basis for the behaviour of microtubule and microfibre cytoskeletons during wood formation. 44 refs., 6 figs.

Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation

DEFF Research Database (Denmark)

Barres, Romain; Grémeaux, Thierry; Gual, Philippe

2006-01-01

a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma m...

Changed Expression of Cytoskeleton Proteins During Lung Injury in a Mouse Model of Streptococcus pneumoniae Infection

Directory of Open Access Journals (Sweden)

Mario Ferrer-Navarro

2018-05-01

Full Text Available Infections by Streptococcus pneumoniae are a major cause of morbidity and mortality worldwide, often causing community-acquired pneumonia, otitis media and also bacteremia and meningitis. Studies on S. pneumoniae are mainly focused on its virulence or capacity to evade the host immune system, but little is known about the injury caused in lungs during a pneumococcal infection. Herein we investigated this issue comparing the proteome profile of lungs from S. pneumoniae-infected mice with control mice by means of difference gel electrophoresis (DIGE technology. In order to obtain reliable results three biological replicas were used, and four technical replicas were carried out in each biological replica. Proteomic comparison was performed at two time points: 24 and 48 h post infection. A total of 91 proteins were identified with different abundance. We found important changes in the protein profiles during pneumococcal infection mainly associated with regulation of vesicle-mediated transport, wound healing, and cytoskeleton organization. In conclusion, the results obtained show that the cytoskeleton of the host cell is modified in S. pneumoniae infection.

Focusing super resolution on the cytoskeleton [version 1; referees: 3 approved

Directory of Open Access Journals (Sweden)

Eric A. Shelden

2016-05-01

Full Text Available Super resolution imaging is becoming an increasingly important tool in the arsenal of methods available to cell biologists. In recognition of its potential, the Nobel Prize for chemistry was awarded to three investigators involved in the development of super resolution imaging methods in 2014. The availability of commercial instruments for super resolution imaging has further spurred the development of new methods and reagents designed to take advantage of super resolution techniques. Super resolution offers the advantages traditionally associated with light microscopy, including the use of gentle fixation and specimen preparation methods, the ability to visualize multiple elements within a single specimen, and the potential to visualize dynamic changes in living specimens over time. However, imaging of living cells over time is difficult and super resolution imaging is computationally demanding. In this review, we discuss the advantages/disadvantages of different super resolution systems for imaging fixed live specimens, with particular regard to cytoskeleton structures.

αII Spectrin Forms a Periodic Cytoskeleton at the Axon Initial Segment and Is Required for Nervous System Function.

Science.gov (United States)

Huang, Claire Yu-Mei; Zhang, Chuansheng; Ho, Tammy Szu-Yu; Oses-Prieto, Juan; Burlingame, Alma L; Lalonde, Joshua; Noebels, Jeffrey L; Leterrier, Christophe; Rasband, Matthew N

2017-11-22

Spectrins form a submembranous cytoskeleton proposed to confer strength and flexibility to neurons and to participate in ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Neuronal spectrin cytoskeletons consist of diverse β subunits and αII spectrin. Although αII spectrin is found in neurons in both axonal and somatodendritic domains, using proteomics, biochemistry, and superresolution microscopy, we show that αII and βIV spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptan1 f/f mice for deletion of CNS αII spectrin. We analyzed αII spectrin-deficient mice of both sexes and found that loss of αII spectrin causes profound reductions in all β spectrins. αII spectrin-deficient mice die before 1 month of age and have disrupted AIS and many other neurological impairments including seizures, disrupted cortical lamination, and widespread neurodegeneration. These results demonstrate the importance of the spectrin cytoskeleton both at the AIS and throughout the nervous system. SIGNIFICANCE STATEMENT Spectrin cytoskeletons play diverse roles in neurons, including assembly of excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. However, the molecular composition and structure of these cytoskeletons remain poorly understood. Here, we show that αII spectrin partners with βIV spectrin to form a periodic cytoskeleton at the AIS. Using a new αII spectrin conditional knock-out mouse, we show that αII spectrin is required for AIS assembly, neuronal excitability, cortical lamination, and to protect against neurodegeneration. These results demonstrate the broad importance of spectrin cytoskeletons for nervous system function and development and have important implications for nervous system injuries and diseases because disruption of the spectrin cytoskeleton is a common molecular pathology. Copyright © 2017 the authors 0270-6474/17/3711311-12$15.00/0.

Active Vertex Model for cell-resolution description of epithelial tissue mechanics.

Science.gov (United States)

Barton, Daniel L; Henkes, Silke; Weijer, Cornelis J; Sknepnek, Rastko

2017-06-01

We introduce an Active Vertex Model (AVM) for cell-resolution studies of the mechanics of confluent epithelial tissues consisting of tens of thousands of cells, with a level of detail inaccessible to similar methods. The AVM combines the Vertex Model for confluent epithelial tissues with active matter dynamics. This introduces a natural description of the cell motion and accounts for motion patterns observed on multiple scales. Furthermore, cell contacts are generated dynamically from positions of cell centres. This not only enables efficient numerical implementation, but provides a natural description of the T1 transition events responsible for local tissue rearrangements. The AVM also includes cell alignment, cell-specific mechanical properties, cell growth, division and apoptosis. In addition, the AVM introduces a flexible, dynamically changing boundary of the epithelial sheet allowing for studies of phenomena such as the fingering instability or wound healing. We illustrate these capabilities with a number of case studies.

Par-4-mediated recruitment of Amida to the actin cytoskeleton leads to the induction of apoptosis

International Nuclear Information System (INIS)

Boosen, Meike; Vetterkind, Susanne; Koplin, Ansgar; Illenberger, Susanne; Preuss, Ute

2005-01-01

Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4. Amida resides mainly in the nucleus but displays nucleo-cytoplasmic shuttling in heterokaryons. Upon coexpression with Par-4 in REF52.2 cells, Amida translocates to the cytoplasm and is recruited to actin filaments by Par-4, resulting in enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the induction of apoptosis is abrogated when either Amida/Par-4 complex formation or association of these complexes with the actin cytoskeleton is impaired, indicating that the Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis

Effects of Silver and Other Metals on the Cytoskeleton

Science.gov (United States)

Conrad, Gary W.

1997-01-01

Directly or indirectly, trace concentrations of silver ion (Ag(+)) stabilize microtubules (Conrad, A.H., et al. Cell Motil. & Cytoskel. 27:117-132), as does taxol (Conrad, A.H., et al. J. Exp. Zool. 262:154-165), an effect with major consequences for cellular shape changes and development. Polymerization of microtubules is gravity-sensitive (Tabony and Job, Proc. Natl. Acad. Sci. USA 89:6948-6952), so trace amounts of Ag(+) may alter cellular ability to respond to gravity. If Ag electrolysis is used to purify water on NASA space vehicles, plants and animals/astronauts will be exposed continuously to Ag(+), a regimen with unknown cellular and developmental consequences. Fertilized eggs of the marine mudsnail, Ilyanassa obsoleta, are the cells in which the effects of A(+) on microtubules were discovered. They distribute visible cytoplasmic contents according to gravity and contain cytoplasmic morphogenetic determinants for heart development. The objectives are to determine if the effects of Ag(+), AU(3+), (of biosensor relevance), or Gd(3+) (inhibitor of some stretch-activated ion channels) on the cytoskeleton (in the presence and absence of mechanical loading) will affect cellular responses to gravity.

Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis.

Science.gov (United States)

Dong, Bo; Deng, Wei; Jiang, Di

2011-04-01

Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.

Focal adhesion kinase maintains, but not increases the adhesion of dental pulp cells.

Science.gov (United States)

Qian, Yuyan; Shao, Meiying; Zou, Wenlin; Wang, Linyan; Cheng, Ran; Hu, Tao

2017-04-01

Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.

Interconnection between actin cytoskeleton and plant defense signaling

Czech Academy of Sciences Publication Activity Database

Janda, Martin; Matoušková, J.; Burketová, Lenka; Valentová, O.

2014-01-01

Roč. 9, č. 11 (2014) ISSN 1559-2316 R&D Projects: GA ČR(CZ) GAP501/11/1654 Institutional support: RVO:61389030 Keywords : Actin * Cytoskeleton * Pathogen Subject RIV: ED - Physiology http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=MEDLINE&DestLinkType=FullRecord&UT=25482795

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

OpenAIRE

Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae; Bae, Chang-Dae; Hong, Kyeong-Man

2008-01-01

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 re...

The microtubule cytoskeleton does not integrate auxin transport and gravitropism in maize roots

Science.gov (United States)

Hasenstein, K. H.; Blancaflor, E. B.; Lee, J. S.

1999-01-01

The Cholodny-Went hypothesis of gravitropism suggests that the graviresponse is controlled by the distribution of auxin. However, the mechanism of auxin transport during the graviresponse of roots is still unresolved. To determine whether the microtubule (MT) cytoskeleton is participating in auxin transport, the cytoskeleton was examined and the movement of 3H-IAA measured in intact and excised taxol, oryzalin, and naphthylphthalamic acid (NPA)-treated roots of Zea mays cv. Merit. Taxol and oryzalin did not inhibit the graviresponse of roots but the auxin transport inhibitor NPA greatly inhibited both auxin transport and graviresponse. NPA had no effect on MT organization in vertical roots, but caused MT reorientation in horizontally placed roots. Regardless of treatment, the organization of MTs in intact roots differed from that in root segments. The MT inhibitors, taxol and oryzalin had opposite effects on the MTs, namely, depolymerization (oryzalin) and stabilization and thickening (taxol), but both treatments caused swelling of the roots. The data indicate that the MT cytoskeleton does not directly interfere with auxin transport or auxin-mediated growth responses in maize roots.

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

Directory of Open Access Journals (Sweden)

Irina V. Gorudko

2013-07-01

Myeloperoxidase (MPO is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist. In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE. Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

Mechanical loading and how it affects bone cells: The role of the osteocyte cytoskeleton in maintaining our skeleton

Directory of Open Access Journals (Sweden)

J Klein-Nulend

2012-09-01

Full Text Available Lack of physical activity causes bone loss and fractures not only in elderly people, but also in bedridden patients or otherwise inactive youth. This is fast becoming one of the most serious healthcare problems in the world. Osteocytes, cells buried within our bones, stimulate bone formation in the presence of mechanical stimuli, as well as bone degradation in the absence of such stimuli. As yet, we do not fully comprehend how osteocytes sense mechanical stimuli, and only know a fraction of the whole range of molecules that osteocytes subsequently produce to regulate bone formation and degradation in response to mechanical stimuli. This dramatically hampers the design of bone loss prevention strategies. In this review we will focus on the first step in the cascade of events leading to adaptation of bone mass to mechanical loading, i.e., on how osteocytes are able to perceive mechanical stimuli placed on whole bones. We will place particular emphasis on the role of the osteocyte cytoskeleton in mechanosensing. Given the crucial importance of osteocytes in maintaining a proper resistance against bone fracture, greater knowledge of the molecular mechanisms that govern the adaptive response of osteocytes to mechanical stimuli may lead to the development of new strategies towards fracture prevention and enhanced bone healing.

The cell wall of Arabidopsis thaliana influences actin network dynamics.

Science.gov (United States)

Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

2017-07-20

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

International Nuclear Information System (INIS)

Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

2013-01-01

The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility

One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

Energy Technology Data Exchange (ETDEWEB)

Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A., E-mail: roberto.perego@unimib.it

2013-08-01

The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

Science.gov (United States)

Hou, Guichuan; Mohamalawari, Deepti R.; Blancaflor, Elison B.

2003-01-01

The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

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

International Nuclear Information System (INIS)

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

2010-01-01

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

Dynamics of a bilayer membrane coupled to a two-dimensional cytoskeleton: Scale transfers of membrane deformations

Science.gov (United States)

Okamoto, Ryuichi; Komura, Shigeyuki; Fournier, Jean-Baptiste

2017-07-01

We theoretically investigate the dynamics of a floating lipid bilayer membrane coupled with a two-dimensional cytoskeleton network, taking into account explicitly the intermonolayer friction, the discrete lattice structure of the cytoskeleton, and its prestress. The lattice structure breaks lateral continuous translational symmetry and couples Fourier modes with different wave vectors. It is shown that within a short time interval a long-wavelength deformation excites a collection of modes with wavelengths shorter than the lattice spacing. These modes relax slowly with a common renormalized rate originating from the long-wavelength mode. As a result, and because of the prestress, the slowest relaxation is governed by the intermonolayer friction. Conversely, and most interestingly, forces applied at the scale of the cytoskeleton for a sufficiently long time can cooperatively excite large-scale modes.

Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

Energy Technology Data Exchange (ETDEWEB)

Lasalvia, Maria [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Castellani, Stefano [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); D’Antonio, Palma [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Perna, Giuseppe [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Carbone, Annalucia [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); Colia, Anna Laura; Maffione, Angela Bruna [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Capozzi, Vito [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Conese, Massimo, E-mail: massimo.conese@unifg.it [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy)

2016-10-15

The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity in

Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

International Nuclear Information System (INIS)

Lasalvia, Maria; Castellani, Stefano; D’Antonio, Palma; Perna, Giuseppe; Carbone, Annalucia; Colia, Anna Laura; Maffione, Angela Bruna; Capozzi, Vito; Conese, Massimo

2016-01-01

The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity in the

RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton.

Science.gov (United States)

Mamon, L A; Ginanova, V R; Kliver, S F; Yakimova, A O; Atsapkina, A A; Golubkova, E V

2017-04-01

The mutual relationship between mRNA and the cytoskeleton can be seen from two points of view. On the one hand, the cytoskeleton is necessary for mRNA trafficking and anchoring to subcellular domains. On the other hand, cytoskeletal growth and rearrangement require the translation of mRNAs that are connected to the cytoskeleton. β-actin mRNA localization may influence dynamic changes in the actin cytoskeleton. In the cytoplasm, long-lived mRNAs exist in the form of RNP (ribonucleoprotein) complexes, where they interact with RNA-binding proteins, including NXF (Nuclear eXport Factor). Dm NXF1 is an evolutionarily conserved protein in Drosophila melanogaster that has orthologs in different animals. The universal function of nxf1 genes is the nuclear export of different mRNAs in various organisms. In this mini-review, we briefly discuss the evidence demonstrating that Dm NXF1 fulfils not only universal but also specialized cytoplasmic functions. This protein is detected not only in the nucleus but also in the cytoplasm. It is a component of neuronal granules. Dm NXF1 marks nuclear division spindles during early embryogenesis and the dense body on one side of the elongated spermatid nuclei. The characteristic features of sbr mutants (sbr 10 and sbr 5 ) are impairment of chromosome segregation and spindle formation anomalies during female meiosis. sbr 12 mutant sterile males with immobile spermatozoa exhibit disturbances in the axoneme, mitochondrial derivatives and cytokinesis. These data allow us to propose that the Dm NXF1 proteins transport certain mRNAs in neurites and interact with localized mRNAs that are necessary for dynamic changes of the cytoskeleton. © 2017 Wiley Periodicals, Inc.

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

Science.gov (United States)

Mazars, C; Thion, L; Thuleau, P; Graziana, A; Knight, M R; Moreau, M; Ranjeva, R

1997-11-01

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

Hepatocyte cytoskeleton during ischemia and reperfusion influence of ANP-mediated p38 MAPK activation

Institute of Scientific and Technical Information of China (English)

Melanie Keller; Alexander L Gerbes; Stefanie Kulhanek-Heinze; Tobias Gerwig; Uwe Grützner; Nico van Rooijen; Angelika M Vollmar; Alexandra K Kiemer

2005-01-01

AIM: To determine functional consequences of this activation, whereby we focused on a potential regulation of the hepatocyte cytoskeleton during ischemia and reperfusion.METHODS: For in vivo experiments, animals received ANP (5 μg/kg) intravenously. In a different experimental setting, isolated rat livers were perfused with KH-buffer ±ANP (200 nmol/L)±SB203580 (2 μmol/L). Liverswere then kept under ischemic conditions for 24 h, and either transplanted or reperfused. Actin, Hsp27, and phosphorylated Hsp27 were determined by Western blotting, p38 MAPK activity by in vitro phosphorylation assay. F-actin distribution was determined by confocal microscopy.RESULTS: We first confirmed that ANP preconditioning leads to an activation of p38 MAPK and observedalterations of the cytoskeleton in hepatocytes of ANPpreconditioned organs. ANP induced an increase of hepatic F-actin after ischemia, which could be prevented by the p38 MAPK inhibitor SB203580 but had no effect on bile flow. After ischemia untreated livers showed a translocation of Hsp27 towards the cytoskeleton and an increase in total Hsp27, whereas ANP preconditioning prohibited translocation but caused an augmentation of Hsp27 phosphorylation. This effect is also mediated via p38 MAPK, since it was abrogated by the p38 MAPK inhibitor SB203580.CONCLUSION: This study reveals that ANP-mediated p38 MAPK activation leads to changes in hepatocyte cytoskeleton involving an elevation of phosphorylated Hsp27 and thereby for the first time shows functional consequences of ANP-induced hepatic p38 MAPK activation.

A novel membrane-bound toxin for cell division, CptA (YgfX), inhibits polymerization of cytoskeleton proteins, FtsZ and MreB, in Escherichia coli.

Science.gov (United States)

Masuda, Hisako; Tan, Qian; Awano, Naoki; Yamaguchi, Yoshihiro; Inouye, Masayori

2012-03-01

Nearly all free-living bacteria carry toxin-antitoxin (TA) systems on their genomes, through which cell growth and death are regulated. Toxins target a variety of essential cellular functions, including DNA replication, translation, and cell division. Here, we identified a novel toxin, YgfX, on the Escherichia coli genome. The toxin, consisting of 135 residues, is composed of the N-terminal membrane domain, which encompasses two transmembrane segments, and the C-terminal cytoplasmic domain. Upon YgfX expression, the cells were initially elongated and then the middle portion of the cells became inflated to form a lemon shape. YgfX was found to interact with MreB and FtsZ, two essential cytoskeletal proteins in E. coli. The cytoplasmic domain [YgfX(C)] was found to be responsible for the YgfX toxicity, as purified YgfX(C) was found to block the polymerization of FtsZ and MreB in vitro. YgfY, located immediately upstream of YgfX, was shown to be the cognate antitoxin; notably, YgfX is the first membrane-associating toxin in bacterial TA systems. We propose to rename the toxin and the antitoxin as CptA and CptB (for Cytoskeleton Polymerization inhibiting Toxin), respectively. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Description of gasket failure in a 7 cell PEMFC stack

Energy Technology Data Exchange (ETDEWEB)

Husar, Attila; Serra, Maria [Institut de Robotica i Informatica Industrial, Parc Tecnologic de Barcelona, Edifici U, C. Llorens i Artigas, 4-6, 2a Planta, 08028 Barcelona (Spain); Kunusch, Cristian [Laboratorio de Electronica Industrial Control e Instrumentacion, Facultad de Ingenieria, UNLP (Argentina)

2007-06-10

This article presents the data and the description of a fuel cell stack that failed due to gasket degradation. The fuel cell under study is a 7 cell stack. The unexpected change in several variables such as temperature, pressure and voltage indicated the possible failure of the stack. The stack was monitored over a 6 h period in which data was collected and consequently analyzed to conclude that the fuel cell stack failed due to a crossover leak on the anode inlet port located on the cathode side gasket of cell 2. This stack failure analysis revealed a series of indicators that could be used by a super visional controller in order to initiate a shutdown procedure. (author)

Remodeling of tick cytoskeleton in response to infection with Anaplasma phagocytophilum

Czech Academy of Sciences Publication Activity Database

Cabezas Cruz, Alejandro; Alberdi, P.; Valdés, James J.; Villar, M.; de la Fuente, J.

2017-01-01

Roč. 22, Jun 1 (2017), s. 1830-1844 ISSN 1093-4715 EU Projects: European Commission(XE) 278976 - ANTIGONE Institutional support: RVO:60077344 Keywords : cytoskeleton * proteomics * transcriptomics * Ixodes scapularis * Anaplasma phagocytophilum Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology

Association of vinculin to the platelet cytoskeleton during thrombin-induced aggregation

NARCIS (Netherlands)

Asyee, G. M.; Sturk, A.; Muszbek, L.

1987-01-01

Vinculin is a protein generally believed to be involved in membrane-cytoskeleton interaction, and its presence in platelets has been verified earlier. Here we show that in resting bovine platelets, vinculin is not associated with the Triton-insoluble cytoskeletal fraction but becomes incorporated

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

Science.gov (United States)

Westendorf, Christian; Negrete, Jose; Bae, Albert J.; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten

2013-01-01

The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments. PMID:23431176

TGF1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton

DEFF Research Database (Denmark)

Elsafadi, Mona; Manikandan, Muthurangan; Almalki, Sami

2018-01-01

MSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFβl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton....... To investigate further, we examined the actin cytoskeleton following treatment with TGFβ1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment...... of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFβ1 and cytochalasin D. Our study demonstrates that TGFβ1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton....

Evaluation of cell behavior on modified polypropylene with swift heavy ion irradiation

International Nuclear Information System (INIS)

Arbeitman, Claudia R.; Ibañez, Irene L.; García Bermúdez, Gerardo; Durán, Hebe; Grosso, Mariela F. del; Salguero, Noelia; Mazzei, Rubén

2012-01-01

Ion beam irradiation is a well known means to change the physico-chemical properties of polymers, and induced bio and citocompatibility in controlled conditions and in selected areas of surface. However, the enhancement of cell adhesion on a modified substrate does not mean that the surface is adequate for functional cells. The purpose of the present work is to study proliferation, changes in cytoskeleton and cell morphology on substrates as a function of irradiation parameters. We irradiated polypropylene with sulfur (S) ion-beam at energies of 110 MeV with fluences between 1 × 10 6 and 2 × 10 10 ions cm −2 . NIH 3T3 cells were cultured on each sample. Cell morphology was observed using phase contrast microscopy and cytoskeleton proteins with fluorescence microscopy. The analysis show different cellular responses as a functions of irradiation parameter, strongly suggests that different underlying substratum can result in distinct types of cytoskeleton reorganization.

Evaluation of cell behavior on modified polypropylene with swift heavy ion irradiation

Energy Technology Data Exchange (ETDEWEB)

Arbeitman, Claudia R., E-mail: arbeitman@tandar.cnea.gov.ar [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Ibanez, Irene L. [CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Garcia Bermudez, Gerardo [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Duran, Hebe [CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Gerencia de Desarrollo Tecnologico y Proyectos Especiales, TANDAR-CNEA (Argentina); Grosso, Mariela F. del [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Salguero, Noelia [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); Mazzei, Ruben [U.A. Tecnologicas y Agropecuarias, CNEA (Argentina)

2012-02-15

Ion beam irradiation is a well known means to change the physico-chemical properties of polymers, and induced bio and citocompatibility in controlled conditions and in selected areas of surface. However, the enhancement of cell adhesion on a modified substrate does not mean that the surface is adequate for functional cells. The purpose of the present work is to study proliferation, changes in cytoskeleton and cell morphology on substrates as a function of irradiation parameters. We irradiated polypropylene with sulfur (S) ion-beam at energies of 110 MeV with fluences between 1 Multiplication-Sign 10{sup 6} and 2 Multiplication-Sign 10{sup 10} ions cm{sup -2}. NIH 3T3 cells were cultured on each sample. Cell morphology was observed using phase contrast microscopy and cytoskeleton proteins with fluorescence microscopy. The analysis show different cellular responses as a functions of irradiation parameter, strongly suggests that different underlying substratum can result in distinct types of cytoskeleton reorganization.

Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba

Science.gov (United States)

Negrete, Jose; Pumir, Alain; Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Beta, Carsten; Bodenschatz, Eberhard

2016-09-01

Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

The Emerging Role of the Cytoskeleton in Chromosome Dynamics

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Maya Spichal

2017-05-01

Full Text Available Chromosomes underlie a dynamic organization that fulfills functional roles in processes like transcription, DNA repair, nuclear envelope stability, and cell division. Chromosome dynamics depend on chromosome structure and cannot freely diffuse. Furthermore, chromosomes interact closely with their surrounding nuclear environment, which further constrains chromosome dynamics. Recently, several studies enlighten that cytoskeletal proteins regulate dynamic chromosome organization. Cytoskeletal polymers that include actin filaments, microtubules and intermediate filaments can connect to the nuclear envelope via Linker of the Nucleoskeleton and Cytoskeleton (LINC complexes and transfer forces onto chromosomes inside the nucleus. Monomers of these cytoplasmic polymers and related proteins can also enter the nucleus and play different roles in the interior of the nucleus than they do in the cytoplasm. Nuclear cytoskeletal proteins can act as chromatin remodelers alone or in complexes with other nuclear proteins. They can also act as transcription factors. Many of these mechanisms have been conserved during evolution, indicating that the cytoskeletal regulation of chromosome dynamics is an essential process. In this review, we discuss the different influences of cytoskeletal proteins on chromosome dynamics by focusing on the well-studied model organism budding yeast.

Resolving the role of actoymyosin contractility in cell microrheology.

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Christopher M Hale

2009-09-01

Full Text Available Einstein's original description of Brownian motion established a direct relationship between thermally-excited random forces and the transport properties of a submicron particle in a viscous liquid. Recent work based on reconstituted actin filament networks suggests that nonthermal forces driven by the motor protein myosin II can induce large non-equilibrium fluctuations that dominate the motion of particles in cytoskeletal networks. Here, using high-resolution particle tracking, we find that thermal forces, not myosin-induced fluctuating forces, drive the motion of submicron particles embedded in the cytoskeleton of living cells. These results resolve the roles of myosin II and contractile actomyosin structures in the motion of nanoparticles lodged in the cytoplasm, reveal the biphasic mechanical architecture of adherent cells-stiff contractile stress fibers interdigitating in a network at the cell cortex and a soft actin meshwork in the body of the cell, validate the method of particle tracking-microrheology, and reconcile seemingly disparate atomic force microscopy (AFM and particle-tracking microrheology measurements of living cells.

Effects of Propofol on Several Membrane Characteristics of Cervical Cancer Cell Lines

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Fan Zhang

2016-11-01

Full Text Available Background: Although significant advances have been made toward understanding the molecular mechanisms underlying the effect of propofol on tumor cell metastasis, less is known regarding how cell membrane and cytoskeletal ultrastructure are affected in this process. Here, we investigated the relationship between cell morphology and cell size, which are features mainly defined by the cytoskeleton. Methods: To confirm the effects of propofol on the migratory ability of human cervical carcinoma cells, cell migration and invasion were examined through scratch wound healing and transwell membrane assays. Furthermore, HeLa cells cultivated with different concentrations of propofol were examined by confocal microscopy and atomic force microscopy (AFM, and the mean optical density and migration ability of these cells were also assessed. In addition, cell membrane morphology was inspected using AFM. Results: The results of the wound healing and transwell membrane assays indicated that propofol decreases the migratory ability of cervical carcinoma cells compared to control cells. A comparative analysis of the test results revealed that short-term (3 h exposure to propofol induced marked changes in cell membrane microstructure and in the cytoskeleton in a dose-dependent manner. These morphological changes in the cell membrane were accompanied by cytoskeleton (F-actin derangement. The present findings demonstrate a close relationship between changes in cell membrane ultrastructure and cytoskeletal alterations (F-actin in propofol-treated HeLa cells. AFM scanning analysis showed that cell membrane ultrastructure was significantly changed, including a clear reduction in membrane roughness. Conclusion: The influence of propofol on the HeLa cell cytoskeleton can be directly reflected by changes in cellular morphology, as assessed by AFM. Moreover, the use of AFM is a good method for investigating propofol-mediated changes within cytoskeletal ultrastructure.

Dynamics of cell area and force during spreading.

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Brill-Karniely, Yifat; Nisenholz, Noam; Rajendran, Kavitha; Dang, Quynh; Krishnan, Ramaswamy; Zemel, Assaf

2014-12-16

Experiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The Role of Molecular Microtubule Motors and the Microtubule Cytoskeleton in Stress Granule Dynamics

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Kristen M. Bartoli

2011-01-01

Full Text Available Stress granules (SGs are cytoplasmic foci that appear in cells exposed to stress-induced translational inhibition. SGs function as a triage center, where mRNAs are sorted for storage, degradation, and translation reinitiation. The underlying mechanisms of SGs dynamics are still being characterized, although many key players have been identified. The main components of SGs are stalled 48S preinitiation complexes. To date, many other proteins have also been found to localize in SGs and are hypothesized to function in SG dynamics. Most recently, the microtubule cytoskeleton and associated motor proteins have been demonstrated to function in SG dynamics. In this paper, we will discuss current literature examining the function of microtubules and the molecular microtubule motors in SG assembly, coalescence, movement, composition, organization, and disassembly.

Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions.

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Canetta, Elisabetta; Duperray, Alain; Leyrat, Anne; Verdier, Claude

2005-01-01

Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell.

Effects of hypergravity on adipose-derived stem cell morphology, mechanical property and proliferation

Energy Technology Data Exchange (ETDEWEB)

Tavakolinejad, Alireza [Medical Nanotechnology and Tissue Engineering Research Center, Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Rabbani, Mohsen, E-mail: m.rabbani@eng.ui.ac.ir [Department of Biomedical Engineering, University of Isfahan, Isfahan (Iran, Islamic Republic of); Janmaleki, Mohsen [Medical Nanotechnology and Tissue Engineering Research Center, Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of)

2015-08-21

Alteration in specific inertial conditions can lead to changes in morphology, proliferation, mechanical properties and cytoskeleton of cells. In this report, the effects of hypergravity on morphology of Adipose-Derived Stem Cells (ADSCs) are indicated. ADSCs were repeatedly exposed to discontinuous hypergravity conditions of 10 g, 20 g, 40 g and 60 g by utilizing centrifuge (three times of 20 min exposure, with an interval of 40 min at 1 g). Cell morphology in terms of length, width and cell elongation index and cytoskeleton of actin filaments and microtubules were analyzed by image processing. Consistent changes observed in cell elongation index as morphological change. Moreover, cell proliferation was assessed and mechanical properties of cells in case of elastic modulus of cells were evaluated by Atomic Force Microscopy. Increase in proliferation and decrease in elastic modulus of cells are further results of this study. Staining ADSC was done to show changes in cytoskeleton of the cells associated to hypergravity condition specifically in microfilament and microtubule components. After exposing to hypergravity, significant changes were observed in microfilaments and microtubule density as components of cytoskeleton. It was concluded that there could be a relationship between changes in morphology and MFs as the main component of the cells. - Highlights: • Hypergravity (10 g, 20 g, 40 g and 60 g) affects on adipose derived stem cells (ADSCs). • ADSCs after exposure to the hypergravity are more slender. • The height of ADSCs increases in all test groups comparing their control group. • Hypergravity decreases ADSCs modulus of elasticity and cell actin fiber content. • Hypergravity enhances proliferation rate of ADSCs.

Effects of hypergravity on adipose-derived stem cell morphology, mechanical property and proliferation

International Nuclear Information System (INIS)

Tavakolinejad, Alireza; Rabbani, Mohsen; Janmaleki, Mohsen

2015-01-01

Alteration in specific inertial conditions can lead to changes in morphology, proliferation, mechanical properties and cytoskeleton of cells. In this report, the effects of hypergravity on morphology of Adipose-Derived Stem Cells (ADSCs) are indicated. ADSCs were repeatedly exposed to discontinuous hypergravity conditions of 10 g, 20 g, 40 g and 60 g by utilizing centrifuge (three times of 20 min exposure, with an interval of 40 min at 1 g). Cell morphology in terms of length, width and cell elongation index and cytoskeleton of actin filaments and microtubules were analyzed by image processing. Consistent changes observed in cell elongation index as morphological change. Moreover, cell proliferation was assessed and mechanical properties of cells in case of elastic modulus of cells were evaluated by Atomic Force Microscopy. Increase in proliferation and decrease in elastic modulus of cells are further results of this study. Staining ADSC was done to show changes in cytoskeleton of the cells associated to hypergravity condition specifically in microfilament and microtubule components. After exposing to hypergravity, significant changes were observed in microfilaments and microtubule density as components of cytoskeleton. It was concluded that there could be a relationship between changes in morphology and MFs as the main component of the cells. - Highlights: • Hypergravity (10 g, 20 g, 40 g and 60 g) affects on adipose derived stem cells (ADSCs). • ADSCs after exposure to the hypergravity are more slender. • The height of ADSCs increases in all test groups comparing their control group. • Hypergravity decreases ADSCs modulus of elasticity and cell actin fiber content. • Hypergravity enhances proliferation rate of ADSCs

Citoesqueleto e mecanotransdução na fisiopatologia da lesão pulmonar induzida por ventilador Cytoskeleton and mechanotransduction in the pathophysiology of ventilator-induced lung injury

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Leandro Utino Taniguchi

2010-06-01

Full Text Available A ventilação mecânica é uma terapia importante, mas pode resultar em complicações. Uma das mais relevantes é a lesão pulmonar induzida por ventilador. Devido à hiperdistensão alveolar, o pulmão inicia um processo inflamatório, com infiltrado neutrofílico, formação de membrana hialina, fibrogênese e prejuízo de troca gasosa. Nesse processo, a mecanotransdução da hiperdistensão celular é mediada através do citoesqueleto da célula e de suas interações com a matriz extracelular e com as células vizinhas, de modo que o estímulo mecânico da ventilação se traduz em sinalização bioquímica intracelular, desencadeando ativação endotelial, permeabilidade vascular pulmonar, quimiotaxia leucocitária, produção de citocinas e, possivelmente, lesão de órgãos à distância. Estudos clínicos demonstram essa relação entre distensão pulmonar e mortalidade em pacientes com lesão pulmonar induzida por ventilador. Entretanto, apesar de o citoesqueleto ter um papel fundamental na patogênese da lesão pulmonar induzida por ventilador, a literatura carece de estudos utilizando modelos in vivo sobre as alterações do citoesqueleto e de suas proteínas associadas durante esse processo patológico.Although mechanical ventilation is an important therapy, it can result in complications. One major complication is ventilator-induced lung injury, which is caused by alveolar hyperdistension, leading to an inflammatory process, with neutrophilic infiltration, hyaline membrane formation, fibrogenesis and impaired gas exchange. In this process, cellular mechanotransduction of the overstretching stimulus is mediated by means of the cytoskeleton and its cell-cell and cell-extracellular matrix interactions, in such a way that the mechanical stimulus of ventilation is translated into an intracellular biochemical signal, inducing endothelial activation, pulmonary vascular permeability, leukocyte chemotaxis, cytokine production and, possibly

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

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Wang, Xiang; Zhao, Fang [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009 (China); Lv, Zhong-ming; Shi, Wei-qin [Jiangsu Provincial Center for Disease Control and Prevention, Nanjing (China); Zhang, Lu-yong, E-mail: lyzhang@cpu.edu.cn [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009 (China); Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Nanjing (China); State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009 (China); Yan, Ming, E-mail: brookming@cpu.edu.cn [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009 (China)

2016-11-01

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit the expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

International Nuclear Information System (INIS)

Wang, Xiang; Zhao, Fang; Lv, Zhong-ming; Shi, Wei-qin; Zhang, Lu-yong; Yan, Ming

2016-01-01

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit the expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.

Cell shape regulates global histone acetylation in human mammaryepithelial cells

Energy Technology Data Exchange (ETDEWEB)

Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.

2007-02-28

Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

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G.D. Godbold

1998-08-01

Full Text Available It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.

Frequent alterations in cytoskeleton remodelling genes in primary and metastatic lung adenocarcinomas

DEFF Research Database (Denmark)

Wu, Kui; Zhang, Xin; Li, Fuqiang

2015-01-01

significantly mutated genes are identified, including the most commonly mutated gene TP53 and novel mutation targets such as RHPN2, GLI3 and MRC2. TP53 mutations are furthermore significantly enriched in tumours from patients harbouring metastases. Genes regulating cytoskeleton remodelling processes are also...

A molecular smart surface for spatio-temporal studies of cell mobility.

Science.gov (United States)

Lee, Eun-ju; Luo, Wei; Chan, Eugene W L; Yousaf, Muhammad N

2015-01-01

Active migration in both healthy and malignant cells requires the integration of information derived from soluble signaling molecules with positional information gained from interactions with the extracellular matrix and with other cells. How a cell responds and moves involves complex signaling cascades that guide the directional functions of the cytoskeleton as well as the synthesis and release of proteases that facilitate movement through tissues. The biochemical events of the signaling cascades occur in a spatially and temporally coordinated manner then dynamically shape the cytoskeleton in specific subcellular regions. Therefore, cell migration and invasion involve a precise but constantly changing subcellular nano-architecture. A multidisciplinary effort that combines new surface chemistry and cell biological tools is required to understand the reorganization of cytoskeleton triggered by complex signaling during migration. Here we generate a class of model substrates that modulate the dynamic environment for a variety of cell adhesion and migration experiments. In particular, we use these dynamic substrates to probe in real-time how the interplay between the population of cells, the initial pattern geometry, ligand density, ligand affinity and integrin composition affects cell migration and growth. Whole genome microarray analysis indicates that several classes of genes ranging from signal transduction to cytoskeletal reorganization are differentially regulated depending on the nature of the surface conditions.

Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

Science.gov (United States)

Laskowski, Denise; Båge, Renée; Humblot, Patrice; Andersson, Göran; Sirard, Marc-André; Sjunnesson, Ylva

2017-10-01

Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL - 1 and 0.1 µg mL - 1 ), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

DEFF Research Database (Denmark)

Evans, Evan; Heinrich, Volkmar; Leung, Andrew

2005-01-01

to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether......, and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane...

Mercury exposure induces cytoskeleton disruption and loss of renal function through epigenetic modulation of MMP9 expression.

Science.gov (United States)

Khan, Hafizurrahman; Singh, Radha Dutt; Tiwari, Ratnakar; Gangopadhyay, Siddhartha; Roy, Somendu Kumar; Singh, Dhirendra; Srivastava, Vikas

2017-07-01

Mercury is one of the major heavy metal pollutants occurring in elemental, inorganic and organic forms. Due to ban on most inorganic mercury containing products, human exposure to mercury generally occurs as methylmercury (MeHg) by consumption of contaminated fish and other sea food. Animal and epidemiological studies indicate that MeHg affects neural and renal function. Our study is focused on nephrotoxic potential of MeHg. In this study, we have shown for the first time how MeHg could epigenetically modulate matrix metalloproteinase 9(MMP9) to promote nephrotoxicity using an animal model of sub chronic MeHg exposure. MeHg caused renal toxicity as was seen by increased levels of serum creatinine and expression of early nephrotoxicity markers (KIM-1, Clusterin, IP-10, and TIMP). MeHg exposure also correlated strongly with induction of MMP9 mRNA and protein in a dose dependent manner. Further, while induction of MMP9 promoted cytoskeleton disruption and loss of cell-cell adhesion (loss of F-actin, Vimentin and Fibronectin), inhibition of MMP9 was found to reduce these disruptions. Mechanistic studies by ChIP analysis showed that MeHg modulated MMP9 by promoting demethylation of its regulatory region to increase its expression. Bisulfite sequencing identified critical CpGs in the first exon of MMP9 which were demethylated following MeHg exposure. ChIP studies also showed loss of methyl binding protein, MeCP2 and transcription factor PEA3 at the demethylated site confirming decreased CpG methylation. Our studies thus show how MeHg could epigenetically modulate MMP9 to promote cytoskeleton disruption leading to loss of renal function. Copyright © 2017 Elsevier B.V. All rights reserved.

Beta adrenergic overstimulation impaired vascular contractility via actin-cytoskeleton disorganization in rabbit cerebral artery.

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Hyoung Kyu Kim

Full Text Available BACKGROUND AND PURPOSE: Beta adrenergic overstimulation may increase the vascular damage and stroke. However, the underlying mechanisms of beta adrenergic overstimulation in cerebrovascular dysfunctions are not well known. We investigated the possible cerebrovascular dysfunction response to isoproterenol induced beta-adrenergic overstimulation (ISO in rabbit cerebral arteries (CAs. METHODS: ISO was induced in six weeks aged male New Zealand white rabbit (0.8-1.0 kg by 7-days isoproterenol injection (300 μg/kg/day. We investigated the alteration of protein expression in ISO treated CAs using 2DE proteomics and western blot analysis. Systemic properties of 2DE proteomics result were analyzed using bioinformatics software. ROS generation and following DNA damage were assessed to evaluate deteriorative effect of ISO on CAs. Intracellular Ca(2+ level change and vascular contractile response to vasoactive drug, angiotensin II (Ang II, were assessed to evaluate functional alteration of ISO treated CAs. Ang II-induced ROS generation was assessed to evaluated involvement of ROS generation in CA contractility. RESULTS: Proteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1 in ISO-CAs. As a cause of dysregulation of actin-cytoskeleton organization, we found decreased level of RhoA and ROCK1, which are major regulators of actin-cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca(2+ efflux and constriction response to angiotensin II and high K(+ in ISO-CAs. ISO also increased basal ROS generation and induced oxidative damage in CA; however, it decreased the Ang II-induced ROS generation rate. These results indicate that ISO disrupted actin cytoskeleton proteome network

Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

NARCIS (Netherlands)

Sieberer, B.; Kieft, H.; Franssen-Verheijen, M.A.W.; Emons, A.M.C.; Vos, J.W.

2009-01-01

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g.

The cytoskeleton is disrupted by the bacterial effector HrpZ, but not by the bacterial PAMP flg22, in tobacco BY-2 cells.

Science.gov (United States)

Guan, Xin; Buchholz, Günther; Nick, Peter

2013-04-01

Plant innate immunity is composed of two layers. Basal immunity is triggered by pathogen-associated molecular patterns (PAMPs) such as the flagellin-peptide flg22 and is termed PAMP-triggered immunity (PTI). In addition, effector-triggered immunity (ETI) linked with programmed cell death and cytoskeletal reorganization can be induced by pathogen-derived factors, such as the Harpin proteins originating from phytopathogenic bacteria. To get insight into the link between cytoskeleton and PTI or ETI, this study followed the responses of actin filaments and microtubules to flg22 and HrpZ in vivo by spinning-disc confocal microscopy in GFP-tagged marker lines of tobacco BY-2. At a concentration that clearly impairs mitosis, flg22 can induce only subtle cytoskeletal responses. In contrast, HrpZ causes a rapid and massive bundling of actin microfilaments (completed in ~20 min, i.e. almost simultaneously with extracellular alkalinization), which is followed by progressive disintegration of actin cables and cytoplasmic microtubules, a loss of cytoplasmic structure, and vacuolar disintegration. Cytoskeletal disruption is proposed as an early event that discriminates HrpZ-triggered ETI-like defence from flg22-triggered PTI.

Models of dynamic extraction of lipid tethers from cell membranes

International Nuclear Information System (INIS)

Nowak, Sarah A; Chou, Tom

2010-01-01

When a ligand that is bound to an integral membrane receptor is pulled, the membrane and the underlying cytoskeleton can deform before either the membrane delaminates from the cytoskeleton or the ligand detaches from the receptor. If the membrane delaminates from the cytoskeleton, it may be further extruded and form a membrane tether. We develop a phenomenological model for this process by assuming that deformations obey Hooke's law up to a critical force at which the cell membrane locally detaches from the cytoskeleton and a membrane tether forms. We compute the probability of tether formation and show that tethers can be extruded only within an intermediate range of force loading rates and pulling velocities. The mean tether length that arises at the moment of ligand detachment is computed as are the force loading rates and pulling velocities that yield the longest tethers

The cytoskeleton as a novel target for treatment of renal fibrosis.

Science.gov (United States)

Parrish, Alan R

2016-10-01

The incidence of chronic kidney disease (CKD) is increasing, with an estimated prevalence of 12% in the United States (Synder et al., 2009). While CKD may progress to end-stage renal disease (ESRD), which necessitates renal replacement therapy, i.e. dialysis or transplantation, most CKD patients never reach ESRD due to the increased risk of death from cardiovascular disease. It is well-established that regardless of the initiating insult - most often diabetes or hypertension - fibrosis is the common pathogenic pathway that leads to progressive injury and organ dysfunction (Eddy, 2014; Duffield, 2014). As such, there has been extensive research into the molecular and cellular mechanisms of renal fibrosis; however, translation to effective therapeutic strategies has been limited. While a role for the disruption of the cytoskeleton, most notably the actin network, has been established in acute kidney injury over the past two decades, a role in regulating renal fibrosis and CKD is only recently emerging. This review will focus on the role of the cytoskeleton in regulating pro-fibrotic pathways in the kidney, as well as data suggesting that these pathways represent novel therapeutic targets to manage fibrosis and ultimately CKD. Copyright © 2016. Published by Elsevier Inc.

Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells

Directory of Open Access Journals (Sweden)

Malgorzata Kloc

2012-10-01

Full Text Available The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
a key regulator of cell cycle, controls actin organization and negatively regulates cell motility via regulation of RhoA
expression. We studied the organization of actin and cytokeratin cytoskeleton and the expression of TCTP, p53,
cyclin A, RhoA and actin in HIO180 non-transformed ovarian epithelial cells, and OVCAR3 and SKOV3 (expressing
low level of inducible p53 ovarian epithelial cancer cells with different metastatic potential. Immunostaining
and ultrastructural analyses illustrated a dramatic difference in the organization of the cytokeratin and actin
filaments in non-transformed versus cancer cell lines. We also determined that there is an inverse relationship between
the level of TCTP/RhoA and actin/p53/cyclin A expression in ovarian cancer cell lines. This previously unidentified
negative relationship between TCTP/RhoA and actin/p53/cyclin A may suggest that this interaction is linked
with the high aggressiveness of ovarian cancers.The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
a key regulator of cell cycle, controls actin organization

[INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

Science.gov (United States)

Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

2015-01-01

To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only

5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

DEFF Research Database (Denmark)

Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

2006-01-01

39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events in the ce......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial...

SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Wu, Feng; Jordan, Ashley; Kluz, Thomas [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States); Shen, Steven [Center for Health Informatics and Bioinformatics, New York University Langone Medical Center, New York, NY 10016 (United States); Sun, Hong; Cartularo, Laura A. [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States); Costa, Max, E-mail: Max.Costa@nyumc.org [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States)

2016-02-15

The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.

SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

International Nuclear Information System (INIS)

Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A.; Costa, Max

2016-01-01

The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.

Imaging and reconstruction of cell cortex structures near the cell surface

Science.gov (United States)

Jin, Luhong; Zhou, Xiaoxu; Xiu, Peng; Luo, Wei; Huang, Yujia; Yu, Feng; Kuang, Cuifang; Sun, Yonghong; Liu, Xu; Xu, Yingke

2017-11-01

Total internal reflection fluorescence microscopy (TIRFM) provides high optical sectioning capability and superb signal-to-noise ratio for imaging of cell cortex structures. The development of multi-angle (MA)-TIRFM permits high axial resolution imaging and reconstruction of cellular structures near the cell surface. Cytoskeleton is composed of a network of filaments, which are important for maintenance of cell function. The high-resolution imaging and quantitative analysis of filament organization would contribute to our understanding of cytoskeleton regulation in cell. Here, we used a custom-developed MA-TIRFM setup, together with stochastic photobleaching and single molecule localization method, to enhance the lateral resolution of TIRFM imaging to about 100 nm. In addition, we proposed novel methods to perform filament segmentation and 3D reconstruction from MA-TIRFM images. Furthermore, we applied these methods to study the 3D localization of cortical actin and microtubule structures in U373 cancer cells. Our results showed that cortical actins localize ∼ 27 nm closer to the plasma membrane when compared with microtubules. We found that treatment of cells with chemotherapy drugs nocodazole and cytochalasin B disassembles cytoskeletal network and induces the reorganization of filaments towards the cell periphery. In summary, this study provides feasible approaches for 3D imaging and analyzing cell surface distribution of cytoskeletal network. Our established microscopy platform and image analysis toolkits would facilitate the study of cytoskeletal network in cells.

The actin homologue MreB organizes the bacterial cell membrane

OpenAIRE

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W.

2014-01-01

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lip...

Analyzing the spatial positioning of nuclei in polynuclear giant cells

International Nuclear Information System (INIS)

Stange, Maike; Hintsche, Marius; Sachse, Kirsten; Gerhardt, Matthias; Beta, Carsten; Valleriani, Angelo

2017-01-01

How cells establish and maintain a well-defined size is a fundamental question of cell biology. Here we investigated to what extent the microtubule cytoskeleton can set a predefined cell size, independent of an enclosing cell membrane. We used electropulse-induced cell fusion to form giant multinuclear cells of the social amoeba Dictyostelium discoideum . Based on dual-color confocal imaging of cells that expressed fluorescent markers for the cell nucleus and the microtubules, we determined the subcellular distributions of nuclei and centrosomes in the giant cells. Our two- and three-dimensional imaging results showed that the positions of nuclei in giant cells do not fall onto a regular lattice. However, a comparison with model predictions for random positioning showed that the subcellular arrangement of nuclei maintains a low but still detectable degree of ordering. This can be explained by the steric requirements of the microtubule cytoskeleton, as confirmed by the effect of a microtubule degrading drug. (paper)

Analyzing the spatial positioning of nuclei in polynuclear giant cells

Science.gov (United States)

Stange, Maike; Hintsche, Marius; Sachse, Kirsten; Gerhardt, Matthias; Valleriani, Angelo; Beta, Carsten

2017-11-01

How cells establish and maintain a well-defined size is a fundamental question of cell biology. Here we investigated to what extent the microtubule cytoskeleton can set a predefined cell size, independent of an enclosing cell membrane. We used electropulse-induced cell fusion to form giant multinuclear cells of the social amoeba Dictyostelium discoideum. Based on dual-color confocal imaging of cells that expressed fluorescent markers for the cell nucleus and the microtubules, we determined the subcellular distributions of nuclei and centrosomes in the giant cells. Our two- and three-dimensional imaging results showed that the positions of nuclei in giant cells do not fall onto a regular lattice. However, a comparison with model predictions for random positioning showed that the subcellular arrangement of nuclei maintains a low but still detectable degree of ordering. This can be explained by the steric requirements of the microtubule cytoskeleton, as confirmed by the effect of a microtubule degrading drug.

Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle.

Science.gov (United States)

Rader, Erik P; Turk, Rolf; Willer, Tobias; Beltrán, Daniel; Inamori, Kei-Ichiro; Peterson, Taylor A; Engle, Jeffrey; Prouty, Sally; Matsumura, Kiichiro; Saito, Fumiaki; Anderson, Mary E; Campbell, Kevin P

2016-09-27

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

La3+ uptake and its effect on the cytoskeleton in root protoplasts of Zea mays L.

Science.gov (United States)

Liu, Min; Hasenstein, Karl H

2005-03-01

La(3+) ions are known to antagonize Ca(2+) and are used as a Ca(2+) channel blocker but little is known on the direct effects of La(3+). Micromolar La(3+) concentrations promoted root growth while higher concentrations were inhibitory. The uptake of La(3+) in maize root protoplasts revealed a membrane binding component (0.14 and 0.44 pmol min(-1) protoplast(-1) for 100 and 1,000 microM La(3+)) followed by a slower concentration and time-dependent uptake. Uptake was reduced by Ca(2+), but had no substantial effect on other ions. La(3+) shifted microtubule organization from random to parallel but caused aggregation of microfilaments. Our data suggest that La(3+) is taken up into plant cells and affects growth via stabilization of the cytoskeleton.

Cell elasticity with altered cytoskeletal architectures across multiple cell types.

Science.gov (United States)

Grady, Martha E; Composto, Russell J; Eckmann, David M

2016-08-01

The cytoskeleton is primarily responsible for providing structural support, localization and transport of organelles, and intracellular trafficking. The structural support is supplied by actin filaments, microtubules, and intermediate filaments, which contribute to overall cell elasticity to varying degrees. We evaluate cell elasticity in five different cell types with drug-induced cytoskeletal derangements to probe how actin filaments and microtubules contribute to cell elasticity and whether it is conserved across cell type. Specifically, we measure elastic stiffness in primary chondrocytes, fibroblasts, endothelial cells (HUVEC), hepatocellular carcinoma cells (HUH-7), and fibrosarcoma cells (HT 1080) subjected to two cytoskeletal destabilizers: cytochalasin D and nocodazole, which disrupt actin and microtubule polymerization, respectively. Elastic stiffness is measured by atomic force microscopy (AFM) and the disruption of the cytoskeleton is confirmed using fluorescence microscopy. The two cancer cell lines showed significantly reduced elastic moduli values (~0.5kPa) when compared to the three healthy cell lines (~2kPa). Non-cancer cells whose actin filaments were disrupted using cytochalasin D showed a decrease of 60-80% in moduli values compared to untreated cells of the same origin, whereas the nocodazole-treated cells showed no change in elasticity. Overall, we demonstrate actin filaments contribute more to elastic stiffness than microtubules but this result is cell type dependent. Cancer cells behaved differently, exhibiting increased stiffness as well as stiffness variability when subjected to nocodazole. We show that disruption of microtubule dynamics affects cancer cell elasticity, suggesting therapeutic drugs targeting microtubules be monitored for significant elastic changes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines.

Science.gov (United States)

Emadi Baygi, Modjtaba; Soheili, Zahra-Soheila; Essmann, Frank; Deezagi, Abdolkhaleg; Engers, Rainer; Goering, Wolfgang; Schulz, Wolfgang A

2010-08-01

Many metastatic cancers recapitulate the epithelial-to-mesenchymal transition (EMT) resulting in enhanced cell motility and invasiveness. The EMT is regulated by several transcription factors, including the zinc finger protein SNAI2, also named Slug, which appears to exert additional functions during development and cancer progression. We have studied the function of SNAI2 in prostate cancer cells. Quantitative RT-PCR analysis showed strong SNAI2 expression particularly in the PC-3 and PC3-16 prostate carcinoma cell lines. Knockdown of SNAI2 by specific siRNA induced changes in EMT markers and inhibited invasion of both cell lines into a matrigel matrix. SNAI2 siRNA-treated cells did not tolerate detachment from the culture plates, likely at least in part due to downregulation of integrin alpha6beta4. SNAI2 knockdown disturbed the microtubular and actin cytoskeletons, especially severely in PC-3 cells, resulting in grossly enlarged, flattened, and sometimes multinuclear cells. Knockdown also decreased cell proliferation, with a prominent G0/G1 arrest in PC3-16. Together, our data imply that SNAI2 exerts strong effects on the cytoskeleton and adhesion of those prostate cancer cells that express it and is necessary for their proliferation and invasiveness.

Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light.

Science.gov (United States)

Rascalou, Adeline; Lamartine, Jérôme; Poydenot, Pauline; Demarne, Frédéric; Bechetoille, Nicolas

2018-05-05

Artificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. The purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. The spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. We developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450 nm, 525 nm and 625 nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99 J/cm 2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the "mitochondria" and "integrin signaling" categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. Artificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede

Metamorphosis of Magnetospirillum magneticum AMB-1 cells

Science.gov (United States)

Zhang, Fengli; Yu-Zhang, Kui; Zhao, Sanjun; Xiao, Tian; Denis, Michel; Wu, Longfei

2010-03-01

Magnetospirillum magneticum strain AMB-1 belongs to the family of magnetotactic bacteria. It possesses a magnetosome chain aligning, with the assistance of cytoskeleton filaments MamK, along the long axis of the spiral cells. Most fresh M. magneticum AMB-1 cells exhibit spiral morphology. In addition, other cell shapes such as curved and spherical were also observed in this organism. Interestingly, the spherical cell shape increased steadily with prolonged incubation time. As the actin-like cytoskeleton protein MreB is involved in maintenance of cell shapes in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, the correlation between MreB protein levels and cell shape was investigated in this study. Immunoblotting analysis showed that the quantity of MreB decreased when the cell shape changed along with incubation time. As an internal control, the quantity of MamA was not obviously changed under the same conditions. Cell shape directs cell-wall synthesis during growth and division. MreB is required for maintaining the cell shape. Thus, MreB might play an essential role in maintaining the spiral shape of M. magneticum AMB-1 cells.

Cytoskeleton in gravisensing and signal transductionof lower plants

Science.gov (United States)

Braun, M.

Characean rhizoids and protonemata are favourable cell types for studying tip growth and gravisensing. Both processes are highly dependent on the actin cytoskeleton. The multiple functions and different arrangements of actin in both cell types are regulated by the concerted action of actin-binding proteins. Monomer- binding profilin is distributed evenly throughout the cytoplasm and is likely to be involved in the regulation of the polymerization state of actin. Actin-severing ADF, spectrin- and actinin-like epitopes concentrate in a central prominent spot in the apex of both cell types, where they colocalize with a dense, spherical actin array and a unique aggregation of endoplasmic reticulum (ER), the structural center of the tip - growth organizing Spitzenkörper. The ER aggregate disintegrates and immuno- localization of the actin-binding proteins fails when tip growth is arrested; the epitopes reappear when tip growth resumes. Actin filaments form a meshwork of axially oriented filaments in the subapical zone and focus in this central apical area which seems to represent their apical polymerization site. The rapid turn-over and rearrangement of actin might be under control of ADF and profilin. Spectrin- and actinin-like proteins are candidates for establishing the actin-mediated anchoring and maintaining of the ER aggregate. They could also provide a mechanism for recruiting specific membrane proteins that create the particular physiological environment for gravity-oriented tip growth. The positioning and sedimentation of statoliths in the subapical region (crucial for gravisensing) is highly coordinated by actomyosin. Non-invasive infrared laser micromanipulation techniques, centri- fugation and experiments in microgravity revealed that reorientation of the growth direction was initiated when at least 2-3 statoliths were directed to specific areas of the plasma membrane by actomyosin and gravitational forces. The statolith-sensitive area is confined to the

Espins and the actin cytoskeleton of hair cell stereocilia and sensory cell microvilli

Science.gov (United States)

Sekerková, Gabriella; Zheng, Lili; Loomis, Patricia A.; Mugnaini, Enrico; Bartles, James R.

2008-01-01

The espins are novel actin-bundling proteins that are produced in multiple isoforms from a single gene. They are present at high concentration in the parallel actin bundle of hair cell stereocilia and are the target of deafness mutations in mice and humans. Espins are also enriched in the microvilli of taste receptor cells, solitary chemoreceptor cells, vomeronasal sensory neurons and Merkel cells, suggesting that espins play important roles in the microvillar projections of vertebrate sensory cells. Espins are potent actin-bundling proteins that are not inhibited by Ca2+. In cells, they efficiently elongate parallel actin bundles and, thereby, help determine the steady-state length of microvilli and stereocilia. Espins bind actin monomer via their WH2 domain and can assemble actin bundles in cells. Certain espin isoforms can also bind phosphatidylinositol 4,5-bisphosphate, profilins or SH3 proteins. These biological activities distinguish espins from other actin-bundling proteins and may make them well-suited to sensory cells. PMID:16909209

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms.

NARCIS (Netherlands)

Schmidt, S.; Friedl, P.H.A.

2010-01-01

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In

A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton

Directory of Open Access Journals (Sweden)

Sarah M. Carpanini

2014-06-01

Full Text Available Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM. Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic

A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton.

Science.gov (United States)

Carpanini, Sarah M; McKie, Lisa; Thomson, Derek; Wright, Ann K; Gordon, Sarah L; Roche, Sarah L; Handley, Mark T; Morrison, Harris; Brownstein, David; Wishart, Thomas M; Cousin, Michael A; Gillingwater, Thomas H; Aligianis, Irene A; Jackson, Ian J

2014-06-01

Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions. �

The costa of trichomonads: A complex macromolecular cytoskeleton structure made of uncommon proteins.

Science.gov (United States)

de Andrade Rosa, Ivone; Caruso, Marjolly Brigido; de Oliveira Santos, Eidy; Gonzaga, Luiz; Zingali, Russolina Benedeta; de Vasconcelos, Ana Tereza R; de Souza, Wanderley; Benchimol, Marlene

2017-06-01

The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the

Acetylated tau destabilizes the cytoskeleton in the axon initial segment and is mislocalized to the somatodendritic compartment.

Science.gov (United States)

Sohn, Peter Dongmin; Tracy, Tara E; Son, Hye-In; Zhou, Yungui; Leite, Renata E P; Miller, Bruce L; Seeley, William W; Grinberg, Lea T; Gan, Li

2016-06-29

Neurons are highly polarized cells in which asymmetric axonal-dendritic distribution of proteins is crucial for neuronal function. Loss of polarized distribution of the axonal protein tau is an early sign of Alzheimer's disease (AD) and other neurodegenerative disorders. The cytoskeletal network in the axon initial segment (AIS) forms a barrier between the axon and the somatodentritic compartment, contributing to axonal retention of tau. Although perturbation of the AIS cytoskeleton has been implicated in neurological disorders, the molecular triggers and functional consequence of AIS perturbation are incompletely understood. Here we report that tau acetylation and consequent destabilization of the AIS cytoskeleton promote the somatodendritic mislocalization of tau. AIS cytoskeletal proteins, including ankyrin G and βIV-spectrin, were downregulated in AD brains and negatively correlated with an increase in tau acetylated at K274 and K281. AIS proteins were also diminished in transgenic mice expressing tauK274/281Q, a tau mutant that mimics K274 and K281 acetylation. In primary neuronal cultures, the tauK274/281Q mutant caused hyperdynamic microtubules (MTs) in the AIS, shown by live-imaging of MT mobility and fluorescence recovery after photobleaching. Using photoconvertible tau constructs, we found that axonal tauK274/281Q was missorted into the somatodendritic compartment. Stabilizing MTs with epothilone D to restore the cytoskeletal barrier in the AIS prevented tau mislocalization in primary neuronal cultures. Together, these findings demonstrate that tau acetylation contributes to the pathogenesis of neurodegenerative disease by compromising the cytoskeletal sorting machinery in the AIS.

Letrozole regulates actin cytoskeleton polymerization dynamics in a SRC-1 dependent manner in the hippocampus of mice.

Science.gov (United States)

Zhao, Yangang; Yu, Yanlan; Zhang, Yuanyuan; He, Li; Qiu, Linli; Zhao, Jikai; Liu, Mengying; Zhang, Jiqiang

2017-03-01

In the hippocampus, local estrogens (E 2 ) derived from testosterone that is catalyzed by aromatase play important roles in the regulation of hippocampal neural plasticity, but the underlying mechanisms remain unclear. The actin cytoskeleton contributes greatly to hippocampal synaptic plasticity; however, whether it is regulated by local E 2 and the related mechanisms remain to be elucidated. In this study, we first examined the postnatal developmental profiles of hippocampal aromatase and specific proteins responsible for actin cytoskeleton dynamics. Then we used aromatase inhibitor letrozole (LET) to block local E 2 synthesis and examined the changes of these proteins and steroid receptor coactivator-1 (SRC-1), the predominant coactivator for steroid nuclear receptors. Finally, SRC-1 specific RNA interference was used to examine the effects of SRC-1 on the expression of these actin remodeling proteins. The results showed a V-type profile for aromatase and increased profiles for actin cytoskeleton proteins in both male and female hippocampus without obvious sex differences. LET treatment dramatically decreased the F-actin/G-actin ratio, the expression of Rictor, phospho-AKT (ser473), Profilin-1, phospho-Cofilin (Ser3), and SRC-1 in a dose-dependent manner. In vitro studies demonstrated that LET induced downregulation of these proteins could be reversed by E 2 , and E 2 induced increase of these proteins were significantly suppressed by SRC-1 shRNA interference. These results for the first time clearly demonstrated that local E 2 inhibition could induce aberrant actin polymerization; they also showed an important role of SRC-1 in the mediation of local E 2 action on hippocampal synaptic plasticity by regulation of actin cytoskeleton dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

2017-12-01

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

Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation

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Roa-Espitia, Ana L.; Hernández-Rendón, Eva R.; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J.; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto

2016-01-01

ABSTRACT Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca2+ dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

Membrane elastic properties and cell function.

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Bruno Pontes

Full Text Available Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.

Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

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Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

2014-02-01

In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

Biophysical Assessment of Single Cell Cytotoxicity: Diesel Exhaust Particle-Treated Human Aortic Endothelial Cells

OpenAIRE

Wu, Yangzhe; Yu, Tian; Gilbertson, Timothy A.; Zhou, Anhong; Xu, Hao; Nguyen, Kytai Truong

2012-01-01

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model ...

Unraveling cell processes: interference imaging interwoven with data analysis

DEFF Research Database (Denmark)

Brazhe, Nadezda; Brazhe, Alexey; Pavlov, A N

2006-01-01

The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglo......The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution...... properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1-0.6 Hz) result from plasma membrane processes and that higher frequency variations (20-26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we...... study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation...

Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa.

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Cowles, Kimberly N; Gitai, Zemer

2010-06-01

Spatial organization of bacterial proteins influences many cellular processes, including division, chromosome segregation and motility. Virulence-associated proteins also localize to specific destinations within bacterial cells. However, the functions and mechanisms of virulence factor localization remain largely unknown. In this work, we demonstrate that polar assembly of the Pseudomonas aeruginosa PAO1 type IV pilus is regulated by surface association in a manner that affects gene transcription, protein levels and protein localization. We also uncover one mechanism for this regulation that acts through the actin homologue MreB. Inactivation of MreB leads to mislocalization of the pilus retraction ATPase PilT, mislocalization of the pili themselves and a reduction in motility. Furthermore, the role of MreB in polar localization of PilT is modulated by surface association, corroborating our results that environmental factors influence the regulation of pilus production. Specifically, MreB mediates both the initiation and maintenance of PilT localization when cells are grown in suspension but only affects the initiation of localization when cells are grown on a surface. Together, these results suggest that the bacterial cytoskeleton provides a mechanism for the polar localization of P. aeruginosa pili and demonstrate that protein localization may represent an important aspect of virulence factor regulation in bacterial pathogens.

Cytoskeleton reorganization/disorganization is a key feature of induced inaccessibility for defence to successive pathogen attacks.

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Moral, Juan; Montilla-Bascón, Gracia; Canales, Francisco J; Rubiales, Diego; Prats, Elena

2017-06-01

In this work, we investigated the involvement of the long-term dynamics of cytoskeletal reorganization on the induced inaccessibility phenomenon by which cells that successfully defend against a previous fungal attack become highly resistant to subsequent attacks. This was performed on pea through double inoculation experiments using inappropriate (Blumeria graminis f. sp. avenae, Bga) and appropriate (Erysiphe pisi, Ep) powdery mildew fungi. Pea leaves previously inoculated with Bga showed a significant reduction of later Ep infection relative to leaves inoculated only with Ep, indicating that cells had developed induced inaccessibility. This reduction in Ep infection was higher when the time interval between Bga and Ep inoculation ranged between 18 and 24 h, although increased penetration resistance in co-infected cells was observed even with time intervals of 24 days between inoculations. Interestingly, this increase in resistance to Ep following successful defence to the inappropriate Bga was associated with an increase in actin microfilament density that reached a maximum at 18-24 h after Bga inoculation and very slowly decreased afterwards. The putative role of cytoskeleton reorganization/disorganization leading to inaccessibility is supported by the suppression of the induced resistance mediated by specific actin (cytochalasin D, latrunculin B) or general protein (cycloheximide) inhibitors. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Morphological changes of nuclear and chromatin architecture after microwave electromagnetic field exposure in 3T3 fibroblast cell cultures

International Nuclear Information System (INIS)

Mircea, D.; Chirila, Lavinia; Ciurea, A. V.; Helm, G.; Hankins, G.; Redrick, Jan; Gavrila, L.; Sheppard, B.; Bloodgoog, R.; Pallin, I.; Nitu, Rozalia; Rusu, I.

2001-01-01

It is already demonstrated in the literature that electromagnetic fields, particularly the microwave irradiation could be a powerful weapon against human tumors , but also against human body itself, depending on the wave parameters and irradiation time. The effects of microwave electromagnetic fields on living systems were studied in detail all over the world and, furthermore, the potential of intracellular damages by cytoskeleton, nuclear, chromatin and DNA alterations were carefully evaluated. In this study, the authors emphasize the morphological changes of nucleus and chromatin in fibroblast cell line 3T3 after microwave exposure with progressive increasing powers and times of irradiation. It was used a pulsed wave with 915 MHz frequency, with forward power ranging between 3 - 10 W, emitted by a helical microwave antenna placed into the cell culture medium, close to the cell monolayer. The authors tried to define certain severity stages of nuclear material alterations following different wave intensities and to compare these effects with other cytoplasmic organelle alterations. It was found that the nuclear material is the most sensitive intracellular structure in microwave electromagnetic field exposure. Also the authors tried to establish a well-defined protocol of irradiation with microwave electromagnetic fields in order to destroy the microtubule system of cytoskeleton in different types of cellular lines, in vitro. The cytoskeleton structure was evaluated by immunofluorescence methods. In non-muscle cells the cytoskeleton stability is achieved by interaction between microtubule system and actin filaments. Microtubule depolymerization by microwave exposure produces a secondary instability of cytoskeleton, the actin filaments coupling and cell contractility. The increasing of fibroblast contractility allows a more efficient treatment of the wounds with low spontaneous healing. Electromagnetic therapy could be an alternative therapy in plastic surgery

Cytoskeleton and nuclear lamina affection in recessive osteogenesis imperfecta: A functional proteomics perspective.

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Gagliardi, Assunta; Besio, Roberta; Carnemolla, Chiara; Landi, Claudia; Armini, Alessandro; Aglan, Mona; Otaify, Ghada; Temtamy, Samia A; Forlino, Antonella; Bini, Luca; Bianchi, Laura

2017-09-07

Osteogenesis imperfecta (OI) is a collagen-related disorder associated to dominant, recessive or X-linked transmission, mainly caused by mutations in type I collagen genes or in genes involved in type I collagen metabolism. Among the recessive forms, OI types VII, VIII, and IX are due to mutations in CRTAP, P3H1, and PPIB genes, respectively. They code for the three components of the endoplasmic reticulum complex that catalyzes 3-hydroxylation of type I collagen α1Pro986. Under-hydroxylation of this residue leads to collagen structural abnormalities and results in moderate to lethal OI phenotype, despite the exact molecular mechanisms are still not completely clear. To shed light on these recessive forms, primary fibroblasts from OI patients with mutations in CRTAP (n=3), P3H1 (n=3), PPIB (n=1) genes and from controls (n=4) were investigated by a functional proteomic approach. Cytoskeleton and nucleoskeleton asset, protein fate, and metabolism were delineated as mainly affected. While western blot experiments confirmed altered expression of lamin A/C and cofilin-1, immunofluorescence analysis using antibody against lamin A/C and phalloidin showed an aberrant organization of nucleus and cytoskeleton. This is the first report describing an altered organization of intracellular structural proteins in recessive OI and pointing them as possible novel target for OI treatment. OI is a prototype for skeletal dysplasias. It is a highly heterogeneous collagen-related disorder with dominant, recessive and X-linked transmission. There is no definitive cure for this disease, thus a better understanding of the molecular basis of its pathophysiology is expected to contribute in identifying potential targets to develop new treatments. Based on this concept, we performed a functional proteomic study to delineate affected molecular pathways in primary fibroblasts from recessive OI patients, carrying mutations in CRTAP (OI type VII), P3H1 (OI type VIII), and PPIB (OI type IX) genes

Elasticity and tumorigenic characteristics of cells in a monolayer after nanosecond pulsed electric field exposure.

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Steuer, A; Wende, K; Babica, P; Kolb, J F

2017-09-01

Nanosecond pulsed electric fields (nsPEFs) applied to cells can induce different biological effects depending on pulse duration and field strength. One known process is the induction of apoptosis whereby nsPEFs are currently investigated as a novel cancer therapy. Another and probably related change is the breakdown of the cytoskeleton. We investigated the elasticity of rat liver epithelial cells WB-F344 in a monolayer using atomic force microscopy (AFM) with respect to the potential of cells to undergo malignant transformation or to develop a potential to metastasize. We found that the elastic modulus of the cells decreased significantly within the first 8 min after treatment with 20 pulses of 100 ns and with a field strength of 20 kV/cm but was still higher than the elasticity of their tumorigenic counterpart WB-ras. AFM measurements and immunofluorescent staining showed that the cellular actin cytoskeleton became reorganized within 5 min. However, both a colony formation assay and a cell migration assay revealed no significant changes after nsPEF treatment, implying that cells seem not to adopt malignant characteristics associated with metastasis formation despite the induced transient changes to elasticity and cytoskeleton that can be observed for up to 1 h.

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

Marginal Iodine Deficiency Affects Dendritic Spine Development by Disturbing the Function of Rac1 Signaling Pathway on Cytoskeleton.

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Min, Hui; Dong, Jing; Wang, Yi; Wang, Yuan; Yu, Ye; Shan, Zhongyan; Xi, Qi; Teng, Weiping; Chen, Jie

2017-01-01

Iodine deficiency (ID)-induced thyroid hormone (TH) insufficient during development leads to impairments of brain function, such as learning and memory. Marginal ID has been defined as subtle insufficiency of TH, characterized as low thyroxine (T 4 ) levels, whether marginal ID potentially had adverse effects on the development of hippocampus and the underlying mechanisms remain unclear. Thus, in the present study, we established Wistar rat models with ID diet during pregnancy and lactation. The effects of marginal ID on long-term potentiation (LTP) were investigated in the hippocampal CA1 region. To study the development of dendritic spines in pyramidal cells, Golgi-Cox staining was conducted on postnatal day (PN) 7, PN14, PN21, and PN28. The activation of Rac1 signaling pathway, which is essential for dendritic spine development by regulating actin cytoskeleton, was also investigated. Our results showed that marginal ID slightly reduced the field-excitatory postsynaptic potential (f-EPSP) slope and the population spike (PS) amplitude. Besides, the density of dendritic spines during the critical period of rat postnatal development was mildly decreased, and we found no significant change of spine morphology in marginal ID group. We also observed decreased activation of the Rac1 signaling pathway in pups subjected to maternal marginal ID. Our study may support the hypothesis that decreased T 4 induced by marginal ID results in slight impairments of LTP and leads to mild damage of dendritic spine development, which may be due to abnormal regulation of Rac1 signaling pathway on cytoskeleton.

The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary.

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Lin, Tzu-Huai; Yeh, Tsung-Han; Wang, Tsu-Wei; Yu, Jenn-Yah

2014-11-01

The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator, Yorkie (Yki). Yes-associated protein (YAP, a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. Here, we use border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. We find that hpo and wts are required for migration of the border cell cluster. In outer border cells, overexpression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo and wts or overexpression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, our data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: (1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; (2) in polar cells, it regulates upd expression to control border cell induction and migration. Copyright © 2014 by the Genetics Society of America.

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

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Yukihiro Nishikawa

Full Text Available Withaferin A (WA, a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD. WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L. Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

Fluctuations of cytoskeleton-bound microbeads-the effect of bead-receptor binding dynamics

International Nuclear Information System (INIS)

Metzner, C; Raupach, C; Mierke, C T; Fabry, B

2010-01-01

The cytoskeleton (CSK) of living cells is a crosslinked fiber network, subject to ongoing biochemical remodeling processes that can be visualized by tracking the spontaneous motion of CSK-bound microbeads. The bead motion is characterized by anomalous diffusion with a power-law time evolution of the mean square displacement (MSD), and can be described as a stochastic transport process with apparent diffusivity D and power-law exponent β: MSD ∼ D (t/t 0 ) β . Here we studied whether D and β change with the time that has passed after the initial bead-cell contact, and whether they are sensitive to bead coating (fibronectin, integrin antibodies, poly-L-lysine, albumin) and bead size (0.5-4.5 μm). The measurements are interpreted in the framework of a simple model that describes the bead as an overdamped particle coupled to the fluctuating CSK network by an elastic spring. The viscous damping coefficient characterizes the degree of bead internalization into the cell, and the spring constant characterizes the strength of the binding of the bead to the CSK. The model predicts distinctive signatures of the MSD that change with time as the bead couples more tightly to the CSK and becomes internalized. Experimental data show that the transition from the unbound to the tightly bound state occurs in an all-or-nothing manner. The time point of this transition shows considerable variability between individual cells (2-30 min) and depends on the bead size and bead coating. On average, this transition occurs later for smaller beads and beads coated with ligands that trigger the formation of adhesion complexes (fibronectin, integrin antibodies). Once the bead is linked to the CSK, however, the ligand type and bead size have little effect on the MSD. On longer timescales of several hours after bead addition, smaller beads are internalized into the cell more readily, leading to characteristic changes in the MSD that are consistent with increased viscous damping by the

Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

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Alleaume-Butaux A

2013-07-01

Full Text Available Aurélie Alleaume-Butaux,1,2 Caroline Dakowski,1,2 Mathéa Pietri,1,2 Sophie Mouillet-Richard,1,2 Jean-Marie Launay,3,4 Odile Kellermann,1,2 Benoit Schneider1,2 1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris Cité, UMR-S 747, 3Public Hospital of Paris, Department of Biochemistry, INSERM UMR-S 942, Lariboisière Hospital, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland Abstract: Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps: (1 neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma; (2 neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and (3 the stability and plasticity of neuronal polarity. In neuronal stem cells, remodeling and activation of focal adhesions (FAs associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42, which are involved in actin turnover and the dynamics of FAs. The cellular prion protein (PrPC, a glycosylphosphatidylinositol (GPI-anchored membrane protein mainly known for its role in a group of fatal neurodegenerative diseases, has emerged as a central player in neuritogenesis. Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the signaling pathways fine-tuned by PrPC to promote neurite sprouting, outgrowth, and maintenance. We emphasize that Pr

Extracellular matrix-dependent myosin dynamics during G1-S phase cell cycle progression in hepatocytes

International Nuclear Information System (INIS)

Bhadriraju, Kiran; Hansen, Linda K.

2004-01-01

Cell spreading and proliferation are tightly coupled in anchorage-dependent cells. While adhesion-dependent proliferation signals require an intact actin cytoskeleton, and some of these signals such as ERK activation have been characterized, the role of myosin in spreading and cell cycle progression under different extracellular matrix (ECM) conditions is not known. Studies presented here examine changes in myosin activity in freshly isolated hepatocytes under ECM conditions that promote either proliferation (high fibronectin density) or growth arrest (low fibronectin density). Three different measures were obtained and related to both spreading and cell cycle progression: myosin protein levels and association with cytoskeleton, myosin light chain phosphorylation, and its ATPase activity. During the first 48 h in culture, corresponding with transit through G1 phase, there was a six-fold increase in both myosin protein levels and myosin association with actin cytoskeleton. There was also a steady increase in myosin light chain phosphorylation and ATPase activity with spreading, which did not occur in non-spread, growth-arrested cells on low density of fibronectin. Myosin-inhibiting drugs blocked ERK activation, cyclin D1 expression, and S phase entry. Overexpression of the cell cycle protein cyclin D1 overcame both ECM-dependent and actomyosin-dependent inhibition of DNA synthesis, suggesting that cyclin D1 is a key event downstream of myosin-dependent cell cycle regulation

Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells

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Kaur Punit

2008-11-01

Full Text Available Abstract Background There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Methods Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Results Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. Conclusion The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.

Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells.

Science.gov (United States)

Urueña, Claudia; Cifuentes, Claudia; Castañeda, Diana; Arango, Amparo; Kaur, Punit; Asea, Alexzander; Fiorentino, Susana

2008-11-18

There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.

GAR22β regulates cell migration, sperm motility, and axoneme structure.

Science.gov (United States)

Gamper, Ivonne; Fleck, David; Barlin, Meltem; Spehr, Marc; El Sayad, Sara; Kleine, Henning; Maxeiner, Sebastian; Schalla, Carmen; Aydin, Gülcan; Hoss, Mareike; Litchfield, David W; Lüscher, Bernhard; Zenke, Martin; Sechi, Antonio

2016-01-15

Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β(-/-) Sertoli cells moved faster than wild-type cells. In addition, GAR22β(-/-) cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β(-/-) cells reduced cell motility and focal adhesion turnover. GAR22β-actin interaction was stronger than GAR22β-microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β-EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes. © 2016 Gamper et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Evidence for tension-based regulation of Drosophila MAL and SRF during invasive cell migration.

Science.gov (United States)

Somogyi, Kálmán; Rørth, Pernille

2004-07-01

Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. We report that this migration requires the activity of the transcriptional factor serum response factor (SRF) and its cofactor MAL-D and present evidence that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration.

A nucleation theory of cell surface capping

International Nuclear Information System (INIS)

Coutsias, E.A.; Wester, M.J.; Perelson, A.S.

1997-01-01

We propose a new theory of cell surface capping based on the principles of nucleation. When antibody interacts with cell surface molecules, the molecules initially form small aggregates called patches that later coalesce into a large aggregate called a cap. While a cap can form by patches being pulled together by action of the cell''s cytoskeleton, in the case of some molecules, disruption of the cytoskeleton does not prevent cap formation. Diffusion of large aggregates on a cell surface is slow, and thus we propose that a cap can form solely through the diffusion of small aggregates containing just one or a few cell surface molecules. Here we consider the extreme case in which single molecules are mobile, but aggregates of all larger sizes are immobile. We show that a set of patches in equilibrium with a open-quotes seaclose quotes of free cell surface molecules can undergo a nucleation-type phase transition in which the largest patch will bind free cell surface molecules, deplete the concentration of such molecules in the open-quotes seaclose quotes and thus cause the other patches to shrink in size. We therefore show that a cap can form without patches having to move, collide with each other, and aggregate

Cell Chirality Drives Left-Right Asymmetric Morphogenesis.

Science.gov (United States)

Inaki, Mikiko; Sasamura, Takeshi; Matsuno, Kenji

2018-01-01

Most macromolecules found in cells are chiral, meaning that they cannot be superimposed onto their mirror image. However, cells themselves can also be chiral, a subject that has received little attention until very recently. In our studies on the mechanisms of left-right (LR) asymmetric development in Drosophila , we discovered that cells can have an intrinsic chirality to their structure, and that this "cell chirality" is generally responsible for the LR asymmetric development of certain organs in this species. The actin cytoskeleton plays important roles in the formation of cell chirality. In addition, Myosin31DF ( Myo31DF ), which encodes Drosophila Myosin ID, was identified as a molecular switch for cell chirality. In other invertebrate species, including snails and Caenorhabditis elegans , chirality of the blastomeres, another type of cell chirality, determines the LR asymmetry of structures in the body. Thus, chirality at the cellular level may broadly contribute to LR asymmetric development in various invertebrate species. Recently, cell chirality was also reported for various vertebrate cultured cells, and studies suggested that cell chirality is evolutionarily conserved, including the essential role of the actin cytoskeleton. Although the biological roles of cell chirality in vertebrates remain unknown, it may control LR asymmetric development or other morphogenetic events. The investigation of cell chirality has just begun, and this new field should provide valuable new insights in biology and medicine.