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Cell Motility (cell + motility)
Kinds of Cell Motility Selected AbstractsCell Motility and the Cytoskeleton in transitionCYTOSKELETON, Issue 8 2009B.R. Brinkley Editor-in-Chief No abstract is available for this article. [source] Mechanical Gradient Cues for Guided Cell Motility and Control of Cell Behavior on Uniform SubstratesADVANCED FUNCTIONAL MATERIALS, Issue 18 2009Barbara Cortese Abstract A novel method for the fabrication and the use of simple uniform poly(dimethylsiloxane) PDMS substrates for controlling cell motility by a mechanical gradient is reported. The substrate is fabricated in PDMS using soft lithography and consists of a soft membrane suspended on top of a patterned PDMS substrate. The difference in the gradient stiffness is related to the underlying pattern. It is shown experimentally that these uniform substrates can modulate the response of cell motility, thus enabling patterning on the surfaces with precise cell motility. Because of the uniformity of the substrate, cells can spread equally and a directional movement to stiffer regions is clearly observed. Varying the geometry underlying the membrane, cell patterning and movement can be quantitatively characterized. This procedure is capable of controlling cell motility with high fidelity over large substrate areas. The most significant advance embodied in this method is that it offers the use of mechanical features to control cell adhesion and not topographical or chemical variations, which has not been reported so far. This modulation of the response of cell motility will be useful for the design and fabrication of advanced planar and 3D biological assemblies suitable for applications in the field of biotechnology and for tissue-engineering purposes. [source] Undirected motility of filamentous cyanobacteria produces reticulate matsGEOBIOLOGY, Issue 3 2010R. N. SHEPARD The roles of biology in the morphogenesis of microbial mats and stromatolites remain enigmatic due to the vast array of physical and chemical influences on morphology. However, certain microbial behaviors produce complex morphological features that can be directly attributed to motility patterns. Specifically, laboratory experiments with a strain of the cyanobacteria Pseudanabaena demonstrate that distinctive morphologies arise from the undirected gliding and colliding of filaments. When filamentous cells collide, they align and clump, producing intersecting ridges surrounding areas with low cell density, i.e. reticulate structures. Cell motility is essential for the development of reticulates and associated structures: filaments organize into reticulates faster than cell division and growth, and conditions that inhibit motility also inhibit reticulate formation. Cell density of the inoculum affects the frequency of cell,cell collisions, and thus the time required for biofilm organization into reticulate structures. This also affects the specific geometry of the reticulates. These patterns are propagated into larger structures as cyanobacterial cell numbers increase and cells remain motile. Thus, cell motility is important for templating and maintaining the morphology of these microbial communities, demonstrating a direct link between a microbial behavior and a community morphology. Reticulate geometries have been identified in natural microbial mats as well as in the fossil record, and these structures can be attributed to the motility of filamentous bacteria. [source] Hepatocyte growth factor stimulates cell motility in cultures of the striatal progenitor cells ST14AJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2003E. Cacci Abstract Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the developing and adult mammalian brain and control neuritogenesis of sympathetic and sensory neurons. We report that the striatal progenitor ST14A cells express the Met receptor, which is activated after binding with HGF/SF. The interaction between Met and HGF/SF triggers a signaling cascade that leads to increased levels of c-Jun, c-Fos, and Egr-1 proteins, in agreement with data reported on the signaling events evoked by HGF in other cellular types. We also studied the effects of the exposure of ST14A cells to HGF/SF. By time-lapse photography, we observed that a 24-hr treatment with 50 ng/ml HGF/SF induced modification in cell morphology, with a decrease in cell-cell interactions and increase of cell motility. In contrast, no effect on cell proliferation was observed. To investigate which intracellular pathway is primarily involved we used PD98059 and LY294002, two specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAP-kinase/ERK-kinase) and phosphoinositide 3-OH kinase (PI3-K), respectively. Cell motility in HGF/SF treated cultures was inhibited by LY294002 but not by PD98059, suggesting that PI3-K plays a key role in mediating the HGF/SF-induced dissociation of ST14A cells. Previous evidence of HGF stimulation of motility in nervous system has been obtained on postmitotic neurons, which have already acquired their specificity. Data reported here of a motogenic response of ST14A cell line, which displays properties of neuronal progenitors, seem of interest because they suggest that HGF could play a role in very early steps of neurogenesis. © 2003 Wiley-Liss, Inc. [source] 3- O -Methylfunicone, a metabolite produced by Penicillium pinophilum, modulates ERK1/2 activity, affecting cell motility of human mesothelioma cellsCELL PROLIFERATION, Issue 2 2010E. Buommino Objectives:, 3- O -methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, affects cell proliferation and motility in a variety of human solid tumours. The aim of this study was to demonstrate whether OMF has the ability to arrest cell division and motility, in a human mesothelioma cell line. Malignant mesothelioma is an aggressive cancer that does not respond to standard therapies the cells of which are considered to be highly resistant to apoptosis. Material and methods:, Cell motility and invasion were measured using a modified Boyden chamber. Gene expression was examined by RT-PCR, while ERK1/2 was investigated by Western blot analysis. All experiments were also performed on primary cultures of mesothelial cells. Results:, The present study shows that OMF inhibited motility of the NCI mesothelioma cell line by modulating ERK signalling activity, and affected ,V,5 integrin and MMP-2 expression, inducing marked downregulation at both mRNA and protein levels. Substantial downregulation of VEGF gene expression was also demonstrated. These effects were not observed in normal mesothelial cell cultures. Conclusion:, OMF may have potential as a naturally derived anti-tumour drug for treatment of mesothelioma. [source] Novel interactors and a role for supervillin in early cytokinesis,CYTOSKELETON, Issue 6 2010Tara C. Smith Abstract Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2-G3 or G4-G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle. © 2010 Wiley-Liss, Inc. [source] Connexins, cell motility, and the cytoskeletonCYTOSKELETON, Issue 11 2009Stephan Olk Abstract Connexins (Cx) comprise a family of transmembrane proteins, which form intercellular channels between plasma membranes of two adjoining cells, commonly known as gap junctions. Recent reports revealed that Cx proteins interact with diverse cellular components to form a multiprotein complex, which has been termed "Nexus". Potential interaction partners include proteins such as cytoskeletal proteins, scaffolding proteins, protein kinases and phosphatases. These interactions allow correct subcellular localization of Cxs and functional regulation of gap junction-mediated intercellular communication. Evidence is accruing that Cxs might have channel-independent functions, which potentially include regulation of cell migration, cell polarization and growth control. In the current review, we summarize recent knowledge on Cx interactions with cytoskeletal proteins and highlight some aspects of their role in cellular motility. Cell Motil. Cytoskeleton 66: 1000,1016, 2009. © 2009 Wiley-Liss, Inc. [source] Fibroblast elongation and dendritic extensions in constrained versus unconstrained microtissuesCYTOSKELETON, Issue 3 2009Dylan M. Dean Abstract Cytoskeletal tension is fundamental to many biological processes, including germ layer sorting during embryogenesis [Krieg et al., 2008]. In vitro, such tension influences cell sorting in self-assembled, 3D microtissues and can be of sufficient magnitude to cause complex-shaped microtissue failure [Dean et al., 2007]. To examine the process of failure under cell-derived tension, we subjected normal human fibroblasts (NHFs) to directed self-assembly [Dean et al., 2007] in micro-molds designed to yield self-constraining microtissues. As cells contracted in this assay, the constrained microtissues narrowed, thinned and ultimately failed at their midpoints. By adding small numbers of GFP+ cells, changes in cell movement and morphology were assessed and compared to those of unconstrained microtissues. We found that cells formed numerous dendritic extensions within an hour of self-assembly and retracted these extensions as they elongated up to 30 times their initial diameter (,600 ,m) just prior to failure. Surprisingly, significant coordination in cell motility was observed over large distances within microtissues. Pharmacologic interventions showed that failure was myosin II and Rho kinase dependent and inhibition of failure resulted in shorter cells with greater numbers of extensions. These findings further our understanding of cellular self-assembly and introduce the use of GFP+ cells with directed self-assembly as a scaffold-free analogue to fibroblast-populated collagen gels (FPCGs). Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Opposite effects of overexpressed myosin Va or heavy meromyosin Va on vesicle distribution, cytoskeleton organization, and cell motility in nonmuscle cellsCYTOSKELETON, Issue 3 2008Robbin D. Eppinga Abstract Myosin Va, an actin-based motor protein that transports intracellular cargos, can bundle actin in vitro. Whether myosin Va regulates cellular actin dynamics or cell migration remains unclear. To address this, we compared Chinese Hamster Ovary (CHO) cells that stably express GFP fused to either full length mouse myosin Va (GFP-M5) or heavy meromyosin Va (GFP-M5,). GFP-M5 and GFP-M5, co-immunoprecipitate with CHO myosin Va and serve as overexpression of wild-type and dominant negative mutants of myosin Va. Compared to non-expressing control cells, GFP-M5-overexpressing cells have peripheral endocytic vesicles, spread slowly after plating, as well as produce robust interior actin stress fibers, myosin II bundles, and focal adhesions. However, these cells display normal cell migration and lamellipodial dynamics. In contrast, GFP-M5,-expressing cells have perinuclear endocytic vesicles, produce thin interior actin and myosin bundles and contain no interior focal adhesions. In addition, these cells spread rapidly, migrate slowly and display reduced lamellipodial dynamics. Similarly, neurite outgrowth is compromised in neurons cultured from transgenic Drosophila that express M5,-dsRed and in neurons cultured from Drosophila that produce a tailless version of endogenous myosin V. Together, these data suggest that myosin Va overexpression induces actin bundles in vivo whereas the tailless version fails to bundle actin and disrupts cell motility. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Role of myosin II activity and the regulation of myosin light chain phosphorylation in astrocytomasCYTOSKELETON, Issue 1 2008Bodour Salhia Abstract The generation of contractile force mediated by actin-myosin interactions is essential for cell motility. Myosin activity is promoted by phosphorylation of myosin light chain (MLC). MLC phosphorylation in large part is controlled by kinases that are effectors of Rho family GTPases. Accordingly, in this study we examined the effects of ROCK and Rac1 inhibition on MLC phosphorylation in astrocytoma cells. We found that low concentrations of the ROCK inhibitor Y27632 increased the phosphorylation state of the Triton X-100 soluble fraction of MLC, whereas higher concentrations of Y27632 decreased soluble phospho-MLC. These effects of Y27632 were dependent on Rac1. The soluble form of phospho-MLC comprises about 10% of total phospho-MLC in control cells. Interestingly, ROCK inhibition led to a decrease in the phosphorylation state of total MLC, whereas Rac1 inhibition had little effect. Thus, the soluble form of MLC is differentially regulated by ROCK and Rac1 compared with MLC examined in a total cell extract. We also observed that astrocytoma migration is stimulated by low concentrations of the myosin II inhibitor blebbistatin. However, higher concentrations of blebbistatin inhibit migration leading us to believe that migration has a biphasic dependence on myosin II activity. Taken together, our data show that modulation of myosin II activity is important in determining optimal astrocytoma migration. In addition, these findings suggest that there are at least two populations of MLC that are differentially regulated. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] A FAK/Src chimera with gain-of-function properties promotes formation of large peripheral adhesions associated with dynamic actin assemblyCYTOSKELETON, Issue 1 2008Priscila M. F. Siesser Abstract Formation of a complex between the tyrosine kinases FAK and Src is a key integrin-mediated signaling event implicated in cell motility, survival, and proliferation. Past studies indicate that FAK functions in the complex primarily as a "scaffold," acting to recruit and activate Src within cell/matrix adhesions. To study the cellular impact of FAK-associated Src signaling we developed a novel gain-of-function approach that involves expressing a chimeric protein with the FAK kinase domain replaced by the Src kinase domain. This FAK/Src chimera is subject to adhesion-dependent activation and promotes tyrosine phosphorylation of p130Cas and paxillin to higher steady-state levels than is achieved by wild-type FAK. When expressed in FAK ,/, mouse embryo fibroblasts, the FAK/Src chimera resulted in a striking cellular phenotype characterized by unusual large peripheral adhesions, enhanced adhesive strength, and greatly reduced motility. Live cell imaging of the chimera-expressing FAK ,/, cells provided evidence that the large peripheral adhesions are associated with a dynamic actin assembly process that is sensitive to a Src-selective inhibitor. These findings suggest that FAK-associated Src kinase activity has the capacity to promote adhesion integrity and actin assembly. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Development of micropost force sensor array with culture experiments for determination of cell traction forcesCYTOSKELETON, Issue 7 2007Bin Li Abstract Cell traction forces (CTFs) are critical for cell motility and cell shape maintenance. As such, they play a fundamental role in many biological processes such as angiogenesis, embryogenesis, inflammation, and wound healing. To determine CTFs at the sub-cellular level with high sensitivity, we have developed high density micropost force sensor array (MFSA), which consists of an array of vertically standing poly(dimethylsiloxane) (PDMS) microposts, 2 ,m in diameter and 6 ,m in height, with a center-to-center distance of 4 ,m. In combination with new image analysis algorithms, the MFSA can achieve a spatial resolution of 40 nm and a force sensitivity of 0.5 nN. Culture experiments with various types of cells showed that this MFSA technology can effectively determine CTFs of cells with different sizes and traction force magnitudes. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPaseCYTOSKELETON, Issue 5 2007Heidi Y. Shi Abstract Rac1 and Cdc42 are members of the Rho family of small GTPases that play essential roles in diverse cellular functions, including cell migration. The activities of these Rho family proteins are controlled by growth factor receptor activation and cell-ECM interactions. Here, we show that maspin, a well-documented tumor suppressor gene, also controls cell motility through inhibiting Rac1/Cdc42 activity. Using the GST-PAK and GST-Rho binding protein pull-down assays for GTP-bound Rac1, Cdc42, and RhoA, we showed that treatment of MDA-MB-231 tumor cells with recombinant maspin for a short time period significantly inhibited the activity of Rac1 and Cdc42, but not RhoA. The reactive site loop (RSL) within maspin protein is the functional domain involved in the inhibition. Maspin mutants with the RSL deleted or a point mutation in the RSL region lost their inhibitory activity. We further examined the ability of maspin to inhibit Rac1- and Cdc42-mediated signaling pathways and transcription factors. Treatment of MDA-MB-231 cells with maspin led to the inhibition of JNK kinase activity as assayed by immuno-kinase assays. In addition, the AP-1 transcription activity downstream of JNK kinase pathway was also reduced. Together, we have identified Rac1 and Cdc42 as the downstream targets that mediate the inhibition of mammary tumor cell migration by maspin. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Molecular characterization of the effects of Y-27632CYTOSKELETON, Issue 2 2007Hassina Darenfed Abstract Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extenstions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Causal mapping as a tool to mechanistically interpret phenomena in cell motility: Application to cortical oscillations in spreading cellsCYTOSKELETON, Issue 9 2006Gabriel E. Weinreb Abstract Biological processes that occur at the cellular level and consist of large numbers of interacting elements are highly nonlinear and generally involve multiple time and spatial scales. The quantitative description of these complex systems is of great importance but presents large challenges. We outline a new systems biology approach, causal mapping (CMAP), which is a coarse-grained biological network tool that permits description of causal interactions between the elements of the network and overall system dynamics. On one hand, the CMAP is an intermediate between experiments and physical modeling, describing major requisite elements, their interactions and paths of causality propagation. On the other hand, the CMAP is an independent tool to explore the hierarchical organization of cell and the role of uncertainties in the system. It appears to be a promising easy-to-use technique for cell biologists to systematically probe verbally formulated qualitative hypotheses. We apply the CMAP to study the phenomenon of contractility oscillations in spreading cells in which microtubules have been depolymerized. The precise mechanism by which these oscillations are governed by a complex mechano-chemical system is not known but the data observed in experiments can be described by a CMAP. The CMAP suggests that the source of the oscillations results from the opposing effects of Rho activation leading to a decreased level of myosin light chain phosphatase and a cyclic calcium influx caused by increased membrane tension and leading to a periodically enhanced activation of myosin light chain kinase. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpainsCYTOSKELETON, Issue 4 2006Germain Mazères Abstract The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced ,- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed. Cell Motil. Cytoskeleton 63: 2006. © 2006 Wiley-Liss, Inc. [source] Description and characterization of a chamber for viewing and quantifying cancer cell chemotaxisCYTOSKELETON, Issue 1 2005Lilian Soon Abstract Direct observations of cancer cell invasion underscore the importance of chemotaxis in invasion and metastasis. Yet, there is to date, no established method for real-time imaging of cancer chemotaxis towards factors clinically correlated with metastasis. A chamber has been designed and tested, called the Soon chamber, which allows the direct observation and quantification of cancer cell chemotaxis. The premise for the design of the Soon chamber is the incorporation of a dam, which creates a steep gradient while retaining stability associated with a pressure-driven system. The design is based on the characteristics of cancer cell motility such as relatively low speeds, and slower motility responses to stimuli compared to classical amoeboid cells like neutrophils and Dictyostelium. We tested MTLn3 breast carcinoma cells in the Soon chamber in the presence of an EGF gradient, obtaining hour-long time-lapses of chemotaxis. MTLn3 cells migrated further, more linearly, and at greater speeds within an EGF gradient compared to buffer controls. Computation of the degree of orientation towards the EGF/buffer source showed that MTLn3 cells were significantly more directional toward the EGF gradient compared to buffer controls. Analysis of the time-lapse data obtained during chemotaxis demonstrated that two populations of cancer cells were present. One population exhibited oscillations in directionality occurring at average intervals of 12 min while the second population exhibited sustained high levels of directionality toward the source of EGF. This result suggests that polarized cancer cells can avoid the need for oscillatory path corrections during chemotaxis. Cell Motil. Cytoskeleton 62:27,34, 2005. © 2005 Wiley-Liss, Inc. [source] Symmetry-breaking in mammalian cell cohort migration during tissue pattern formation: Role of random-walk persistenceCYTOSKELETON, Issue 4 2005S. Huang Abstract Coordinated, cohort cell migration plays an important role in the morphogenesis of tissue patterns in metazoa. However, individual cells intrinsically move in a random walk-like fashion when studied in vitro. Hence, in the absence of an external orchestrating influence or template, the emergence of cohort cell migration must involve a symmetry-breaking event. To study this process, we used a novel experimental system in which multiple capillary endothelial cells exhibit spontaneous and robust cohort migration in the absence of chemical gradients when cultured on micrometer-scale extracellular matrix islands fabricated using microcontact printing. A computational model suggested that directional persistence of random-walk and dynamic mechanical coupling of adjacent cells are the critical control parameters for this symmetry-breaking behavior that is induced in spatially-constrained cell ensembles. The model predicted our finding that fibroblasts, which exhibit a much shorter motility persistence time than endothelial cells, failed to undergo symmetry breaking or produce cohort migration on the matrix islands. These findings suggest that cells have intrinsic motility characteristics that are tuned to match their role in tissue patterning. Our results underscore the importance of studying cell motility in the context of cell populations, and the need to address emergent features in multicellular organisms that arise not only from cell-cell and cell-matrix interactions, but also from properties that are intrinsic to individual cells. Cell Motil. Cytoskeleton 61:201,213, 2005. © 2005 Wiley-Liss, Inc. [source] Analysis of force generation during flagellar assembly through optical trapping of free-swimming Chlamydomonas reinhardtiiCYTOSKELETON, Issue 3 2005Rachel Patton McCord Abstract Many studies have used velocity measurements, waveform analyses, and theoretical flagella models to investigate the establishment, maintenance, and function of flagella of the biflagellate green algae Chlamydomonas reinhardtii. We report the first direct measurement of Chlamydomonas flagellar swimming force. Using an optical trap ("optical tweezers") we detect a 75% decrease in swimming force between wild type (CC124) cells and mutants lacking outer flagellar dynein arms (oda1). This difference is consistent with previous estimates and validates the force measurement approach. To examine mechanisms underlying flagella organization and function, we deflagellated cells and examined force generation during flagellar regeneration. As expected, fully regenerated flagella are functionally equivalent to flagella of untreated wild type cells. However, analysis of swimming force vs. flagella length and the increase in force over regeneration time reveals intriguing patterns where increases in force do not always correspond with increases in length. These investigations of flagellar force, therefore, contribute to the understanding of Chlamydomonas motility, describe phenomena surrounding flagella regeneration, and demonstrate the advantages of the optical trapping technique in studies of cell motility. Cell Motil. Cytoskeleton 61:137,144, 2005. © 2005 Wiley-Liss, Inc. [source] RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisationCYTOSKELETON, Issue 3 2005Elen Griffith Abstract TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil. Cytoskeleton 60:140,152, 2005. © 2005 Wiley-Liss, Inc. [source] The motility of glioblastoma tumour cells is modulated by intracellular cofilin expression in a concentration-dependent mannerCYTOSKELETON, Issue 3 2005Celestial T. Yap Abstract The invasive behaviour of tumour cells has been attributed in part to dysregulated cell motility. Members of the ADF/Cofilin family of actin-binding proteins are known to increase microfilament dynamics by increasing the rate at which actin monomers leave the pointed end of the filament and by a filament-severing activity. As depolymerisation is a rate-limiting step in actin dynamics, ADF/Cofilins are suspected to facilitate the motility of cells. To test this, we investigated the influence of cofilin on tumour motility by transient and stably overexpressing cofilin in the human glioblastoma cell line, U373 MG. Several different methods were used to ascertain the level of cofilin in overexpressing clones and this was correlated with their rate of random locomotion. A biphasic relationship between cofilin level and locomotory rate was found. Clones that displayed a moderate amount of overproduction of cofilin were found to have increased rates of locomotion approximately linear to the overproduction of cofilin up to an optimal cofilin level of about 4.5 times that of wild type cells at which the cells were almost twice as fast. However, clones producing more than this optimal amount were found to locomote at progressively reduced speeds. Cells that overexpress cofilin have reduced stress fibres compared to control cells showing that the excess cofilin affects the actin cytoskeleton. We conclude that overexpression of cofilin enhances the motility of glioblastoma tumour cells in a concentration-dependent fashion, which is likely to contribute to their invasiveness. Cell Motil. Cytoskeleton 60:153,165, 2005. © 2005 Wiley-Liss, Inc. [source] Simultaneous quantification of cell motility and protein-membrane-association using active contoursCYTOSKELETON, Issue 4 2002Dirk Dormann Abstract We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns. Cell Motil. Cytoskeleton 52:221,230, 2002. © 2002 Wiley-Liss, Inc. [source] Mathematical and experimental insights into the development of the enteric nervous system and Hirschsprung's DiseaseDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2007Kerry A. Landman The vertebrate enteric nervous system is formed by a rostro-caudally directed invasion of the embryonic gastrointestinal mesenchyme by neural crest cells. Failure to complete this invasion results in the distal intestine lacking intrinsic neurons. This potentially fatal condition is called Hirschsprung's Disease. A mathematical model of cell invasion incorporating cell motility and proliferation of neural crest cells to a carrying capacity predicted invasion outcomes to imagined manipulations, and these manipulations were tested experimentally. Mathematical and experimental results agreed. The results show that the directional invasion is chiefly driven by neural crest cell proliferation. Moreover, this proliferation occurs in a small region at the wavefront of the invading population. These results provide an understanding of why many genes implicated in Hirschsprung's Disease influence neural crest population size. In addition, during in vivo development the underlying gut tissues are growing simultaneously as the neural crest cell invasion proceeds. The interactions between proliferation, motility and gut growth dictate whether or not complete colonization is successful. Mathematical modeling provides insights into the conditions required for complete colonization or a Hirschsprung's-like deficiency. Experimental evidence supports the hypotheses suggested by the modeling. [source] Reprogramming of genetic networks during initiation of the Fetal Alcohol Syndrome,DEVELOPMENTAL DYNAMICS, Issue 2 2007Maia L. Green Abstract Fetal Alcohol Spectrum Disorders (FASD) are birth defects that result from maternal alcohol use. We used a non a priori approach to prioritize candidate pathways during alcohol-induced teratogenicity in early mouse embryos. Two C57BL/6 substrains (B6J, B6N) served as the basis for study. Dosing pregnant dams with alcohol (2× 2.9 g/kg ethanol spaced 4 hr on day 8) induced FASD in B6J at a higher incidence than B6N embryos. Counter-exposure to PK11195 (4 mg/kg) significantly protected B6J embryos but slightly promoted FASD in B6N embryos. Microarray transcript profiling was performed on the embryonic headfold 3 hr after the first maternal alcohol injection (GEO data series accession GSE1074). This analysis revealed metabolic and cellular reprogramming that was substrain-specific and/or PK11195-dependent. Mapping ethanol-responsive KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed down-regulation of ribosomal proteins and proteasome, and up-regulation of glycolysis and pentose phosphate pathway in B6N embryos; and significant up-regulation of tight junction, focal adhesion, adherens junction, and regulation of the actin cytoskeleton (and near-significant up-regulation of Wnt signaling and apoptosis) pathways in both substrains. Expression networks constructed computationally from these altered genes identified entry points for EtOH at several hubs (MAPK1, ALDH3A2, CD14, PFKM, TNFRSF1A, RPS6, IGF1, EGFR, PTEN) and for PK11195 at AKT1. Our findings are consistent with the growing view that developmental exposure to alcohol alters common signaling pathways linking receptor activation to cytoskeletal reorganization. The programmatic shift in cell motility and metabolic capacity further implies cell signals and responses that are integrated by the mitochondrial recognition site for PK11195. Developmental Dynamics 236:613,631, 2007. © 2007 Wiley-Liss, Inc. [source] Synthetic matrix metalloproteinase inhibitor decreases early cardiac neural crest migration in chicken embryosDEVELOPMENTAL DYNAMICS, Issue 4 2002D.H. Cai Abstract During early embryonic development, cardiac neural crest (NC) cells emerge from the forming neural tube, migrate beneath the ectoderm, enter the pharyngeal arches, and subsequently participate in the septation of the heart. Like tumor cells, NC cells penetrate through basement membranes and invade extracellular matrix during their emigration and migration and, therefore, are liable to use similar invasive mechanisms. Matrix metalloproteinases (MMPs) are a family of zinc proteolytic enzymes known to be important in cell migration and invasion of normal and metastatic cells. In an earlier study, we found that the spatial and temporal distribution pattern of MMP-2 positively correlates with cardiac NC migration, suggesting MMP enzymatic activity may be important in mediating cardiac cell NC migration. To test this hypothesis, a synthetic MMP inhibitor, KB8301, was used to block MMP enzymatic activity during in vitro and in vivo cardiac NC cell migration in chick embryos. Injection of KB8301 into the cell-free space adjacent to the neural tube at the level of the second somite before the NC cells emigrated caused major morphologic anomalies in embryos and disrupted cardiac NC morphogenesis. Unilateral injection of KB8301 at lower concentrations, significantly decreased cardiac NC migration on the injected side compared with the noninjected side and compared with that of the injected controls. This decrease correlated with a decrease in MMP activity in the embryos and was not attributable to differences in embryo size or rate of embryonic development after injection. KB8301 also significantly decreased the rate of NC cell motility and distance NC cells migrated from explanted neural tubes and increased cell area and perimeter. These data suggest that MMP enzymatic activity is an important mediator of early cardiac NC migration and that perturbation of endogenous MMP activity may lead to NC-related congenital defects. © 2002 Wiley-Liss, Inc. [source] Effect of elevated homocysteine on cardiac neural crest migration in vitroDEVELOPMENTAL DYNAMICS, Issue 2 2002Philip R. Brauer Abstract A positive correlation between elevated maternal homocysteine (Hcys) and an increased risk of neural tube, craniofacial, and cardiac defects is well known. Studies suggest Hcys perturbs neural crest (NC) development and may involve N-methyl-D-aspartate (NMDA) receptors (Rosenquist et al., 1999). However, there is no direct evidence that Hcys alters NC cell behavior. Here, we evaluated the effect of Hcys on cardiac NC cell migratory behavior in vitro. Neural tube segments from chick embryos treated in ovo with or without Hcys were placed in culture and the migratory behavior of emigrating NC cells was monitored. Hcys significantly increased in vitro NC cell motility at all embryonic stages examined. NC cell surface area and perimeter were also increased. However, the relative distance NC cells migrated from their original starting point only increased in NC cells treated in ovo at stage 6 or at the time neural tube segments were cultured. Cysteine had no effect. NMDA mimicked Hcys' effect on NC motility and migration distance but had no effect on cell area or perimeter. The noncompetitive inhibitor of NMDA receptors, MK801+, significantly inhibited NC cell motility, reduced migration distance, and also blocked the effects of NMDA and Hcys on NC motility and migratory distance in vitro. A monoclonal antibody directed against the NMDA receptor immunostained NC cells in vitro and, in western blots, bound a single protein with the appropriate molecular weight for the NMDA receptor in NC cell lysates. These data are consistent with the hypothesis that a Hcys-sensitive NMDA-like receptor is expressed by early emigrating NC cells or their precursors, which is important in mediating their migratory behavior. Perturbation of this receptor may be related to some of the teratogenic effects observed with elevated Hcys. © 2002 Wiley-Liss, Inc. [source] Polysialic acid controls NCAM-induced differentiation of neuronal precursors into calretinin-positive olfactory bulb interneuronsDEVELOPMENTAL NEUROBIOLOGY, Issue 9 2008Iris Röckle Abstract Understanding the mechanisms that regulate neurogenesis is a prerequisite for brain repair approaches based on neuronal precursor cells. One important regulator of postnatal neurogenesis is polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule NCAM. In the present study, we investigated the role of polySia in differentiation of neuronal precursors isolated from the subventricular zone of early postnatal mice. Removal of polySia promoted neurite induction and selectively enhanced maturation into a calretinin-positive phenotype. Expression of calbindin and Pax6, indicative for other lineages of olfactory bulb interneurons, were not affected. A decrease in the number of TUNEL-positive cells indicated that cell survival was slightly improved by removing polySia. Time lapse imaging revealed the absence of chain migration and low cell motility, in the presence and absence of polySia. The changes in survival and differentiation, therefore, could be dissected from the well-known function of polySia as a promoter of precursor migration. The differentiation response was mimicked by exposure of cells to soluble or substrate-bound NCAM and prevented by the C3d-peptide, a synthetic ligand blocking NCAM interactions. Moreover, a higher degree of differentiation was observed in cultures from polysialyltransferase-depleted mice and after NCAM exposure of precursors from NCAM-knockout mice demonstrating that the NCAM function is mediated via heterophilic binding partners. In conclusion, these data reveal that polySia controls instructive NCAM signals, which direct the differentiation of subventricular zone-derived precursors towards the calretinin-positive phenotype of olfactory bulb interneurons. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008 [source] Proteomic analysis of osteogenic differentiation of dental follicle precursor cellsELECTROPHORESIS, Issue 7 2009Christian Morsczeck Abstract Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins is associated with actin-bundling and defence against oxidative cellular stress, whereas down-regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation. The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic differentiation. We also identified regulatory proteins, such as the transcription factors TP53 and Sp-1, associated with the differentiation process. Further studies will investigate the impact of identified regulatory proteins for cell proliferation and osteogenic differentiation in DFPCs. [source] Aqueous films limit bacterial cell motility and colony expansion on partially saturated rough surfacesENVIRONMENTAL MICROBIOLOGY, Issue 5 2010Gang Wang Summary Bacterial motility is a key mechanism for survival in a patchy environment and is important for ecosystem biodiversity maintenance. Quantitative description of bacterial motility in soils is hindered by inherent heterogeneity, pore-space complexity and dynamics of microhydrological conditions. Unsaturated conditions result in fragmented aquatic habitats often too small to support full bacterial immersion thereby forcing strong interactions with mineral and air interfaces that significantly restrict motility. A new hybrid model was developed to study hydration effects on bacterial motility. Simulation results using literature parameter values illustrate sensitivity of colony expansion rates to hydration conditions and are in general agreement with measured values. Under matric potentials greater than ,0.5 kPa (wet), bacterial colonies grew fast at colony expansion rates exceeding 421 ± 94 µm h,1; rates dropped significantly to 31 ± 10 µm h,1 at ,2 kPa; as expected, no significant colony expansion was observed at ,5 kPa because of the dominance of capillary pinning forces in the submicrometric water film. Quantification of hydration-related constraints on bacterial motion provides insights into optimal conditions for bacterial dispersion and spatial ranges of resource accessibility important for bioremediation and biogeochemical cycles. Results define surprisingly narrow range of hydration conditions where motility confers ecological advantage on natural surfaces. [source] Positively Charged Material Surfaces Generated by Plasma Polymerized Allylamine Enhance Vinculin Mobility in Vital Human Osteoblastss,ADVANCED ENGINEERING MATERIALS, Issue 8 2010Henrike Rebl Abstract Several studies suggest that the modification of an implant surface by chemical means plays an important role in bone tissue engineering. Previously we have shown that osteoblast cell adhesion and spreading can strongly be increased by a positively charged surface. Cell adhesion and migration are two vital processes that are completely dependent on coordinated formation of focal adhesions. Changes in the organization of the actin cytoskeleton and the focal adhesions are essential for numerous cellular processes including cell motility and tissue morphogenesis. We examined the mobility of the cytoskeletally associated protein vinculin on functionalized surfaces using plasma polymerized allylamine (PPAAm), a homogenous plasma polymer layer with randomly distributed amino groups. In living, GFP,vinculin transfected osteoblastic cells we determined a significant increase in vinculin mobility and vinculin contact length on PPAAm compared to collagen I coated surfaces during the initial adhesion phase. We suggest that positive charges control the cell physiology which seems to be dominant over the integrin receptor binding to collagen I. The results emphasize the role of the surface charge for the design of artificial scaffolds in bone repair. [source] | |||||||||||||||||||||||||||||||||||||