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Cytoskeleton
Kinds of Cytoskeleton Terms modified by Cytoskeleton Selected AbstractsDOES THE CYTOSKELETON OF INTESTINAL EPITHHELIAL CELLS FUNCTION AS A CELLULAR ALARM TO IDENTIFY THE E. COLI INFECTIONJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2001Zhe Li Intestinal epithelial cells play an important role in regulating host immunity in response to intestinal infection. Pathogenic bacteria (EPEC and EHEC) cause profound cytoskeletal rearrangement in intestinal epithelial cells during attachment or invasion. Rearrangement of cytoskeletal proteins could be a signal to up-regulate host defence response. Aims, To determine the role of actin cytoskeleton and microtubles in IL-8 mRNA response to E. coli infection. Methods, T84 cell monolayers in 6-well plates were infected with HB101, EPEC and EHEC (105 CFU/well) and compared with uninfected control at 3, 6 and 12 h post infection. Control and infected monolayers were treated with nocodazole (Noc, microtubule disrupter, 30 mm), taxol (Tax, microtubule stabiliser, 10 mm), cytochalasin D (CytoD, actin depolymeriser, 100 nm) and Jasplakinolide (Jasp, actin polymeriser, stabilise actin filaments, 1 mm) and studied 6 h post infection. IL-8 gene expression was measured by semiquantitative RT,PCR in control and uninfected monolayers with and without drug treatment and IL8 protein secretion by ELISA. The morphology of F-actin and ,-tubulin was examined by FITC-phaloidin staining (FAS), immunohistochemistry and confocal microscopy. Results, IL-8 mRNA and IL-8 were increased by infection with all bacterial strains at 3 and 6 h but both IL-8 mRNA and IL-8 in EHEC and EPEC infection were decreased compared with control and HB101 at 12 h. Disruption of microfilaments by Noc increased IL-8 (2.7 fold) while preservation of microfilaments by Tax inhibited IL8 response (0.5 fold) to HB101 infection only. CytoD decreased (0.1,0.5 fold) IL8 expression at all time points in all infections while stabilising actin by Jasp markedly increased the IL8 response (2,6 fold) in control, HB101, EHEC and EPEC at 3 and 6 h. CytoD inhibited Noc-induced IL8 gene expression. Confocal microscopy demonstrated that CytoD and Noc caused major morphological damage to the actin and ,-tubulin by 6 h. Similar changes were also observed in EPEC and EHEC infection at 12 h but not HB101. Jasp preserved actin stress filaments in both EPEC and EHEC. Conclusions, Disruption of microtubules and exogenous rearrangement of actin by pathogenic organism may be primary stimuli to up-regulate proinflammatory cytokine gene expression. Preservation of actin filaments is required for this response and may be necessary for signal transduction to the nucleus. [source] THE EPITHELIAL BRUSH BORDER Na+/H+ EXCHANGER NHE3 ASSOCIATES WITH THE ACTIN CYTOSKELETON BY BINDING TO EZRIN DIRECTLY AND VIA PDZ DOMAIN-CONTAINING Na+/H+ EXCHANGER REGULATORY FACTOR (NHERF) PROTEINSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2008Boyoung Cha SUMMARY 1The Na+/H+ exchanger NHE3 associates with the actin cytoskeleton by binding ezrin both directly and indirectly. Both types of interaction are necessary for acute regulation of NHE3. Most acute regulation of NHE3 occurs by changes in trafficking via effects on exocytosis and/or endocytosis. However, NHE3 activity can also be regulated without changing the surface expression of NHE3 (change in turnover number). 2A positive amino acid cluster in the a-helical juxtamembrane region in the COOH-terminus of NHE3 (amino acids K516, R520 and R527) is necessary for binding to the protein 4.1, ezrin, radixin, moesin (FERM) domain III of ezrin. Direct binding of NHE3 to ezrin is necessary for many aspects of basal trafficking, including basal exocytosis, delivery from the synthetic pathway and movement of NHE3 in the brush border (BB), which probably contributes to endocytosis over a prolonged period of time. 3In addition, NHE3 binds indirectly to ezrin. The PDZ domain-containing proteins Na+/H+ exchanger regulatory factor (NHERF) 1 and NHERF2, as intermediates in linking NHE3 to ezrin, are necessary for many aspects of NHE3 regulation. The binding of NHERF,ezrin/radixin/moesin to NHE3 occurs in the cytosolic domain of NHE3 between amino acids 475 and 689. This NHERF binding is involved in the formation of the NHE3 complex and restricts NHE3 mobility in the BB. However, it is dynamic; for example, changing in some cases of signalling. Furthermore, NHERF binding is necessary for lysophosphatidic acid stimulation of NHE3 and inhibition of NHE3 by Ca2+, cAMP and cGMP. [source] Profilin-1 overexpression restores adherens junctions in MDA-MB-231 breast cancer cells in R-cadherin-dependent mannerCYTOSKELETON, Issue 12 2009Li Zou Abstract Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, is downregulated in several different types of adenocarcinoma and elicits tumor-suppressive effect on breast cancer cell lines. MDA-MB-231 (MDA-231), a breast cancer cell line that displays all the characteristics of post-epithelial-to-mesenchymal transition and does not form cell,cell adhesion, can be reverted to an epithelioid phenotype by Pfn1 overexpression. This morphological transition is caused by restoration of adherens junctions (AJ) requiring Pfn1's interaction with actin. Pfn1 overexpression increases the expression level of R-cadherin (a type of cadherin that is endogenously expressed in the parental cell line) and restores AJ in MDA-231 cells in R-cadherin-dependent manner. These findings highlight important role of Pfn1 in the regulation of epithelial cell,cell adhesion. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Myosin Vb localises to nucleoli and associates with the RNA polymerase I transcription complexCYTOSKELETON, Issue 12 2009Andrew J. Lindsay Abstract It is becoming increasingly clear that the mammalian class V myosins are involved in a wide range of cellular processes such as receptor trafficking, mRNA transport, myelination in oligodendrocytes and cell division. Using paralog-specific antibodies, we observed significant nuclear localisation for both myosin Va and myosin Vb. Myosin Vb was present in nucleoli where it co-localises with RNA polymerase I, and newly synthesised ribosomal RNA (rRNA), indicating that it may play a role in transcription. Indeed, its nucleolar pattern was altered upon treatment with RNA polymerase I inhibitors. In contrast, myosin Va is largely excluded from nucleoli and is unaffected by these inhibitors. Myosin Vb was also found to physically associate with RNA polymerase I and actin in co-immunoprecipitation experiments. We propose that myosin Vb serves a role in rRNA transcription. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Dynamics of bacterial cytoskeletal elementsCYTOSKELETON, Issue 11 2009Peter L. Graumann Abstract Bacterial cytoskeletal elements are involved in an astonishing spectrum of cellular functions, from cell shape determination to cell division, plasmid segregation, the positioning of membrane-associated proteins and membrane structures, and other aspects of bacterial physiology. Interestingly, these functions are not necessarily conserved, neither between different bacterial species nor between bacteria and eukaryotic cells. The flexibility of cytoskeletal elements in performing different tasks is amazing and emphasises their very early development during evolution. This review focuses on the dynamics of cytoskeletal elements from bacteria. Cell Motil. Cytoskeleton 2009. © 2009 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] The heel and toe of the cell's foot: A multifaceted approach for understanding the structure and dynamics of focal adhesionsCYTOSKELETON, Issue 11 2009Haguy Wolfenson Abstract Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic "plaque" proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary FAs defines a boundary between the dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of the entire actin network. The major "mechanical" and "sensory" functions of FAs; namely, the nucleation and regulation of the contractile, myosin-II-containing stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central element in FA regulation concerns the positive feedback loop, based on the most intriguing feature of FAs; that is, their dependence on mechanical tension developing by the growing stress fibers. FAs grow in response to such tension, and rapidly disassemble upon its relaxation. In this article, we address the mechanistic relationships between the process of FA development, maturation and dissociation and the dynamic molecular events, which take place in different regions of the FA, primarily in the distal end of this structure (the "toe") and the proximal "heel," and discuss the central role of local mechanical forces in orchestrating the complex interplay between FAs and the actin system. Cell Motil. Cytoskeleton 66: 1017,1029, 2009. © 2009 Wiley-Liss, Inc. [source] An in vitro model system for cytoskeletal confinementCYTOSKELETON, Issue 10 2009Sarah Köster Abstract The motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins - mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the complete contour of the filament which makes the technique applicable to a broad range of biological polymers, including those with a persistence length smaller than the optical resolution. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Helicobacter pylori activates protein kinase C delta to control Raf in MAP kinase signalling: Role in AGS epithelial cell scattering and elongationCYTOSKELETON, Issue 10 2009Sabine Brandt Abstract Helicobacter pylori is a major etiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. Virulent H. pylori strains harbor a type IV secretion system (T4SS) encoded by the cag pathogenicity island. This T4SS injects the CagA protein into gastric epithelial cells leading to actin-cytoskeletal rearrangements followed by cell elongation and scattering. Here we report that PMA (4,-phorbol-12-myristate-13-acetate), a well-known cell-permeable activator of protein kinase C (PKC), induces a remarkably similar cellular phenotype as compared to infection with H. pylori. PKCs comprise a large family of serine/threonine kinases which are important for multiple physiological processes of host cells. We therefore investigated the role of individual PKC members and the signalling pathways involved in phenotypical outcome. Using isoform-specific silencing RNAs and pharmacological inhibitors we found that two isoforms, PKC-, and PKC-,, were essential for both PMA- and H. pylori -induced elongation phenotype. Furthermore, we provide evidence that PKC-, activity is profoundly stimulated during the course of infection using activation-specific antibodies against PKC phosphorylated at threonine residue 505 or serine residue 660. Infection with H. pylori wild-type and mutants showed that at least two bacterial factors activate PKC-, in a time-dependent manner, one of which is CagA. Immunofluorescence microscopy studies further demonstrated that phosphorylated PKC-, is accumulated and recruited to dynamic actin-structures at the cell membrane. Finally, we show that PKC-, specifically targets Raf kinase to stimulate the Erk1/2 kinase pathway, which is also crucial for phenotypical outcome. Thus, PKC-, is another important mediator of H. pylori -induced pathogenesis. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] AKAP-independent localization of type-II protein kinase A to dynamic actin microspikesCYTOSKELETON, Issue 9 2009Robert L. Rivard Abstract Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RII, subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RII, and a GFP-RII, fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RII,-associated microspikes are highly dynamic and that the coupling of RII, to actin is tight, as the movement of both actin and RII, are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RII, and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RII, is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RII, fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] A cytoskeletal tropomyosin can compromise the structural integrity of skeletal muscleCYTOSKELETON, Issue 9 2009Anthony J. Kee Abstract We have identified a number of extra-sarcomeric actin filaments defined by cytoskeletal tropomyosin (Tm) isoforms. Expression of a cytoskeletal Tm (Tm3) not normally present in skeletal muscle in a transgenic mouse resulted in muscular dystrophy. In the present report we show that muscle pathology in this mouse is late onset (between 2 and 6 months of age) and is predominately in the back and paraspinal muscles. In the Tm3 mice, Evans blue dye uptake in muscle and serum levels of creatine kinase were markedly increased following downhill exercise, and the force drop following a series of lengthening contractions in isolated muscles (extensor digitorum longus) was also significantly increased in these mice. These results demonstrate that expression of an inappropriate Tm in skeletal muscle results in increased susceptibility to contraction-induced damage. The extra-sarcomeric actin cytoskeleton therefore may have an important role in protecting the muscle from contractile stress. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Cell Motility and the Cytoskeleton in transitionCYTOSKELETON, Issue 8 2009B.R. Brinkley Editor-in-Chief No abstract is available for this article. [source] Thinking about flagellar oscillationCYTOSKELETON, Issue 8 2009Charles J. Brokaw Abstract Bending of cilia and flagella results from sliding between the microtubular outer doublets, driven by dynein motor enzymes. This review reminds us that many questions remain to be answered before we can understand how dynein-driven sliding causes the oscillatory bending of cilia and flagella. Does oscillation require switching between two distinct, persistent modes of dynein activity? Only one mode, an active forward mode, has been characterized, but an alternative mode, either inactive or reverse, appears to be required. Does switching between modes use information from curvature, sliding direction, or both? Is there a mechanism for reciprocal inhibition? Can a localized capability for oscillatory sliding become self-organized to produce the metachronal phase differences required for bend propagation? Are interactions between adjacent dyneins important for regulation of oscillation and bend propagation? Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Fascin1 is dispensable for mouse development but is favorable for neonatal survivalCYTOSKELETON, Issue 8 2009Yoshihiko Yamakita Abstract Fascin1, an actin-bundling protein, has been demonstrated to be critical for filopodia formation in cultured cells, and thus is believed to be vital in motile activities including neurite extension and cell migration. To test whether fascin1 plays such essential roles within a whole animal, we have generated and characterized fascin1-deficient mice. Unexpectedly, fascin1-deficient mice are viable and fertile with no major developmental defect. Nissl staining of serial coronal brain sections reveals that fascin1-deficient brain is grossly normal except that knockout mouse brain lacks the posterior region of the anterior commissure neuron and has larger lateral ventricle. Fascin1-deficient, dorsal root ganglion neurons are able to extend neurites in vitro as well as those from wild-type mice, although fascin1-deficient growth cones are smaller and exhibit fewer and shorter filopodia than wild-type counterparts. Likewise, fascin1-deficient, embryonic fibroblasts are able to assemble filopodia, though filopodia are fewer, shorter and short-lived. These results indicate that fascin1-mediated filopodia assembly is dispensable for mouse development. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instabilityCYTOSKELETON, Issue 8 2009Lynne Cassimeris Abstract TOGp, a member of the XMAP215 MAP family, is required for bipolar mitotic spindle assembly. To understand how TOGp contributes to spindle assembly, we examined microtubule dynamics after depleting TOGp by siRNA. Fluorescence recovery after photobleaching of GFP-tubulin demonstrated that spindle microtubule turnover is slowed two-fold in the absence of TOGp. Consistent with photobleaching results, microtubule regrowth after washout of the microtubule depolymerizing drug nocodazole was slower at the centrosomes and in the vicinity of mitotic chromatin in cells depleted of TOGp. The slower microtubule turnover is likely due to either nucleation or the transitions of dynamic instability because TOGp depletion did not effect the rate of plus end growth, measured by tracking EB1-GFP at microtubule ends. In contrast, microtubule regrowth after nocodazole washout was unaffected by prior depletion of TACC3, a centrosomal protein that interacts with TOGp. Kinetochore fibers in both untreated and TOGp-depleted cells were stable to incubation at 4°C or lysis in buffer containing calcium indicating that stable kinetochore-microtubule attachments are formed in the absence of TOGp. Depletion of TOGp, but not TACC3, reduced kinetochore oscillations during prometaphase/metaphase. Defects in oscillations are not due simply to multipolarity or loss of centrosome focus in the TOGp-depleted cells, since kinetochore oscillations appear normal in cells treated with the proteosome inhibitor MG132, which also results in multipolar spindles and centrosome fragmentation. We hypothesize that TOGp is required for chromosome motility as a downstream consequence of reduced microtubule dynamics and/or density. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Protein methylation in full length Chlamydomonas flagellaCYTOSKELETON, Issue 8 2009Roger D. Sloboda Abstract Post-translational protein modification occurs extensively in eukaryotic flagella. Here we examine protein methylation, a protein modification that has only recently been reported to occur in flagella [Schneider MJ, Ulland M, Sloboda RD.2008. Mol Biol Cell 19(10):4319,4327.]. The cobalamin (vitamin B12) independent form of the enzyme methionine synthase (MetE), which catalyzes the final step in methionine production, is localized to flagella. Here we demonstrate, using immunogold scanning electron microscopy, that MetE is bound to the outer doublets of the flagellum. Methionine can be converted to S-adenosyl methionine, which then serves as the methyl donor for protein methylation reactions. Using antibodies that recognize symmetrically or asymmetrically methylated arginine residues, we identify three highly methylated proteins in intact flagella: two symmetrically methylated proteins of about 30 and 40 kDa, and one asymmetrically methylated protein of about 75 kDa. Several other relatively less methylated proteins could also be detected. Fractionation and immunoblot analysis shows that these proteins are components of the flagellar axoneme. Immunogold thin section electron microscopy indicates that the symmetrically methylated proteins are located in the central region of the axoneme, perhaps as components of the central pair complex and the radial spokes, while the asymmetrically methylated proteins are associated with the outer doublets. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Interactions of MAP/microtubule affinity regulating kinases with the adaptor complex AP-2 of clathrin-coated vesiclesCYTOSKELETON, Issue 8 2009Gerold Schmitt-Ulms Abstract MARK [microtubule-associated proteins (MAPs)/microtubule affinity regulating kinase]/Par-1 (partition defective) phosphorylate MAPs tau, MAP2 and MAP4 at KXGS motifs and thereby regulate microtubule dynamics and transport processes in cells [Drewes et al., Cell1997;89:297,308]. We report here that MARK copurifies with clathrin-coated vesicles (CCVs) via interaction with the adaptor complex AP-2. The adaptin binding site on MARK includes the regulatory loop of its catalytic domain. Immunofluorescence demonstrates the colocalization of MARK with AP-2 and clathrin, as well as other MARK-interacting proteins such as PAK5. The results are consistent with an observed influence of MARK on the trafficking of CCVs. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] The roles of actin cytoskeleton and microtubules for membrane recycling of a food vacuole in Tetrahymena thermophilaCYTOSKELETON, Issue 7 2009Maki Sugita Abstract Phagocytosis is a fundamental cellular event for the uptake of nutrients from the environment in several kinds of eukaryote. Most ciliates egest waste and undigested materials in food vacuoles (FVs) through a cytoproct, which is a specific organelle for defecation. It is considered that FV egestion is initiated by fusion between the FV membrane and plasma membrane in a cytoproct and completed with retrieval of the membrane into a cytoplasmic space. In addition, electron microscopy indicated that microfilaments might be involved in the recycling process of the FV membrane in ciliates over 30 years ago; however, there is no conclusive evidence. Here we demonstrated actin organization on FV near a cytoproct in Tetrahymena thermophila by using a marker for a cytoproct. Moreover, it was revealed that cells treated with actin cytoskeletal inhibitor, Latrunculin B, might be suppressed for membrane retrieval in a cytoproct following FV egestion. On the other hand, the actin structures, likely to be the site of membrane retrieval, were frequently observed in the cells treated with cytoplasmic microtubules inhibitor, Nocodazole. We concluded that actin filaments were probably required for recycling of the FV membrane in a cytoproct although the role was not essential for FV egestion. In addition, it was possible that microtubules might be involved in transportation of recycling vesicles of FV coated with F-actin. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] ,3-Tubulin is induced by estradiol in human breast carcinoma cells through an estrogen-receptor dependent pathwayCYTOSKELETON, Issue 7 2009Jennifer Saussede-Aim Abstract Microtubules are involved in a variety of essential cell functions. Their role during mitosis has made them a target for anti-cancer drugs. However development of resistance has limited their use. It has been established that enhanced ,3-tubulin expression is correlated with reduced response to antimicrotubule agent-based chemotherapy or worse outcome in a variety of tumor settings. However little is known regarding the regulation of ,3-tubulin expression. We investigated the regulatory mechanisms of expression of ,3-tubulin in the MCF-7 cell line, a model of hormone-dependent breast cancer. Exposure of MCF-7 cells to estradiol was found to induce ,3-tubulin mRNA as well as ,3-tubulin protein expression. Conversely, we did not observe induction of ,3-tubulin mRNA by estradiol in MDA-MB-231 cells which are negative for the estrogen receptor (ER). In order to determine whether ,3-tubulin up-regulation is mediated through the ER pathway, MCF-7 cells were exposed to two ER modulators. Exposure to tamoxifen, a selective estrogen receptor modulator, completely abolished the ,3-tubulin mRNA induction due to estradiol in MCF-7 cells. This result was confirmed with fulvestrant, a pure antagonist of ER. These results demonstrate that the effect of estradiol on ,3-tubulin transcription is mediated through an ER dependent pathway. Cell Motil. Cytoskeleton 66:378,388, 2009. © 2009 Wiley-Liss, Inc. [source] The geometry and motion of nematode sperm cellsCYTOSKELETON, Issue 6 2009Evgeny Demekhin Abstract The nematode sperm cell crawls by recycling major sperm protein (MSP) from dimers into subfilaments, filaments, and filament complexes, as a result of thermal writhing in the presence of hydrophobic patches. Polymerization near leading edges of the cell intercolates MSP dimers onto the tips of growing filament complexes, forcing them against the cell boundary, and extending the cytoskeleton in the direction of motion. Strong adhesive forces attach the cell to the substrate in the forward part of the lamellipod, while depolymerization in the rearward part of the cell breaks down the cytoskeleton, contracting the lamellipod and pulling the cell body forward. The movement of these cells, then, is caused by coordinated protrusive, adhesive and contractile forces, spatially separated across the lamellipod. This paper considers a phenomenological model that tracks discrete elements of the cytoskeleton in curvilinear coordinates. The pseudo-two dimensional model primarily considers protrusion and rotation of the cell, along with the evolution of the cell boundary. General assumptions are that pH levels within the lamellipod regulate protrusion, contraction and adhesion, and that growth of the cytoskeleton, over time, is perpendicular to the evolving cell boundary. The model follows the growth and contraction of a discrete number of MSP fiber complexes, since they appear to be the principle contributors for force generation in cell boundary protrusion and contraction, and the backbone for the dynamic geometry and motion. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Neutrophils display biphasic relationship between migration and substrate stiffnessCYTOSKELETON, Issue 6 2009Kimberly M. Stroka Abstract Neutrophils are one type of migrating cell in the body's innate immune system and are the first line of defense against inflammation or infection. While extensive work exists on the effect of adhesive proteins on neutrophil motility, little is known about how neutrophil motility is affected by the mechanical properties of their physical environment. This study investigated the effects of substrate stiffness on the morphology, random motility coefficient, track speed (v), spreading area, and distribution of turning angles of neutrophils during chemokinesis. Human neutrophils were plated onto polyacrylamide gels of varying stiffness, ranging from 3 to 13 kPa, and coated with the extracellular matrix protein fibronectin, and timelapse images were taken with phase contrast microscopy. Our results show a biphasic behavior between neutrophil motility and substrate stiffness, with the optimum stiffness for motility depending on the concentration of fibronectin on the surface of the gel. On 100 ,g/mL fibronectin, the optimum stiffness is 4 kPa (v = 6.9 ± 0.6 ,m/min) while on 10 ,g/mL fibronectin, the optimum stiffness increases to 7 kPa (v = 4.5 ± 2.0 ,m/min). This biphasic behavior most likely arises because neutrophils on soft gels are less adherent, preventing production of traction forces, while neutrophils on stiff gels adhere strongly, resulting in decreased migration. At intermediate stiffness, however, neutrophils can attain optimal motility as a function of extracellular matrix coating. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera)CYTOSKELETON, Issue 5 2009Maria Giovanna Riparbelli Abstract In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, ,-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Directional change produced by perpendicularly-oriented microgrooves is microtubule-dependent for fibroblasts and epitheliumCYTOSKELETON, Issue 5 2009Douglas W. Hamilton Abstract Anisotropic substrata such as micromachined grooves can control cell shape, orientation, and the direction of cell movement, a phenomena termed topographic guidance. Although many types of cells exhibit topographic guidance, little is known regarding cell responses to conflicting topographic cues. We employed a substratum with intersecting grooves in order to present fibroblasts and epithelial cells with conflicting topographic cues. Using time-lapse and confocal microscopy, we examined cell behavior at groove intersections. Migrating fibroblasts and epithelial cells typically extended a cell process into the intersection ahead of the cell body. After travelling along the "X" groove to enter the intersection, the leading lamellipodia of the cell body encountered the perpendicular "Y" groove, and spread latterly along the "Y" groove. The formation of lateral lamellipodia resulted in cells forming "T" or "L" morphologies, which were characterized by the formation of phosphotyrosine-rich focal adhesions at the leading edges. The "Y" groove did not prove an absolute barrier to cell migration, particularly for epithelial cells. Analysis of cytoskeletal distribution revealed that F-actin bundles did not adapt closely to the groove patterns, but typically did align to either the "X" or "Y" grooves. In contrast microtubules (MT) adapted closely to the walls. Inhibition of microtubule nucleation attenuated fibroblast and epithelial cell orientation within the intersection of the perpendicular grooves. We conclude that MT may be the prime determinant of fibroblast and epithelial cell conformation to conflicting topographies. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Compaction of cell shape occurs before decrease of elasticity in CHO-K1 cells treated with actin cytoskeleton disrupting drug cytochalasin DCYTOSKELETON, Issue 4 2009Christian Schulze Abstract The actin filaments of the cytoskeleton form a highly dynamic polymer scaffold which is actively involved in many essential mechanisms such as cell migration, transport, mitosis, and mechanosensitivity. We treated CHO-K1 cells with different concentrations of the actin cytoskeleton disrupting drug cytochalasin D. Then investigating the cells' elastic behaviour by scanning force microscopy-based rheology we confirmed for high cytochalasin D concentrations (,1.5 ,M) a significant decrease of mechanical stability. At lower concentrations we measured no significant softening, but flattening and a horizontal contraction was observable even at low concentrations (,0.3 ,M) of cytochalasin D. The observed changes in cell shape resulted in a lower cell volume, showing that there is compensation by volume for small decreases in cytoskeletal strength resulting from reduced numbers or lengths of actin filaments. These results suggest that the characteristic functions defining a cell's mechanical stability such as mechanosensitivity can be maintained via small changes in cell volume in order to counter fluctuations in cytoskeletal composition. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumberCYTOSKELETON, Issue 4 2009Masaya Morita Abstract The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (>7.8) solution without cAMP, and a phosphorylation assay using (,-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+ -binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+ -sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+ -sensitive flagellar protein phosphorylation. Cell Motil. Cytoskeleton 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] Microtubule-dependent motility and orientation of the cortical endoplasmic reticulum in elongating characean internodal cellsCYTOSKELETON, Issue 3 2009Ilse Foissner Abstract Motility of the endoplasmic reticulum (ER) is predominantly microtubule- dependent in animal cells but thought to be entirely actomyosin-dependent in plant cells. Using live cell imaging and transmission electron microscopy to examine ER motility and structural organization in giant internodal cells of characean algae, we discovered that at the onset of cell elongation, the cortical ER situated near the plasma membrane formed a tight meshwork of predominantly transverse ER tubules that frequently coaligned with microtubules. Microtubule depolymerization increased mesh size and decreased the dynamics of the cortical ER. In contrast, perturbing the cortical actin array with cytochalasins did not affect the transverse orientation but decreased mesh size and increased ER dynamics. Our data suggest that myosin-dependent ER motility is confined to the ER strands in the streaming endoplasm, while the more sedate cortical ER uses microtubule-based mechanisms for organization and motility during early stages of cell elongation. We show further that the ER has an inherent, NEM-sensitive dynamics which can be altered via interaction with the cytoskeleton and that tubule formation and fusion events are cytoskeleton-independent. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] In vivo analysis of MT-based vesicle transport by confocal reflection microscopyCYTOSKELETON, Issue 2 2009Imre Gáspár Abstract The use of confocal reflection microscopy (CRM) for the in vivo analysis of microtubule (MT) mediated transport of lipid droplets in the developing Drosophila egg primordia is described here. The developing Drosophila oocytes are ideal objects to study MT-mediated transport in vivo: transport of e.g. the lipid droplets can be conveniently, selectively and sensitively monitored through CRM and the egg primordia are readily available for physical, chemical and/or genetic manipulations. CRM is a non-destructive way to follow vesicle movement and allows high frame rate image recording. When combined with fluorescence imaging, CRM offers simultaneous visualization of the cargo and the protein(s) of interest, i.e. a motor or a cargo adapter, thus allowing a better understanding of MT-mediated transport and spatiotemporal coordination of the transport machinery. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unitCYTOSKELETON, Issue 1 2009Vijay S. Rao Abstract Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Functional studies of an evolutionarily conserved, cytochrome b5 domain protein reveal a specific role in axonemal organisation and the general phenomenon of post-division axonemal growth in trypanosomesCYTOSKELETON, Issue 1 2009Helen Farr Abstract Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Several recent proteomic studies of cilia and flagella have been published, including a proteome of the flagellum of the protozoan parasite Trypanosoma brucei. Comparing proteomes reveals many novel proteins that appear to be widely conserved in evolution. Amongst these, we found a previously uncharacterised protein which localised to the axoneme in T. brucei, and therefore named it Trypanosome Axonemal protein (TAX)-2. Ablation of the protein using RNA interference in the procyclic form of the parasite has no effect on growth but causes a reduction in motility. Using transmission electron microscopy, various structural defects were seen in some axonemes, most frequently with microtubule doublets missing from the 9+2 arrangement. RNAi knockdown of TAX-2 expression in the bloodstream form of the parasite caused defects in growth and cytokinesis, a further example of the effects caused by loss of flagellar function in bloodstream form T. brucei. In procyclic cells we used a new set of vectors to ablate protein expression in cells expressing a GFP:TAX-2 fusion protein, which enabled us to easily quantify protein reduction and visualise axonemes made before and after RNAi induction. This establishes a useful generic technique but also revealed a specific observation that the new flagellum on the daughter trypanosome continues growth after cytokinesis. Our results provide evidence for TAX-2 function within the axoneme, where we suggest that it is involved in processes linking the outer doublet microtubules and the central pair. Cell Motil. 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