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PubMed1985

Mechanisms of thrombin-induced modifications of human platelet cytoskeleton.

Thrombin treated with phenylmethanesulphonyl fluoride, like active enzyme, promotes modifications to human platelet cytoskeleton. The removal of active thrombin by hirudin partially reverses this process. Chymotrypsin-treated platelets do not modify their cytoskeleton after thrombin stimulus, but are still able to increase their adhesiveness to collagen. It is concluded that thrombin influences the cytoskeleton and adhesion by non-enzymic mechanisms which may be mediated by different modulators.

Blood Platelets↗
PubMed1991

Oxidation of thiol in the vimentin cytoskeleton.

Sublethal doses of H2O2, which induces oxidative stress, cause substantial alteration to the vimentin cytoskeleton in various cell types. We have used a thiol-blot assay to assess thiol status in individual proteins from cell extracts. Vimentin thiol is oxidized in preference to other cytoskeleton proteins. Immunoblot analysis also demonstrated a loss of reactivity to an anti-vimentin monoclonal antibody under non-reducing conditions, possibly due to thiol-group oxidation. During induced oxidative stress a number of proteins become associated with the cytoskeleton extracts.

Cells, Cultured↗
PubMed2002

Chromium(VI)-induced damage to the cytoskeleton and cell death in isolated hepatocytes.

Cr(VI) is a known human carcinogen. Although it has been investigated widely, the mechanism(s) of its action is/are not fully understood. The aim of this study was to evaluate Cr(VI)-induced damage to the cell cytoskeleton and the mode of cell death in primary cultures of hepatocytes. Exposure of the cultured cells (10(5)/cm(2)) to 1 and 5 microM Cr(VI) for 24 h resulted in loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Staining of the cells with annexin V and propidium iodide showed that Cr(VI) induces apoptosis at low concentrations (5 microM), whereas at higher concentrations (25 microM) it induces necrosis. This study shows that Cr(VI) causes damage to the cell cytoskeleton, and induces apoptosis at low concentrations. However, the importance of necrosis and apoptosis in vivo, and the effects of longer exposure times, which simulate environmental and occupational exposure to Cr(VI), remain to be investigated.

Animals↗
PubMed2005

SH3 domain-containing proteins and the actin cytoskeleton in yeast.

SH3 (Src homology-3) domains are involved in protein-protein interactions through proline-rich domains. Many SH3-containing proteins are implicated in actin cytoskeleton organization. The aim of our ongoing work is to study the functions of the SH3-containing proteins in actin cytoskeleton regulation. The yeast Saccharomyces cerevisiae proteome includes 29 SH3 domains distributed in 25 proteins. We have examined the direct involvement of these SH3 domains in actin polymerization using an in vitro polymerization assay on GST (glutathione S-transferase)-SH3-coated beads. As expected, not all SH3 domains show polymerization activity, and many recruit distinct partners as assessed by microscopy and pull-down experiments. One such partner, Las17p, the yeast homologue of WASP (Wiskott-Aldrich syndrome protein), was assayed because it stimulates actin nucleation via the Arp2/3 (actin-related protein 2/3) complex. Ultimately, proteins involved in specific biological processes, such as membrane trafficking, may also be recruited by some of these SH3 domains, shedding light on the SH3-containing proteins and actin cytoskeleton functions in these processes.

Actins↗
PubMed2001

The voltage-sensitive motor protein and the Ca2+-sensitive cytoskeleton in developing rat cochlear outer hair cells.

Cochlear outer hair cells (OHCs) possess a unique fast voltage-driven motility associated with a voltage-sensitive motor protein embedded in the basolateral membrane. This mechanism is believed to underlie the cochlear amplification in mammals. OHCs also have a Ca2+/calmodulin-dependent mechanical pathway which involves a submembranous circumferential cytoskeleton. The purpose of this study was to compare the functional appearance of the voltage-sensitive motor proteins with that involving the Ca2+-sensitive cytoskeleton during postnatal development of rat OHCs. We demonstrate that whole-cell electromotility and Ca2+-voked mechanical responses, by ionomycin, develop concomitantly after postnatal day 5 (P5). These two mechanical properties also develop simultaneously in OHCs isolated from two-week-old cultures of P0-P1 organs of Corti. This excludes the participation of neural innervation in the postnatal maturation of the OHCs' motile properties. In addition, we show that the expression of the membranous voltage-sensitive motor protein precedes, by several days, the appearance of whole-cell electromotility. The concomitant development of whole-cell electromotility and Ca2+-sensitive motility, both in vivo and in vitro, underlines the cytoskeleton as an important factor in the functional organization of the voltage-sensitive motor proteins within the plasma membrane.

Aging↗
PubMed2000

The cytoskeleton in plant and fungal cell tip growth.

Tip-growing cells have a particular lifestyle that is characterized by the following features: (1) the cells grow in one direction, forming a cylindrical tube; (2) tip-growing cells are able to penetrate their growth environment, thus having to withstand considerable external forces; (3) the growth velocity of tip-growing cells is among the fastest in biological systems. Tip-growing cells therefore appear to be a system well suited to investigating growth processes. The cytoskeleton plays an important role in cell growth in general, which is why tip-growing cells provide an excellent model system for studying this aspect. The cytoskeletal system comprises structural elements, such as actin filaments and microtubules, as well as proteins that link these elements, control their configuration or are responsible for transport processes using the structural elements as tracks. Common aspects as well as differences in configuration and function of the cytoskeleton in various types of tip-growing cells reveal the general principles that govern the relationship between the cytoskeleton and cell growth.

Cytoskeleton↗
PubMed2001

A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization.

A central feature of Salmonella pathogenicity is the bacterium's ability to enter into non-phagocytic cells. Bacterial internalization is the consequence of cellular responses characterized by Cdc42- and Rac-dependent actin cytoskeleton rearrangements. These responses are triggered by the co-ordinated function of bacterial proteins delivered into the host cell by a specialized protein secretion system termed type III. We report here that SopB, a Salmonella inositol polyphosphatase delivered to the host cell by this secretion system, mediates actin cytoskeleton rearrangements and bacterial entry in a Cdc42-dependent manner. SopB exhibits overlapping functions with two other effectors of bacterial entry, the Rho family GTPase exchange factors SopE and SopE2. Thus, Salmonella strains deficient in any one of these proteins can enter into cells at high efficiency, whereas a strain lacking all three effectors is completely defective for entry. Consistent with an important role for inositol phosphate metabolism in Salmonella-induced cellular responses, a catalytically defective mutant of SopB failed to stimulate actin cytoskeleton rearrangements and bacterial entry. Furthermore, bacterial infection of intestinal cells resulted in a marked increase in Ins(1,4,5,6)P4, a consumption of InsP5 and the activation of phospholipase C. In agreement with the in vivo findings, purified SopB specifically dephosphorylated InsP5 to Ins(1,4,5,6)P4 in vitro. Surprisingly, the inositol phosphate fluxes induced by Salmonella were not caused exclusively by SopB. We show that the SopB-independent inositol phosphate fluxes are the consequence of the SopE-dependent activation of an endogenous inositol phosphatase. The ability of Salmonella to stimulate Rho GTPases signalling and inositol phosphate metabolism through alternative mechanisms is an example of the remarkable ability of this bacterial pathogen to manipulate host cellular functions.

Actins↗
PubMed2003

Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton.

A family of nine Salmonella typhimurium type III secretion effectors with a conserved amino-terminus have been defined. Three family members (SifA, SifB and SseJ) have previously been demonstrated to localize to the Salmonella-containing vacuole and to Salmonella-induced filaments. In contrast, we demonstrate that two other family members, SspH2 and SseI, co-localized with the polymerizing actin cytoskeleton. These proteins also interacted with the mammalian actin cross-linking protein filamin in the yeast two-hybrid assay through their highly conserved amino-terminal domains. This amino-terminus was sufficient to direct localization to the polymerizing actin cytoskeleton, suggesting that the interaction with filamin is important for this subcellular localization. In addition, SspH2 co-localized with vacuole-associated actin polymerizations (VAP) induced by intracellular bacteria through the Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS). SspH2 interacted with the actin-binding protein profilin in the yeast two-hybrid assay and by affinity chromatography. This interaction was highly specific to SspH2 and was mediated by its carboxy-terminus. Furthermore, SspH2 inhibited the rate of actin polymerization in vitro, suggesting that it functions to reduce or remodel VAP. Strains with mutations in sspH2 and sseI retained the ability to form VAP. However, a third intracellular virulence factor, spvB, which ADP-ribosylates actin, strongly inhibited VAP formation in HeLa cells, suggesting a more subtle effect for SspH2 and SseI on the actin cytoskeleton.

Actins↗
PubMed2001

"Caged cytoskeletons": a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells.

In the cytoskeleton method for isolating microtubule-associated proteins MAP65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY-2 cells for only several minutes allows extraction of detergent-soluble proteins, leaving synchronized "caged cytoskeletons" for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti-microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin-related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.

Aphidicolin↗
PubMed1998

Dialysis neutropenia: the role of the cytoskeleton.

Dialysis neutropenia is the result of pulmonary sequestration of neutrophils after complement activation by the dialyzer membrane. Increased expression of neutrophil adhesion receptors, such as CD11b/CD18, suggests that neutrophil adhesion to the capillary endothelium is a possible mechanism. An alternative hypothesis is that the complement fragment C5a modulates neutrophil mechanical properties via the cytoskeleton-largely filamentous actin (F-actin)-stiffening them and thereby slowing their passage through the pulmonary capillaries. To investigate this hypothesis, we developed an assay to measure the F-actin content of neutrophils in whole blood using flow cytometry and the stain NBD-phallacidin. We measured neutrophil F-actin content during hemodialysis of patients with polysulfone (N = 6), Hemophan (N = 6), and Cuprophan membranes sterilized with either ethylene oxide (N = 5) or steam (N = 6). Cell counts, neutrophil and monocyte CD11b expression and plasma C5a concentrations were also measured. The results confirm the strong relationship between the degree of neutropenia, increases in CD11b expression and plasma C5a levels reported by previous researchers. Modulation of the F-actin content of neutrophils was also strongly related to C5a levels, indicating that the neutrophil cytoskeleton is active during dialysis. Modeling of cell counts suggests that with Cuprophan a substantial fraction of neutrophils and monocytes are sequestered before they even pass through the dialyzer, suggesting some form of systemic activation of these cells. Evidence for systemic activation was also seen in measurements of F-actin content, but not CD11b expression, a finding that strengthens the case for the involvement of the cytoskeleton in dialysis neutropenia.

Actins↗
PubMed2003

Redistribution of glycoprotein Ib within platelets in response to protease-activated receptors 1 and 4: roles of cytoskeleton and calcium.

Thrombin activates human platelets by hydrolyzing the protease-activated receptors PAR-1 and PAR-4, exposing new N-terminal sequences which act as tethered ligands, and binding to glycoprotein (GP) Ib, whose surface accessibility transiently decreases when platelets are stimulated by the enzyme. In an attempt to better understand this latter process, we used the peptides SFLLRNPNDKYEPF (PAR-1-AP or TRAP) and AYPGKF (PAR-4-AP) to study whether hydrolysis of both PAR receptors leads to GPIb redistribution. Both peptides induced surface clearance of GPIb with a maximum at 2 min and 5 min for PAR-1-AP and PAR-4-AP, respectively, followed by a slow return to the surface with levels normalizing between 30 and 60 min. Translocation was associated with the formation of clusters of GPIb as revealed by fluorescence microscopy. This transient redistribution of GPIb was blocked by cytochalasin D and in large part by the membrane permeable Ca2+ chelator, BAPTA. The inhibitor of phosphatidylinositol 3-kinase and myosin light chain kinase, wortmannin, did not significantly modify internalization of GPIb, although its return to the surface was delayed for PAR-1-AP. PAR receptor-mediated association of GPIb to the insoluble cytoskeleton was blocked by cytochalasin D, while BAPTA alone increased and stabilized the presence of GPIb. Globally, immunoprecipitation experiments and analysis of the cytoskeleton confirmed that GPIb translocation is powered by a contractile mechanism involving Ca2+ mobilization, actin polymerization, and myosin incorporation into the cytoskeleton and that both PAR-1 and PAR-4 can activate this process.

Actins↗
PubMed2001

Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through rho-kinase-mediated alterations in the acto-myosin cytoskeleton.

Although migration of stellate cells during hepatic injury is essential for wound-healing and fibrosis of the liver, the extracellular and intracellular signals that regulate stellate cell migration are incompletely understood. In this study we tested the hypothesis that wound-induced migration of stellate cells is modulated by endothelin-1 (ET-1) through rho-kinase-mediated alterations in the acto-myosin cytoskeleton. To address this hypothesis, a method was established for direct visualization of wound-induced migration of culture-activated stellate cells with subcellular resolution. Migration in response to wounding was characterized by (1) plasma membrane ruffling and protrusion into the wound, (2) lamellipodia formation at the leading edge, (3) focal adhesion and stress fiber assembly, and (4) myosin reorganization. Exogenous ET-1 accelerated wound-induced migration of stellate cells, but did not alter wound-induced proliferation. Experiments using ET-1 antagonists in the absence of exogenous ET-1 showed that wound-induced migration was also stimulated by endogenous ET-1. Selective inhibition of rho-associated kinase decelerated migration in response to wounding. Moreover, inhibition of rho-associated kinase was distinguished by abrogation of focal adhesion formation, stress fiber assembly, and myosin reorganization. This study shows that rho-kinase-dependent alterations in the acto-myosin cytoskeleton contribute to wound-induced stellate cell migration, which is accelerated by both exogenous and endogenous ET-1. Consequently, these results provide important new evidence suggesting that, migration of stellate cells is modulated by paracrine and autocrine ET-1 stimulation via the action of rho-kinase on the acto-myosin cytoskeleton.

Actomyosin↗
PubMed2000

Hormone-stimulated calcium release is inhibited by cytoskeleton-disrupting toxins in AR4-2J cells.

We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP(3))-production and Ca(2+)release in the pancreatic acinar tumour cell line AR4-2J. Intracellular and extracellular free Ca(2+)concentrations were measured in cell suspensions, using Fura-2. Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 microM), cytochalasin D (10 microM) or toxin B from Clostridium difficile (20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP(3)-production and Ca(2+)release. The toxins had no effect on binding of bombesin to its receptor, on Ca(2+)uptake into intracellular stores and on resting Ca(2+)levels. Ca(2+)mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP(3)(1 microM) was not impaired by latrunculin B. In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP(3)- production and Ca(2+)release was partly suspended in the presence of aluminum fluoride, an activator of G-proteins. Aluminum fluoride had no effect on basal IP(3)and Ca(2+)levels of control and toxin-pretreated cells. We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP(3)-production and Ca(2+)release.

Aluminum Compounds↗
PubMed2002

Emerging roles of the actin cytoskeleton in cholangiocyte function and disease.

Actin is a ubiquitous, abundant protein whose monomers polymerize into polarized actin filaments. Within epithelial cells, filamentous actin is concentrated at the plasma membrane where a wide variety of actin-associated proteins harness the potential and structure of actin filaments to moderate functions at the plasma membrane. These functions include structural support of the plasma membrane, establishing and maintaining cell polarity, regulation of membrane protein distribution and activity and enhancing membrane vesicle trafficking. Consequently, the actin cytoskeleton contributes significantly to the cellular pathogenesis in a number of disease states. In recent years, the actin cytoskeleton has been found to contribute significantly to cholangiocyte function and disease. This includes directing cyclic adenosine monophosphate (cAMP)-mediated Cl(-) secretion, modulating vesicular trafficking at the apical membrane and initiating structural and functional alterations in ischemic bile ducts. Although much remains to be discovered, this article will highlight observations that indicate that the actin cytoskeleton plays a central role in the physiology and diseases of the intrahepatic bile duct.

Actins↗
PubMed2005

The Abl/Arg substrate ArgBP2/nArgBP2 coordinates the function of multiple regulatory mechanisms converging on the actin cytoskeleton.

ArgBP2, and its brain-specific splice variant, nArgBP2, are interactors and substrates of Abl/Arg tyrosine kinases and of the ubiquitin ligase Cbl. They are members of a family of adaptor proteins that colocalize with actin on stress fibers and at cell-adhesion sites, including neuronal synapses. We show here that their NH2-terminal region, which contains a sorbin homology domain domain, interacts with spectrin, and we identify binding proteins for their COOH-terminal SH3 domains. All these binding partners participate in the regulation of the actin cytoskeleton. These include dynamin, synaptojanin, and WAVE isoforms, as well as WAVE regulatory proteins. At least two of the ArgBP2/nArgBP2 binding partners, synaptojanin 2B and WAVE2, undergo ubiquitination and Abl-dependent tyrosine phosphorylation. ArgBP2/nArgBP2 knockdown in astrocytes produces a redistribution of focal adhesion proteins and an increase in peripheral actin ruffles, whereas nArgBP2 overexpression produces a collapse of the actin cytoskeleton. Thus, ArgBP2/nArgBP2 is a scaffold protein that control the balance between adhesion and motility by coordinating the function of multiple signaling pathways converging on the actin cytoskeleton.

Actins↗
PubMed2001

Epstein-Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors.

Epstein-Barr virus encodes integral membrane proteins LMP1 and LMP2A in transformed lymphoblastoid cell lines. We now find that LMP1 associates with the cell cytoskeleton through a tumor necrosis factor receptor-associated factor-interacting domain, most likely mediated by tumor necrosis factor receptor-associated factor 3. LMP1 is palmitoylated, and the transmembrane domains associate with lipid rafts. Mutation of LMP1 cysteine-78 abrogates palmitoylation but does not affect raft association or NF-kappaB or c-Jun N-terminal kinase activation. LMP2A also associates with rafts and is palmitoylated but does not associate with the cell cytoskeleton. The associations of LMP1 and LMP2A with rafts and of LMP1 with the cell cytoskeleton are likely to effect interactions with cell proteins involved in shape, motility, signal transduction, growth, and survival.

Cell Line, Transformed↗
PubMed1993

Stimulus-induced dissociation of alpha subunits of heterotrimeric GTP-binding proteins from the cytoskeleton of human neutrophils.

Previous studies on the mechanism responsible for terminating the generation of second messengers induced by chemotactic factor-receptor complexes have, on one hand, suggested a direct role of a GTP-binding protein(s) (G protein), and, on the other hand, proposed that there is a lateral segregation of the ligand-receptor complexes into G protein-depleted domains of the plasma membrane. In the present investigation, which addresses these apparently contradictory findings, we found that a substantial part of the alpha subunits of the Gn protein (Gn alpha) in unstimulated neutrophils were associated with a cytoskeletal fraction and that release of these subunits occurred upon stimulation with the chemotactic factor fMet-Leu-Phe. An identical Gn alpha release could also be induced by direct activation of G proteins with guanosine 5'-[gamma-thio]triphosphate or AIF4-. In contrast, the alpha subunits of the stimulatory G protein (Gs alpha) also found associated with the cytoskeletal fraction of unstimulated cells were not released by fMet-Leu-Phe stimulation. However, they were effectively released by direct G-protein activation with guanosine 5'-[gamma-thio]triphosphate. In addition, inhibition of the fMet-Leu-Phe-stimulated modulation of the actin network by pertussis toxin did not affect the fMet-Leu-Phe-induced release of Gn alpha from the cytoskeletal fraction. These observations indicate that fMet-Leu-Phe-induced activation of neutrophils involves a specific dissociation of Gn alpha from the cytoskeleton and that this release is not a consequence of the well-known effect of fMet-Leu-Phe on the cytoskeleton of neutrophils. The present data contribute ideas concerning the transducing properties of G proteins in cellular signaling and seem to reconcile the apparently contradictory concepts of how the cytoskeleton participates in the termination of the chemotactic-factor-induced generation of second messengers in human neutrophils.

Cell Membrane Permeability↗
PubMed1993

Association of the low molecular weight GTP-binding protein rap2B with the cytoskeleton during platelet aggregation.

The intracellular distribution of the low-molecular-weight GTP-binding protein rap2B was investigated in resting and agonist-activated human platelets. In both cases, platelets were lysed by Triton X-100, and cell fractions were obtained by differential centrifugations. Using a specific polyclonal antiserum, we found that rap2B in resting platelets was completely detergent-soluble. When platelets were aggregated with thrombin, the thromboxane analogue U46619, or the Ca(2+)-ATPase inhibitor thapsigargin, a significant amount of rap2B became associated with the cytoskeleton. This association was paralleled by a decrease of rap2B in the Triton X-100-soluble fraction. Translocation of rap2B to the cytoskeleton strictly depended on platelet aggregation, and maximal incorporation was found when approximately 50% aggregation was measured. Inhibition of fibrinogen binding to the glycoprotein IIb-IIIa complex completely prevented the interaction of rap2B with the cytoskeleton. These results clearly demonstrate that changes in the intracellular localization of rap2B occur during platelet activation and represent evidence that this low molecular weight GTP-binding protein may be involved in platelet function.

Blood Platelets↗