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PubMed1987

Translational control and the cytoskeleton in Physarum polycephalum.

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.

Cytoskeletal Proteins↗
PubMed2004

Raft and cytoskeleton associations of an ABC transporter: P-glycoprotein.

BACKGROUND: A novel flow cytometric assay has been described in an accompanying report (Gombos et al., METHODS: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents. RESULTS: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping. CONCLUSIONS: By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Cytoskeleton antibodies in chronic active hepatitis, primary biliary cirrhosis, and alcoholic liver disease.

Antibodies to cytoplasmic microfilaments, intermediate filaments (vimentin filaments), and microtubules which comprise the cytoskeleton of the cell were assayed in sera from 23 patients with HBsAg-negative chronic active hepatitis and 1 with HBsAg-positive chronic active hepatitis, 15 patients with primary biliary cirrhosis, 20 patients with alcoholic liver disease, and 32 healthy controls. The cytoskeleton antibodies were assayed by indirect immunofluorescence technique using vinblastine-treated cultured human embryonic fibroblasts as substrates. There was a significantly increased incidence of cytoskeleton antibodies in patients with liver disease as compared to the control group. Antibodies to microfilaments were found frequently in sera from patients with chronic active hepatitis (67%) and primary biliary cirrhosis (53%) but were rare in sera from patients with alcoholic liver disease (25%) and in control sera (3%). On the other hand, antibodies to microtubules were found in 50% of sera from patients with alcoholic liver disease but in only 7 to 13% of sera from other groups. Intermediate filament antibodies of IgG or IgA class were found only in patient sera whereas intermediate filament antibodies of IgM class were found in the majority of sera in all groups including control sera. The highest titers of intermediate filament antibodies were seen in primary biliary cirrhosis and chronic active hepatitis. The production of cytoskeleton antibodies may be due to the reorganization or destruction of cytoskeletal structures in the liver.

Adult↗
PubMed2003

Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C-dependent p38 mitogen-activated protein kinase signaling.

Disruption of the actin cytoskeleton in subconfluent mesenchymal cells induces chondrogenic differentiation via protein kinase C (PKC) alpha signaling. In this study, we investigated the role of p38 mitogen-activated protein (MAP) kinase in the chondrogenic differentiation of mesenchymal cells that is induced by depolymerization of the actin cytoskeleton. Treatment of mesenchymal cells derived from chick embryonic limb buds with cytochalasin D (CD) disrupted the actin cytoskeleton with concomitant chondrogenic differentiation. The chondrogenesis was accompanied by an increase in p38 MAP kinase activity and inhibition of p38 MAP kinase with SB203580 blocked chondrogenesis. Together these results suggest an essential role for p38 MAP kinase in chondrogenesis. In addition, inhibition of p38 MAP kinase did not alter CD-induced increased expression and activity of PKC alpha, whereas down-regulation of PKC by prolonged exposure of cells to phorbol ester inhibited CD-induced p38 MAP kinase activation. Our results therefore suggest that PKC is involved in the regulation of chondrogenesis induced by disruption of the actin cytoskeleton via a p38 MAP kinase signaling pathway.

Actins↗
PubMed2001

Bruton's tyrosine kinase associates with the actin-based cytoskeleton in activated platelets.

Bruton's tyrosine kinase (Btk) plays a crucial role in the maturation and differentiation of B-lymphocytes and immunoglobulin synthesis. Recently Btk has been described to be present in significant amount in human platelets. To investigate the regulation of this kinase in the platelets we studied its subcellular redistribution in the resting and activated cells. In the resting platelets Btk was almost absent from the actin-based cytoskeleton. Upon challenge of the platelet thrombin receptor upto 30% of total Btk appeared in the cytoskeleton and the protein underwent phosphorylation on tyrosine. Translocation of Btk to the cytoskeleton but not aggregation was prevented by cytochalasin B, which inhibits actin polymerization. Wortmannin and genistein (inhibitors of phosphoinositide 3-kinase and protein tyrosine kinase, respectively) decreased while phenylarsine oxide (a tyrosine phosphatase inhibitor) increased the cytoskeletal content of Btk. The association of Btk with the cytoskeleton was regulated by integrin alpha(IIb)beta(3) and partly reversible. Taken together, these data suggest that Btk might be a component of a signaling complex containing specific cytoskeletal proteins in the activated platelets.

Actins↗
PubMed2006

Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure.

Our recently published data suggested the involvement of protein phosphatase 2A (PP2A) in endothelial cell (EC) barrier regulation (Tar et al. [2004] J Cell Biochem 92:534-546). In order to further elucidate the role of PP2A in the regulation of EC cytoskeleton and permeability, PP2A catalytic (PP2Ac) and A regulatory (PP2Aa) subunits were cloned and human pulmonary arterial EC (HPAEC) were transfected with PP2A mammalian expression constructs or infected with PP2A recombinant adenoviruses. Immunostaining of PP2Ac or of PP2Aa + c overexpressing HPAEC indicated actin cytoskeleton rearrangement. PP2A overexpression hindered or at least dramatically reduced thrombin- or nocodazole-induced F-actin stress fiber formation and microtubule (MT) dissolution. Accordingly, it also attenuated thrombin- or nocodazole-induced decrease in transendothelial electrical resistance indicative of barrier protection. Inhibition of PP2A by okadaic acid abolished its effect on agonist-induced changes in EC cytoskeleton; this indicates a critical role of PP2A activity in EC cytoskeletal maintenance. The overexpression of PP2A significantly attenuated thrombin- or nocodazole-induced phosphorylation of HSP27 and tau, two cytoskeletal proteins, which potentially could be involved in agonist-induced cytoskeletal rearrangement and in the increase of permeability. PP2A-mediated dephosphorylation of HSP27 and tau correlated with PP2A-induced preservation of EC cytoskeleton and barrier maintenance. Collectively, our observations clearly demonstrate the crucial role of PP2A in EC barrier protection.

Blood-Air Barrier↗
PubMed1985

Membrane-cytoskeleton interactions and the regulation of chemotactic peptide-induced activation of human granulocytes: the effects of dihydrocytochalasin B.

When N-formyl chemotactic peptides bind to granulocyte receptors at 37 degrees C they rapidly form a high-affinity ligand-receptor complex whose coisolation with cytoskeletal residues of Triton X-100-extracted cells is under cellular control [Jesaitis et al: J Cell Biol 98:1378, 1984]. Experiments were performed to investigate the significance of this coisolation. When the granulocytes were preincubated with dihydrocytochalasin B (dhCB) for 10 min at 37 degrees C and then stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe, the rate of uptake of the radioligand by the cells was inhibited. Colocalization of the retained peptide with the Triton X-100 fraction of these cells was also reduced relative to this fraction of the untreated cells. This inhibition was apparent before the onset of FMLP endocytosis. The inhibition was 50% effective at 0.25 microgram dhCB/ml. Maximal inhibition (80-90%) occurred at doses of dhCB greater than 1 microgram/ml. The 90% retention of two plasma membrane markers by the cytoskeleton was marginally affected. These results support the hypothesis that coisolation of the high-affinity receptor-peptide complexes with granulocyte cytoskeletons occurs because of specific association of the complexes with the cytoskeleton at the cell surface. In addition, since these events precede internalization, they suggest that formation of the association between the ligand-receptor complex and cytoskeleton may be necessary for ligand-receptor endocytosis. Experiments were also performed to evaluate other functional consequences of cytoskeletal disruption on chemotactic peptide-stimulated functions. f-Met-Leu-Phe stimulation of O2- production was potentiated due to a prolongation of and an increase in the rate of O2- production. This potentiation had the same dose dependency as the inhibition of receptor modulation. The possible relationship of these various functions is discussed.

Cell Membrane↗
PubMed1990

Multiple types of mRNA-cytoskeleton interactions.

Nearly all actively translated mRNAs are associated with the cytoskeleton in HeLa cells and the nature of this association is poorly understood. To gain insight into this association, we have examined and compared the cytoskeleton-mRNA interactions of a signal peptide-histone fusion mRNA (membrane-bound polysomal mRNA) to those of endogenous histone mRNA (nonmembrane-bound polysomal mRNA). We report here the detection of a cytoskeleton attachment site within the signal peptide-histone fusion mRNP/mRNA nucleotide sequence that is not present in wild-type histone mRNA or in HLA-B7 and chorionic gonadotropin-alpha membrane-bound polysomal mRNAs. These results support the possibility that there are multiple mechanisms for the attachment of specific classes of mRNAs to the cytoskeleton.

Base Sequence↗
PubMed1988

Role of the cytoskeleton in laminin induced mammary gene expression.

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, alpha-casein, and alpha-lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of alpha-casein, transferrin, and alpha-lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of alpha-casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of alpha-casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in alpha-casein, alpha-lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.

Animals↗
PubMed2004

Disruption of the actin cytoskeleton results in nuclear factor-kappaB activation and inflammatory mediator production in cultured human intestinal epithelial cells.

The cytoskeleton in eukaryotic cells is composed of two major filament systems, the microtubule system and the actin cytoskeleton. The microtubule system has recently emerged as an important regulator of NF-kappaB function. However, the role that the actin microfilament system plays in controlling NF-kappaB activation is incompletely understood. In this study, we examined the effect of actin cytoskeleton disruption on NF-kappaB activation in human intestinal epithelial cells. Treatment of HT-29 or Caco-2 cells with the prototypic actin disrupting agents cytochalasin D or latrunculin B resulted in increased NF-kappaB DNA binding and NF-kappaB-dependent transcriptional activity. This NF-kappaB activation by cytochalasin D was secondary to an effect on IkappaB, because cytochalasin D-induced IkappaB degradation and the cytochalasin D-induced increase in NF-kappaB-dependent transcriptional activity was prevented by a dominant negative IkappaB mutant. Exposure of the cells to cytochalasins or latrunculin B increased gene expression and release of the NF-kappaB-dependent chemokines IL-8 and GRO-alpha. Cytochalasin D also activated p38 MAP kinase, which pathway contributed to the cytochalasin D-induced increase in IL-8 production. These results demonstrate that the actin cytoskeleton plays an important role in the regulation of NF-kappaB activation and inflammatory events in intestinal epithelial cells.

Actins↗
PubMed2006

FHL2 interacts with both ADAM-17 and the cytoskeleton and regulates ADAM-17 localization and activity.

ADAM-17 is a metalloprotease-disintegrin responsible for the ectodomain shedding of several transmembrane proteins. Using the yeast two-hybrid system, we showed that ADAM-17 interacts with the Four and Half LIM domain 2 protein (FHL2), a LIM domain protein that is involved in multiple protein-protein interaction. We demonstrated that this interaction involved the amino-acid sequence of ADAM-17 from position 721 to739. In the cardiomyoblast cells H9C2, ADAM-17 and FHL2 colocalize with the actin-based cytoskeleton and we showed that FHL2 binds both ADAM-17 and the actin-based cytoskeleton. We found that mainly the mature form of ADAM-17 associates with the cytoskeleton, although the maturation of ADAM-17 by furin is not necessary for its binding to the cytoskeleton. Interestingly, less ADAM-17 was detected at the surface of wild-type mouse macrophages compared to FHL2 deficient macrophages. However, wild-type cells have a higher ability to release ADAM-17 substrates under PMA stimulation. Altogether, these results demonstrate a physical and functional interaction between ADAM-17 and FHL2 that implies that FHL2 has a role in the regulation of ADAM-17.

ADAM Proteins↗
PubMed1987

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy.

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1-2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.

Cytoskeleton↗
PubMed2003

RhoA and lysophosphatidic acid are involved in the actin cytoskeleton reorganization of astrocytes exposed to ethanol.

Astroglial cells play an important role in maintaining neuronal function in the adult and in the developing nervous system. Ethanol exposure induces profound alterations in the astrogliogenesis process, affecting important cell functions, including intracellular protein trafficking. Because the actin cytoskeleton plays a crucial role in intracellular protein transport, the aim of the present study was to analyze the effects of ethanol on actin cytoskeleton organization and the involvement of the RhoA signaling pathway in these effects. We show that RhoA and lysophosphatidic acid (LPA), an upstream activator of RhoA, stimulate the formation of stress fibers and focal adhesion in cortical astrocytes in primary culture. Exposure of cultured astrocytes to different concentrations of ethanol profoundly disorganizes the actin cytoskeleton, leading to the formation of actin rings at the cell periphery and decreasing the content of focal adhesion proteins. Furthermore, LPA treatment or RhoA transfection revert the ethanol-induced actin alterations in astrocytes, whereas transfection with an inactive mutant of RhoA is unable to revert the actin ring organization. In addition, inhibition of endogenous RhoA by C3 exoenzyme effectively blocks ethanol-induced actin ring formation. These results suggest that the effects of alcohol on actin cytoskeleton organization are mediated by the RhoA signaling pathway. Disruptions in actin organization may impair important astrocyte functions, participating in ethanol-induced astroglial and brain damage during development.

Actins↗
PubMed2000

Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-alpha signaling.

Disruption of actin cytoskeleton with cytochalasin D has been known to induce chondrogenic differentiation of chick embryo limb bud mesenchymal cells. However, the mechanism(s) for the induction of chondrogenesis by cytochalasin D is not yet clearly known. In the present study, we examined possible involvement of protein kinase C (PKC) and extracellular signal-regulated protein kinase (Erk-1) in chondrogenesis of mesenchymal cells induced by disruption of actin cytoskeleton. Disruption of actin cytoskeleton with cytochalasin D or latrunculin B induced chondrogenesis of mesenchymal cells cultured at subconfluent cell density, as determined by type II collagen expression. Among the expressed PKC isoforms, cytochalasin D dramatically increased expression and activation of PKCalpha in a dose-dependent manner, and inhibition or downregulation of PKCalpha blocked cytochalasin D-induced chondrogenesis. Cytochalasin D also downregulated Erk-1 phosphorylation that is associated with chondrogenesis. Our results, therefore, suggest that disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating PKCalpha and by inhibiting Erk-1 signaling.

Actins↗
PubMed2001

ATP-binding cassette transporter-1 induces rearrangement of actin cytoskeletons possibly through Cdc42/N-WASP.

Positional cloning approaches revealed that Tangier disease (TD), a genetic high density lipoprotein deficiency, is associated with mutations in the ATP-binding cassette transporter-1 (ABCA1) gene. However, the biological function of ABCA1 is still not fully investigated. Recently, we have reported that the cells from the patients with TD had abnormal actin cytoskeletons in association with decreased expression of Cdc42, a member of RhoGTPases family. In the present study, we have found that actin cytoskeletons were altered in HEK293 cells transfected with human ABCA1 (hABCA1) cDNA. Cells expressing hABCA1 were divided into the following two groups by the distinct morphology with altered actin cytoskeletons: one had increased formation of filopodia (designated as Type I) and the other had long protrusions (designated as Type II). Type I cells had morphology similar to that of cells transfected with dominant active form of Cdc42 (Cdc42-DA, V12Cdc42Hs-DA). Type II cells had morphology similar to that of cells transfected with neural Wiskott-Aldrich Syndrome Protein (N-WASP),one of the established downstream effector molecules of Cdc42. We have obtained the data showing a possible pathway of ABCA1/Cdc42/N-WASP by the following experiments. Introduction of mutant of Cdc42 (dominant negative form of Cdc42, N17Cdc42Hs-DN) and N-WASP (N-WASP lacking verprolin homology domain, N-WASPDeltaVPH), both of which are supposed to have potential to inhibit rearrangement of actin cytoskeletons, significantly inhibited the morphological changes induced by expression of hABCA1. Immunoprecipitation study with FLAG-tagged ABCA1 (hABCA1-FLAG) revealed that Cdc42 was coimmunoprecipitated with hABCA1-FLAG. In addition, we have demonstrated possible intracellular colocalization of these two molecules in the overexpressing cells by the confocal laser microscopy. These results may suggest that hABCA1 regulates actin organization through the possible interaction with Cdc42Hs.

ATP Binding Cassette Transporter 1↗
PubMed1995

Protein kinase M, the cytosolic counterpart of protein kinase C, remodels the internal cytoskeleton of the mammalian egg during activation.

We have investigated mechanisms by which intracellular signals act to restructure the spatial organization of the cytoskeleton as the mammalian egg is converted into the zygote. Four distinct approaches (one cytological, two biochemical, and one pharmacological) demonstrate protein kinase C (PKC) and its cytosolic active counterpart, PKM, act in succession at the time of egg activation. PKM serves to remodel the internal cytoskeleton. The cytological approach mapped the distribution of kinase over time using the PKC reporter dye, Rim-1, which demonstrated a temporal shift in kinase distribution from its initial site of activation at the plasma membrane to its subsequent association with a cross-linked network of intermediate filaments referred to as sheets. The first of two biochemical analyses, Western blot analysis, demonstrated that eggs activated with calcium ionophore contained PKC in the detergent-soluble fraction and PKM in the sheet enriched fraction. Prior to egg activation, PKM is not detected in the sheet-enriched fraction; only PKC is detected in the detergent-soluble fraction of these eggs. The second biochemical analysis demonstrated, via [32P]ATP labeling of a known PKC substrate, that the PKM generated from activation of PKC in response to activation of eggs with calcium ionophore is an active kinase and is located in the sheet-enriched fraction. In addition, purified forms of PKM, but not PKC, could be shown to act on the internal cytoskeleton when perfused into a permeabilized egg system. Pharmacological treatments demonstrate that elevation of [Ca2+]i does not act directly to alter the internal cytoskeleton of the egg. Our results suggest that this kinase is employed at the time of fertilization to provide an internal chronometer acting first at the cell periphery as PKC and subsequently in the cell interior as PKM.

Animals↗
PubMed1998

Eps8, a tyrosine kinase substrate, is recruited to the cell cortex and dynamic F-actin upon cytoskeleton remodeling.

Eps8 is a recently identified substrate of receptor and nonreceptor tyrosine kinases implicated in the control of cell proliferation. To investigate potential functions of Eps8, its intracellular localization has been examined in several cell types. In cycling fibroblasts immunolabeling with antibodies to Eps8 reveals a punctate pattern within the perinuclear region and staining of motile peripheral cell extensions and cell-cell contact regions. Stimulation of quiescent Swiss 3T3 fibroblasts with serum induces a striking reorganization of the actin cytoskeleton which is accompanied by the enrichment of Eps8 and cortactin in membrane ruffles and lamellipodia. A similar accumulation of Eps8 to membrane ruffles is observed in cells treated with phorbol esters, which also induce marked changes of the F-actin cytoskeleton. The localization of Eps8 at the cell cortex is largely independent from the binding of Eps8 to an EGFR/ErbB-2 chimeric receptor. Moreover, fractionation studies reveal that a portion of the Eps8 molecules present in the cell periphery, unlike cortactin and the receptor, is resistant to mild extraction with detergent. Upon cellular transformation by the tyrosine kinase v-Src, a pool of Eps8 is recruited to newly formed specialized regions of the cytoskeleton, such as actin bodies in terminally differentiated myotubes and podosomes in fibroblasts, where cortactin and a variety of cytoskeletal proteins are also found. Extraction with Triton X-100 preserves the association of Eps8 to podosomes and leaves the majority of the v-Src tyrosine-phosphorylated Eps8 in the detergent-resistant fraction. The observed recruitment of Eps8 to highly dynamic cytoskeletal structures of normal and transformed cells suggests that Eps8 may play a role in the reorganization of the cytoskeleton, perhaps acting as a docking site for other signaling molecules.

3T3 Cells↗
PubMed1998

Hepatocyte growth factor enables enhanced integrin-cytoskeleton linkage by affecting integrin expression in subconfluent epithelial cells.

Hepatocyte growth factor (HGF) exerts mitogenic and motogenic effects in different cell types. In the epithelial cell line mHepR1 we found that HGF induced pronounced alterations in cell morphology and promoted cell adhesion and spreading. To analyze the mechanisms how HGF affects these integrin mediated functions we studied the physical linkage of integrins with the cytoskeleton. First we found that HGF increased the expression of different integrin subunits in subconfluent cells and influenced the distribution of integrins on the cell surface. To address the physical association of integrins with the cytoskeleton we analyzed Triton X-100-extracted cell fractions using flow cytometry. Here we show that cultivation of the cells with HGF for 24 h prior to integrin cross-linking significantly enhanced the cytoskeletal anchorage of integrins. To further find out whether HGF directly induces an integrin-cytoskeleton link without subsequent cross-linking we added HGF to suspended cells but failed to detect cytoskeletally immobilized integrins in the detergent-insoluble cell fraction which could be related to the absence of a calcium response induced by HGF. Overall, the results indicate that HGF promotes the physical linkage of integrins to the cytoskeleton which requires additional stimulation of integrins.

Animals↗