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PubMed1998

Image processing and analysis of fluorescent labelled cytoskeleton.

Cytoskeleton elements play an important role in cellular processes including normal and pathological conditions or even cell death. Although immunofluorescence microscopy techniques have proved useful to study the cytoskeleton, description and comparative results commonly rely on the direct visual analysis of the targeted cells. We report here an objective and quantitative method to analyze the cytoskeletal pattern of cultured HeLa cells based on image processing and analysis (IPA) of fluorescent labelled cytoskeleton components. Different structural quantifiable characteristics using IPA facilities (densitometric profile, number of branched points, Euler number, convexity, orientation value) allow accurate characterization and differentiation of the three types of fibre nets studied (microtubules, actin microfilament, and cytokeratin intermediate filaments). The application of the analytical methods presented here provides information concerning the organization and modification of cytoskeleton components, and could aid in the further understanding of structural and functional aspects of the cytoskeleton under normal and pathological conditions.

Actins↗
PubMed2003

Cytoskeletons in prokaryotes.

Not only eukaryotes, but also prokaryotes possess a cytoskeleton. Tubulin-related bacterial protein FtsZ, and actin-related bacterial proteins MreB/Mbl have recently been described as constituents of bacterial cytoskeletons. Genes coding for MreB/Mbl could only be found in elongated bacteria, not in coccoid forms. It was speculated that constituents of today's eukaryotic cytoskeleton (tubulin, actin) may have evolved from prokaryotic precursor proteins closely related to today's bacterial proteins FtsZ and MreB/Mbl. Prior to the description of proteins MreB/Mbl, evidence had been obtained for the existence of a shape-preserving cytoskeleton ubiquitously present in all bacteria. In the meantime, structural studies allow to speculate on a possible role of bacterial elongation factor Tu (EF-Tu) as a structural element in such a "cytoskeletal web". EF-Tu was long known to form fibrillar structures in vitro; now experimental data accumulate, pointing towards formation of intracellular protofilaments containing EF-Tu, and networks thereof as well. In addition, results of these structural studies suggest a so far unknown mode of complex formation of EF-Tu with active ribosomes: ribosomes/polysomes were seen to be attached to intracellular protofilaments. Implications for the understanding of EF-Tu-ribosome interaction, and a role of such a kind of putative protofilaments as a general site of attachment for cellular functional macromolecules are discussed. The notion is discussed that an EF-Tu-containing cytoskeletal web might have been the "primary" or "basic" kind of prokaryotic cytoskeleton, already in existence prior to the "invention" of precursors of today's MreB.

Bacterial Proteins↗
PubMed2001

Plasmodesmata and plant cytoskeleton.

Plant cell-to-cell communication is achieved by membranous conduits called plasmodesmata, which bridge the cytoplasm of neighboring cells. A growing body of immunolocalization data shows an association of the cytoskeleton machinery with plasmodesmata. The role of the cytoskeleton in the plasmodesmata-mediated transport has been well documented for virus movement. Because viruses are known to exploit existing host pathways and because the cytoskeleton is involved in intracellular trafficking, the cytoskeleton is thought to drive and target macromolecules to plasmodesmata. It is this link between plasmodesmata and the cytoskeleton that will be described here.

Biological Transport↗
PubMed2000

Dystrophin and the cardiomyocyte membrane cytoskeleton in the healthy and failing heart.

The cardiomyocyte membrane cytoskeleton consists of the costameric proteins that mediate force transduction from the cell to the extracellular matrix, and a sub-membrane network composed of dystrophin and associated proteins. Studies of the precise cellular distribution of dystrophin and of the consequences of genetic mutations leading to abnormal expression of the dystrophin molecule, as occurs in Duchenne and Becker's muscular dystrophies, highlight potential functional roles of this sub-membrane protein complex in cardiomyocytes. Detailed investigation of dystrophin distribution using the complementary cell imaging techniques of immunoconfocal microscopy and freeze-fracture cytochemistry at the electron-microscopical level show that, in contrast to rat cardiomyocytes, the dystrophin network in human cardiomyocytes is locally enriched at costameres. Thus located, the dystrophin network appears to have a mechanical role, involving stabilization of the peripheral plasma membrane during the repetitive distortion associated with cardiac contraction and, in the human myocyte, contributing to lateral force-transduction. Evidence from animal models of muscular dystrophy and from investigation of the interactions of the sub-membrane cytoskeleton with other membrane-associated proteins including ion channels, receptors and enzymes, further suggests a role for dystrophin in organization and regulation of membrane domains. The relative preservation of the membrane cytoskeleton in non-dystrophic dilated cardiomyopathy and in ischemic cardiomyopathy, conditions in which the myocyte contractile apparatus and internal desmin-based cytoskeleton are commonly disrupted, emphasizes the vital role of the membrane cytoskeleton in cell survival. Continued cardiomyocyte survival despite loss of contractile protein organization has implications in the potential for reversibility of left ventricular remodeling that can be achieved in the clinical setting.

Animals↗
PubMed2000

Cardiomyocyte cytoskeleton and myofibrillogenesis in healthy and diseased heart.

The unique cytoarchitecture of cardiomyocytes arises by complex interactions of different filamentous structures of the cytoskeleton. Intermediate filaments of the non-sarcomeric cytoskeleton are not essential for development but important for maintenance of myofibrils. Myofibrils consist of contractile proteins involved in force generation and the muscle cytoskeleton framework. The latter is essential for proper assembly and maintenance as well as for interaction with other cardiomyocytes or the extracellular matrix, thus being involved in force transmission. The information for sarcomere assembly is encoded in the proteins and some domains essential for faithful incorporation have been identified by epitope tagging experiments. Many KO mutations result in embryonic lethal phenotypes and new techniques e.g. using cardiomyocytes derived from ES cell-lines will have to be developed that allow to study such mutations in cardiomyocytes rather than whole organisms. Alterations in the expression levels of several proteins of the muscle cytoskeleton or impairment of their function by point mutations can result in increased mechanical stress in the cardiomyocytes which finally leads to cellular responses such as the development of dilated cardiomyopathy (DCM). MLP (muscle-LIM-protein) deficient mice develop DCM and changes in the mechanical coupling of cardiomyocytes result in alterations at the intercalated disks and enhanced accumulation of adherens junction proteins. Therefore, controlled interactions between proteins of the muscle cytoskeleton and contractile proteins are essential to ensure proper cardiac function and a more detailed insight in these processes might provide new tools to improve the contractile efficiency of the cardiomyocytes and thus working output in cardiomyopathies.

Animals↗
PubMed2002

Mechanical response of single filamin A (ABP-280) molecules and its role in the actin cytoskeleton.

Filamin A produces isotropic cross-linked three-dimensional orthogonal networks with actin filaments in the cortex and at the leading edge of cells. Filamin A also links the actin cytoskeleton to the plasma membrane via its association with various kinds of membrane proteins. Recent new findings strongly support that filamin A plays important roles in the mechanical stability of plasma membrane and cortex, formation of cell shape, mechanical responses of cells, and cell locomotion. To elucidate the mechanical properties of the actin/filamin A network and the complex of membrane protein-filamin A-actin cytoskeleton, the mechanical properties of single human filamin A (hsFLNa) molecules in aqueous solution were investigated using atomic force microscopy. Ig-fold domains of filamin A can be unfolded by the critical external force (50-220 pN), and this unfolding is reversible, i.e., the refolding of the unfolded chain of the filamin A occurs when the external force is removed. Due to this reversible unfolding of Ig-fold domains, filamin A molecule can be stretched to several times the length of its native state. Based on this new feature of filamin A as the 'large-extensible linker', we describe our hypothesis for the mechanical role of filamin A in the actin cytoskeletons in cells and discuss its biological implications. In this review, function of filamin A in actin cytoskeleton, mechanical properties of single filamin A proteins, and the hypothesis for the mechanical role of filamin A in the actin cytoskeletons are discussed.

Actins↗
PubMed2003

All roads lead to actin: the intimate relationship between TCR signaling and the cytoskeleton.

Regardless of cell type, the regulation of the actin cytoskeleton is tightly linked to vital biological properties such as polarity, motility, cell-cell contact, exocytosis and proliferation. In the immune system, where rapid and efficient response to antigen-provoked stimuli is crucial, an overwhelming amount of data implicate the actin cytoskeleton and its regulators as central to immune function. Increasingly, the cytoskeleton is considered an essential amplification step in T cell receptor (TCR)-, costimulatory-, and integrin-mediated signaling. Advances in genetic manipulation and confocal imaging have led to a keener appreciation of the importance of TCR signal integration by the actin cytoskeleton. This review outlines recent advances in elucidating the regulation of T cell function through the actin cytoskeleton. We also examine intriguing parallels between the immune system and other models of cytoskeletal regulation.

Actins↗
PubMed2003

Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin.

Mechanical forces on matrix-integrin-cytoskeleton linkages are crucial for cell viability, morphology and organ function. The production of force depends on the molecular connections from extracellular-matrix-integrin complexes to the cytoskeleton. The minimal matrix complex causing integrin-cytoskeleton connections is a trimer of fibronectin's integrin-binding domain FNIII7-10 (ref. 4). Here we report a specific, molecular slip bond that was broken repeatedly by a force of 2 pN at the cellular loading rate of 60 nm x s(-1); this occurred with single trimer beads but not with monomer. Talin1, which binds to both integrins and actin filaments in vitro, is required for the 2-pN slip bond and rapid cytoskeleton binding. Further, inhibition of fibronectin binding to alpha(v)beta3 and deletion of beta3 markedly decreases the 2-pN force peak. We suggest that talin1 initially forms a molecular slip bond between closely packed fibronectin-integrin complexes and the actin cytoskeleton, which can apply a low level of force to fibronectin until many bonds form or a signal is received to activate a force response.

3T3 Cells↗
PubMed1998

The cytoskeleton of the vertebrate smooth muscle cell.

Smooth muscle cells possess a structural lattice composed of two primary parts: the 'cytoskeleton' that pervades the cytoplasm and the 'membrane skeleton' that provides anchorage for the cytoskeleton and contractile apparatus at the cell surface. The cytoskeleton contains two major components: first, a complement of actin filaments that links the cytoplasmic dense bodies at equispaced intervals in longitudinal fibrils; and second, a network of desmin intermediate filaments that co-distributes with the cytoskeletal actin. The actin filaments of the contractile apparatus are presumed to interface with the cytoskeleton at the cytoplasmic dense bodies and with the longitudinal rib-like arrays of dense plaques of the membrane skeleton that couple to the extracellular matrix. The present report focuses attention on the functional role of intermediate filaments and on the molecular domain structure of the protein calponin, which is found both in the cytoskeleton and the contractile apparatus. New information about the role of intermediate filaments in smooth muscle has come from studies of transgenic mice in which desmin expression has been ablated. These have shown that while desmin is dispensable for normal development and viability its absence has significant consequences for the mechanical properties of muscle tissue. Thus, the visceral smooth muscles develop only 40% of the normal contractile force and the maximal shortening velocity is reduced by 25-40%. Intermediate filaments therefore play an active role in force transmission and do not contribute solely to cell shape maintenance, as has hitherto been presumed. Recent studies on calponin have revealed a second actin binding domain at the C-terminus of the molecule and have also pinpointed an N-terminal domain that shares homology with a growing family of actin binding and signalling molecules. How these newly identified features of calponin relate to its function in vivo remains to be established.

Animals↗
PubMed2000

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton.

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr(774) in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Czeta, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1983

Synthesis of spectrin in avian erythroid cells: association of nascent polypeptide chains with the cytoskeleton.

The site of synthesis of spectrin was investigated in erythroid cells from 10-day chicken embryos. After various periods of [35S]methionine incorporation the cells were lysed in a Triton X-100 (TX-100)-containing buffer and were separated into a TX-100-soluble and -insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis after a short pulse-labeling period reveals that alpha-spectrin nascent polypeptides are present predominantly in the TX-100-insoluble fraction. These polypeptides can be immunoprecipitated with alpha-spectrin antisera and the [35S]methionine incorporated into them during a short pulse can be chased into mature alpha-spectrin molecules. The alpha-spectrin nascent polypeptide chains are released quantitatively from the TX-100 cytoskeleton by treatment of lysed cells with puromycin, suggesting that they themselves are not associated with the cytoskeleton. A small fraction of the newly synthesized mature alpha-spectrin molecules is rapidly incorporated into the cytoskeleton, as shown by the fact that they are not released by the puromycin treatment; the rest are recovered in the soluble fraction. These results suggest that alpha-spectrin is synthesized in association with the cytoskeleton during chicken erythropoiesis and assembles onto the membrane-cytoskeleton posttranslationally.

Animals↗
PubMed1995

Integrin alpha IIb beta 3-mediated translocation of CDC42Hs to the cytoskeleton in stimulated human platelets.

To investigate the function of the human Ras-related CDC42 GTP-binding protein (CDC42Hs) we studied its subcellular redistribution in platelets stimulated by thrombin-receptor activating peptide (TRAP) or ADP. In resting platelets CDC42Hs was detected exclusively in the membrane skeleton (9.6 +/- 1.5% of total) and the detergent soluble fraction (90 +/- 4%). When platelets were aggregated with TRAP or ADP, CDC42Hs (10% of total) appeared in the cytoskeleton and decreased in the membrane skeleton, whereas RhoGDI (guanine-nucleotide dissociation inhibitor) and CDC42HsGAP (GTPase-activating protein) remained exclusively in the detergent-soluble fraction. Upon prolonged platelet stimulation CDC42Hs disappeared from the cytoskeleton and reappeared in the membrane skeleton. Rac translocated to the cytoskeleton with a similar time course as CDC42Hs. When platelets were stimulated under conditions that precluded the activation of the alpha IIb beta 3 integrin and platelet aggregation, cytoskeletal association of CDC42Hs was abolished. Translocation of CDC42Hs to the cytoskeleton but not aggregation was also prevented by cytochalasins B or D or the protein tyrosine kinase inhibitor genistein. Platelet secretion and thromboxane formation were not required but facilitated the cytoskeletal association of CDC42Hs. The results indicate that in platelets stimulated by TRAP or ADP, a fraction of CDC42Hs translocates from the membrane skeleton to the cytoskeleton. This process is reversible and is mediated by activation of the alpha IIb beta 3 integrin and subsequent actin polymerization and protein-tyrosine kinase stimulation. CDC42Hs might be a new component of a signaling complex containing specific cytoskeletal proteins and protein-tyrosine kinases that forms after activation of the alpha IIb beta 3 integrin in platelets.

Actins↗
PubMed1996

The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.

The glycoprotein (GP) Ib-IX complex is one of the major platelet membrane glycoproteins. Its extracellular domain binds von Willebrand factor at a site of injury, an interaction that leads to activation of intracellular pathways. Its intracellular domain associates tightly with the platelet cytoskeleton through actin-binding protein. The goal of the present study was to investigate the role of the cytoplasmic domain of the GP Ib-IX complex and its interaction with the cytoskeleton. Cultured cells were transfected with the cDNAs coding for GP Ib(beta), GP IX, and full-length or truncated forms of GP Ib(alpha). Western blots of detergent-insoluble fractions of Triton X-100-lysed cells showed that deletion of amino acids Trp-570 to Ser-590 from the cytoplasmic domain of GP IB(alpha) abolished the interaction of the entire GP Ib-IX complex with the cytoskeleton. Truncated GP Ib(alpha) that was unable to associate with the cytoskeleton retained its ability to associate with GP Ib(beta), to be inserted into the membrane, and to bind von Willebrand factor. Cells expressing GP Ib(alpha) changed their shape following adhesion to immobilized von Willebrand factor. Cells expressing truncated GP Ib(alpha) also changed their shape following adhesion but showed a very different morphology as compared to cells expressing full-length GP Ib(alpha). These results show that GP Ib-IX-von Willebrand factor interactions lead to cytoskeletal reorganizations, that the cytoplasmic domain of GP Ib(alpha) regulates these reorganizations, and that the cytoplasmic domain of GP Ib(alpha) is absolutely required for attachment of the GP Ib-IX complex to the cytoskeleton.

Amino Acid Sequence↗
PubMed1997

Reversible translocation of phosphoinositide 3-kinase to the cytoskeleton of ADP-aggregated human platelets occurs independently of Rho A and without synthesis of phosphatidylinositol (3,4)-bisphosphate.

The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha-p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3, 4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60(c-)src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.

Adenosine Diphosphate↗
PubMed1999

Rap1B and Rap2B translocation to the cytoskeleton by von Willebrand factor involves FcgammaII receptor-mediated protein tyrosine phosphorylation.

Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcgammaII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72(syk), the phospholipase Cgamma2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcgammaII receptor in the mechanism of platelet activation by vWF.

Blood Platelets↗
PubMed2000

The low M(r) protein-tyrosine phosphatase is involved in Rho-mediated cytoskeleton rearrangement after integrin and platelet-derived growth factor stimulation.

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. In fact, LMW-PTP is able to specifically bind and dephosphorylate activated PDGF receptor, thus modulating PDGF-induced mitogenesis. In particular, LMW-PTP is involved in pathways that regulate the transcription of the immediately early genes myc and fos in response to growth factor stimulation. Recently, we have found that LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its phosphorylation, LMW-PTP increases its catalytic activity about 20-fold. In this study, our interest was to investigate the role of LMW-PTP phosphorylation in cellular response to PDGF stimulation. To address this issue, we have transfected in NIH-3T3 cells a mutant form of LMW-PTP in which the c-Src phosphorylation sites (Tyr(131) and Tyr(132)) were mutated to alanine. We have established that LMW-PTP phosphorylation by c-Src after PDGF treatment strongly influences both cell adhesion and migration. In addition, we have discovered a new LMW-PTP substrate localized in the cytoskeleton that becomes tyrosine-phosphorylated after PDGF treatment: p190Rho-GAP. Hence, LMW-PTP plays multiple roles in PDGF receptor-mediated mitogenesis, since it can bind and dephosphorylate PDGF receptor, and, at the same time, the cytoskeleton-associated LMW-PTP, through the regulation of the p190Rho-GAP phosphorylation state, controls the cytoskeleton rearrangement in response to PDGF stimulation.

3T3 Cells↗
PubMed2000

T lymphocyte-triggering factor of african trypanosomes is associated with the flagellar fraction of the cytoskeleton and represents a new family of proteins that are present in several divergent eukaryotes.

The trypanosome cytoskeleton consists almost entirely of microtubule-based structures. Although alpha- and beta-tubulin from Trypanosoma brucei have been well characterized, much less is known about other cytoskeleton-associated proteins in trypanosomes. Using biochemical fractionation, we demonstrate here that T lymphocyte-triggering factor (TLTF) from T. brucei is a component of the detergent-resistant and Ca(2+)-resistant fraction of the parasite cytoskeleton. This fraction contains the flagellar apparatus and a subset of cytoskeletal protein complexes that together function in cell motility, cytokinesis, and organelle inheritance. We also show that TLTF-related genes are present in several highly divergent eukaryotic organisms. Although the function of the corresponding proteins is not known, the mammalian TLTF-like gene (GAS11; growth arrest-specific gene 11) is up-regulated in growth-arrested cells and is a candidate tumor suppressor (Whitmore, S. A., Settasatian, C., Crawford, J., Lower, K. M., McCallum, B., Seshadri, R., Cornelisse, C. J., Moerland, E. W., Cleton-Jansen, A. M., Tipping, A. J., Mathew, C. G., Savnio, M., Savoia, A., Verlander, P., Auerbach, A. D., Van Berkel, C., Pronk, J. C., Doggett, N. A., and Callen, D. F. (1998) Genomics 52, 325-331), suggestive of a role in coordinating cytoskeleton activities. Consistent with this possibility, we show that the human GAS11 protein contains a 144-amino acid domain that co-localizes with microtubules when fused to the green fluorescent protein and expressed in mammalian cells. These findings suggest that TLTF represents a newly defined protein family, whose members contribute to cytoskeleton function in species as diverse as protozoa and mammals.

Amino Acid Sequence↗
PubMed2002

Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion.

Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259-27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71Delta(110)) in the membrane cytoskeleton of human platelets. Both Dp71Delta(110) and utrophin sediment from lysed platelets along with the high speed detergent-insoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71Delta(110) and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombin-stimulated platelets. Immunoelectron microscopy provided further evidence that Dp71Delta(110) was localized to the submembranous cytoskeleton. In addition to Dp71Delta(110), platelets contained several components of the dystrophin-associated protein complex, including beta-dystroglycan and syntrophin. To better understand the potential function of Dp71Delta(110), collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx(3cv)) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx(3cv) mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71Delta(110) is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion.

Animals↗