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Recently we showed that signaling across beta3-integrin leads to activation of calpain and formation of integrin clusters that are involved in Rac activation. The subsequent activation of Rac and Rho leads to the formation of focal complexes and focal adhesions, respectively. The goal of the present study was to determine whether different proteins link the integrin to the cytoskeleton in the different complexes. We show that talin is present in focal adhesions but not in the calpain-induced clusters. alpha-Actinin colocalized with integrin at various sites, including the calpain-induced clusters. Skelemin, a protein shown recently to interact with beta1- and beta3-integrin in vitro, colocalized with integrin in calpain-induced clusters but was absent from focal adhesions. Cells transiently expressing skelemin C2 motifs, which contain the integrin binding site, failed to form integrin clusters or to spread on a substrate for beta1- and beta3-integrins. These results 1) suggest a dynamic reorganization of integrin complexes during cell spreading, 2) show that different cytoskeletal proteins link integrins in different complexes, and 3) demonstrate that skelemin is responsible for linking integrin to the calpain-induced clusters, and 4) show that the integrin-skelemin interaction is essential for transmission of signals leading to the initial steps of cell spreading.

Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration, spreading, proliferation, and differentiation. The details of the signaling events that induce these changes in cell behavior are not well understood but they appear to involve activation of Rho family members which activate signaling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. The result is the formation of focal complexes, focal adhesions, and bundles and networks of actin filaments that allow the cell to spread. The present study shows that mu-calpain is active in adherent cells, that it cleaves proteins known to be present in focal complexes and focal adhesions, and that overexpression of mu-calpain increased the cleavage of these proteins, induced an overspread morphology and induced an increased number of stress fibers and focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by expression of a dominant negative form of mu-calpain resulted in an inability of cells to spread or to form focal adhesions, actin filament networks, or stress fibers. Cells expressing constitutively active Rac1 could still form focal complexes and actin filament networks (but not focal adhesions or stress fibers) in the presence of calpain inhibitors; cells expressing constitutively active RhoA could form focal adhesions and stress fibers. Taken together, these data indicate that calpain plays an important role in regulating the formation of focal adhesions and Rac- and Rho-induced cytoskeletal reorganizations and that it does so by acting at sites upstream of both Rac1 and RhoA.

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.

Previously, we showed that a subpopulation of the major platelet integrin, alphaIIbbeta3, co-sediments from detergent lysates with talin and other membrane skeleton proteins. Once alphaIIbbeta3 has bound adhesive ligand in a platelet aggregate, the detergent-insoluble alphaIIbbeta3 redistributes (along with the detergent-insoluble membrane skeleton proteins and a variety of signaling molecules) to a fraction that contains cytoplasmic actin filaments. Concomitantly, certain signaling molecules are activated. The present study shows that, in intact platelets, alphaIIbbeta3 forms clusters when occupied by ligand and is selectively moved into the open canalicular system; alphaIIbbeta3 that has not bound ligand remains diffusely distributed at the periphery of the cell. When cytoplasmic actin filaments are depolymerized by cytochalasins, the ability of alphaIIbbeta3 to bind ligand is decreased, and the movement of ligand-occupied alphaIIbbeta3 is prevented. Together with the previous findings, these results suggest that (i) membrane skeleton-associated alphaIIbbeta3 is selectively induced to bind ligand in activated platelets, (ii) ligand-induced transmembrane signaling causes an altered association of membrane skeleton-associated alphaIIbbeta3 with the cytoplasmic component of the cytoskeleton, (iii) ligand-induced cytoskeletal reorganizations stabilize the interaction between ligand and integrin, and (iv) ligand-occupancy triggers cytoskeletal reorganizations that result in selective movements of occupied ligand.

The platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments which fill the cytoplasm and mediate contractile events. The other is the membrane skeleton, which coats the plasma membrane and regulates properties of the membrane such as its contours and stability. In the unstimulated platelet, only 30-40% of the actin is polymerized into filaments; the rest is thought to be prevented from polymerizing by the association of thymosin beta 4 with monomeric actin and by the association of gelsolin with the barbed ends of pre-existing actin filaments. When platelets are activated, there is a rapid increase in actin polymerization; new filaments fill the extending filopodia and form a network at the periphery of the platelet. As a result of activation, myosin binds to cytoplasmic actin filaments, causing them to move towards the center of the platelet. As platelets aggregate, additional cytoskeletal reorganizations occur: GP IIb-IIIa associates with adhesive ligand in a platelet aggregate; this results in the association of GP IIb-IIIa, membrane skeleton proteins, and signaling molecules with cytoplasmic actin. Future studies should help to elucidate the significance of the cytoskeleton in regulating signal transduction events in platelets.

The platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments that fill the cytoplasm and mediate contractile events. The other is the membrane skeleton that coats the plasma membrane and regulates properties of the membrane such as its contours and stability and the lateral distribution of membrane glycoproteins. Recent work reviewed in this article indicates that the GP IIb-IIIa complex can associate with the membrane skeleton. Upon platelet activation, GP IIb-IIIa becomes competent to bind its adhesive ligand, fibrinogen. This induces a reorganization of the cytoskeleton such that the membrane skeletal proteins with which GP IIb-IIIa is associated become associated with underlying cytoplasmic filaments. As in focal contacts of cultured cells, this ligand-induced association of GP IIb-IIIa with cytoplasmic actin filaments regulates the ability of GP IIb-IIIa to bind adhesive ligand. Intracellular enzymes that are activated as a consequence of ligand binding to the GP IIb-IIIa complex include tyrosine kinase(s) and calpain, making these potential candidates for enzymes inducing the two-way signaling across the membrane. Additional candidates include phosphoinositide 3-kinase and protein kinase C, other enzymes that have been detected in focal contacts of aggregating platelets. Future studies identifying interactions between the GP IIb-IIIa complex and membrane skeletal proteins should help to further elucidate the significance of the GP IIb-IIIa in cytoskeleton interaction in regulating integrin-mediated transmembrane signaling in platelets.

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)-polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X-100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N-ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.

Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane.

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.