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M C Beckerle

Publications and source records attributed to M C Beckerle.

53 records · Page 3Linked to original sources
PubMed1993

The LIM motif defines a specific zinc-binding protein domain.

The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.

Amino Acid Sequence↗
PubMed1993

Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets.

Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) (also referred to as alpha IIb beta 3 integrin), becomes incorporated into the detergent-insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-IIIa is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, approximately 22% of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, alpha-actinin, or vinculin in the complex. We found that the cytoskeleton-associated GPIIb-IIIa is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPIIb-IIIa associated with the alpha-granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, alpha-granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPIIb-IIIa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPIIb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-IIIa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.

Actins↗
PubMed1993

Talin distribution and phosphorylation in thrombin-activated platelets.

We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333-3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with thrombin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are deficient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.

Biological Transport↗
PubMed1992

An interaction between zyxin and alpha-actinin.

Zyxin is an 82-kD protein first identified as a component of adhesion plaques and the termini of stress fibers near where they associate with the cytoplasmic face of the adhesive membrane. We report here that zyxin interacts with the actin cross-linking protein alpha-actinin. Zyxin cosediments with filamentous actin in an alpha-actinin-dependent manner and an association between zyxin and alpha-actinin is observed in solution by analytical gel filtration. The specificity of the interaction between zyxin and alpha-actinin was demonstrated by blot overlay experiments in which 125I-zyxin recognizes most prominently alpha-actinin among a complex mixture of proteins extracted from avian smooth muscle. By these blot overlay binding studies, we determined that zyxin interacts with the NH2-terminal 27-kD domain of alpha-actinin, a region that also contains the actin binding site. Solid phase binding assays were performed to evaluate further the specificity of the binding and to determine the affinity of the zyxin-alpha-actinin interaction. By these approaches we have demonstrated a specific, saturable, moderate-affinity interaction between zyxin and alpha-actinin. Furthermore, double-label immunofluorescence reveals that zyxin and alpha-actinin exhibit extensive overlap in their subcellular distributions in both chicken embryo fibroblasts and pigmented retinal epithelial cells. The significant colocalization of the two proteins is consistent with the possibility that the interaction between zyxin and alpha-actinin has a biologically relevant role in coordinating membrane-cytoskeletal interactions.

Actinin↗
PubMed1992

Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton.

Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847-5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2-terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine-rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Purification and characterization of zyxin, an 82,000-dalton component of adherens junctions.

We describe here the purification and characterization of a recently identified adherens junction protein that has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels (Beckerle, M. C. (1986) J. Cell Biol. 103, 1679-1687). The 82-kDa protein was isolated from avian smooth muscle by a low ionic strength alkaline pH extraction followed by ammonium sulfate fractionation. Sequential chromatographic separation using DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite resins results in a purified 82-kDa protein. The 82-kDa protein has a Stokes radius of 5.6 nm and a relative sedimentation coefficient of 3.0 S. The calculated native molecular mass of the protein based on its hydrodynamic properties is 69 kDa, and the derived frictional ratio (f/fo) is 2.1. The protein does not focus discretely by isoelectric-focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis; there are numerous isoelectric point variants in the range of 6.4-7.2, with the average isoelectric point being 6.9. The 82-kDa protein is phosphorylated in vivo and appears to be a cytoplasmic component of adherens junctions. The properties of the 82-kDa protein distinguish it from other known adherens junction proteins of this molecular mass. In fibroblasts, the 82-kDa protein is found in adhesion plaques as well as along actin-containing stress fibers near where they terminate at sites of cell-substratum adhesion. It is also found in the cell-cell adherens junctions of pigmented retinal epithelial cells and the dense plaques of smooth muscle cells. Since the 82-kDa protein is found at both cell-substratum and cell-cell adherens junctions, we propose to call it zyxin, meaning a joining, to indicate that it is found at regions where extracellular ligands are structurally and functionally joined to the cytoskeleton.

Animals↗
PubMed1990

The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.

Animals↗
PubMed1989

Activation-dependent redistribution of the adhesion plaque protein, talin, in intact human platelets.

Talin is a high molecular weight protein localized at adhesion plaques in fibroblasts. It binds vinculin and integrin and appears to participate in generating a transmembrane connection between the extracellular matrix and the cytoskeleton. We have recently shown that talin is an abundant protein in platelets, cells highly specialized for regulated adhesion. Although talin constitutes greater than 3% of the total protein in intact human platelets, its location within the cells had not been defined. In the work reported here, we have investigated the distribution of talin in resting and activated human platelets by immunofluorescence and immunoelectron microscopy. We have found that talin undergoes an activation-dependent change in its subcellular location. In resting platelets, which are nonadhesive, talin is uniformly distributed throughout the cytoplasm. In contrast, in thrombin- and glass-activated, substratum-adherent platelets, talin is concentrated at the cytoplasmic face of the plasma membrane. This dramatic, regulated redistribution of talin raises the possibility that talin plays a role in the controlled development of platelet adhesion.

Blood Platelets↗
PubMed1987

Colocalization of calcium-dependent protease II and one of its substrates at sites of cell adhesion.

Adhesion plaques, specialized regions of the plasma membrane where a cell contacts its substratum, are dynamic structures. However, little is known about how the protein-protein interactions that occur at adhesion plaques are controlled. One mechanism by which a cell might modulate its associations with the substratum is by selective, regulated proteolysis of an adhesion plaque component. Here we show that the catalytic subunit of the calcium-dependent protease type II (CDP-II) is localized in adhesion plaques of several cell types (BS-C-1, EBTr, and MDBK). We have compared the susceptibility of the adhesion plaque constituents vinculin, talin, and alpha-actinin to calcium-dependent proteolysis in vitro and have found talin to be the preferred substrate for CDP-II. The colocalization of a calcium-requiring proteolytic enzyme and talin in adhesion plaques raises the possibility that calcium-dependent proteolytic activity provides a mechanism for regulating some aspect of adhesion plaque physiology and function via cleavage of talin.

Actinin↗
PubMed1986

Demonstration of a relationship between talin and P235, a major substrate of the calcium-dependent protease in platelets.

Talin is a 225,000-Dalton protein we have purified from smooth muscle. In chick embryo fibroblasts talin is found in adhesion plaques (focal contacts), areas where the cell is closely apposed to the substratum. In comparison with other cytoskeletal proteins, we found talin to be unusually susceptible to proteolysis and have identified a 190,000-Dalton proteolytic fragment of talin in the immunoblots of many tissues. These observations raised the possibility that the cleavage of talin to this fragment has physiological relevance. One system that we have investigated in which significant proteolysis occurs is platelets. During platelet activation several high-molecular-weight proteins are cleaved to lower-molecular-weight forms. Here we demonstrate that talin is closely related to one of these platelet high-molecular-weight proteins, P235. The purification of talin is comparable to that developed for P235, and the two proteins have similar biophysical properties. In addition, antibodies raised against chicken gizzard talin recognize P235 in purified form as well as in crude platelet extracts. The platelet protein also resembles smooth-muscle talin in its susceptibility to endogenous proteolysis: P235 is rapidly cleaved to a 190-200 kD polypeptide by a calcium-activated protease found in platelet extracts. Moreover, partial proteolysis of P235 and talin with chymotrypsin, elastase, or trypsin also generates remarkably similar one-dimensional peptide maps. Because of their similar biophysical properties, immunological crossreactivity, and similar one-dimensional partial peptide maps, we conclude that P235 is the platelet form of talin.

Animals↗
PubMed1986

Identification of a new protein localized at sites of cell-substrate adhesion.

A new protein found at sites of cell-substrate adhesion has been identified by analysis of a nonimmune rabbit serum. By indirect immunofluorescence this serum stains focal contacts (adhesion plaques) and the associated termini of actin filament bundles in cultured chicken cells. Western immunoblot analysis of total chick embryo fibroblast protein demonstrated an 82-kD polypeptide to be the major protein recognized by the unfractionated serum. This 82-kD protein is immunologically distinct from other known adhesion plaque proteins such as vinculin, talin, alpha-actinin, and fimbrin. Antibody affinity-purified against the electrophoretically isolated, nitrocellulose-bound 82-kD protein retained the ability to stain the area of the adhesion plaque, which confirms that the 82-kD protein is indeed a constituent of the focal contact. The 82-kD polypeptide has a basic isoelectric point relative to actin and fibronectin, and it appears to be very low in abundance. The 82-kD protein is ubiquitous in chicken embryo tissues. However, it appears to be more abundant in fibroblasts and smooth muscle than in brain or liver. Intermediate levels of the protein were detected in skeletal and cardiac muscle. The subcellular distribution of the 82-kD protein raises the possibility that this polypeptide is involved in linking actin filaments to the plasma membrane at sites of substrate attachment or regulating these dynamic interactions.

Actins↗
PubMed1984

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells.

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.

Animals↗
PubMed1983

Analysis of the role of microtubules and actin in erythrophore intracellular motility.

The Holocentrus erythrophore, a red pigment cell, represents a model system for the study of organized intracellular transport. We have investigated the possibility that microtubules and actin are integral components of the pigment translocating motility machine. By creating cells that have total or partial loss of the microtubule framework we have demonstrated that the presence of microtubules is essential for organized, radial transport of the pigment granules. However, in the absence of microtubules, some undirected movement of the pigment can be stimulated; this suggests that a nonmicrotubular component of the cytoplast is responsible, at least in part, for the generation of motive force. In order to test the hypothesis that this component consists of actin or actomyosin, we examined the effects of probes for these classical motility proteins. Neither microinjection of phalloidin, DNase I or N-ethylmaleimide-modified heavy meromyosin nor exogenous application of cytochalasin B has any effect on pigment motion, although these materials do block the actin-mediated motility of other systems in our hands. Therefore, intracellular particle transport in erythrophores does not appear to be actin or actomyosin-based.

Actins↗
PubMedDate not supplied

Identification of talin as a major cytoplasmic protein implicated in platelet activation.

During platelet activation there is a major reorganization in the platelet cytoskeleton that accompanies a rapid change in platelet shape. Many of the events associated with activation are attributed to a rise in calcium concentration within the platelet cytoplasm. One direct consequence of the elevated calcium is the activation of a calcium-dependent protease that cleaves a major platelet protein of relative molecular mass (Mr) approximately 235,000 (235K) to 200K. This protein, P235, has been purified and reported to interact with actin, but the significance of the proteolytic cleavage is unknown. Talin, a cytoskeletal protein in smooth muscle and fibroblasts, binds vinculin and, together with vinculin, is localized in fibroblasts at sites of actin-membrane attachment. Talin and P235 have similar purification procedures, sedimentation coefficients and Stokes' radii (ref. 6 and Molony et al., unpublished observations). Of particular significance, talin is readily cleaved by proteases from approximately 215K to a fragment of approximately 190K. Given these similarities we have investigated the possible relationship between these proteins. Here we demonstrate that platelet P235 is recognized by anti-talin antibody and that it binds vinculin. Both proteins are cleaved in vitro by the calcium-activated protease to yield similar fragments. We conclude that P235 corresponds to the platelet form of talin.

Actins↗
PubMedDate not supplied

Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage.

Many observations suggest the presence of transmembrane linkages between the cytoskeleton and the extracellular matrix. In fibroblasts both light and electron microscopic observations reveal a co-alignment between actin filaments at the cell surface and extracellular fibronectin. These associations are seen at sites of cell matrix interaction, frequently along stress fibres and sometimes where these bundles of microfilaments terminate at adhesion plaques (focal contacts). Non-morphological evidence also indicates a functional linkage between the cytoskeleton and extracellular matrix. Addition of fibronectin to transformed cells induces flattening of the cells and a reorganization of the actin cytoskeleton, with the concomitant appearance of arrays of stress fibres. Conversely, disruption of the actin cytoskeleton by treatment with cytochalasin B leads to release of fibronectin from the cell surface. As yet, there is no detailed knowledge of the molecules involved in this transmembrane linkage, although several proteins have been suggested as candidates in the chain of attachment between bundles of actin filaments and the cytoplasmic face of the plasma membrane: these include vinculin, alpha-actinin and talin, each one having been identified at regions where bundles of actin filaments interact with the plasma membrane and underlying cell-surface fibronectin. Recently, the cell-substrate attachment (CSAT) antigen has been identified as a plasma membrane receptor for fibronectin, raising the possibility that this glycoprotein complex may serve as a bridge between fibronectin and one or more of the underlying cytoskeletal components mentioned. Here we have investigated the interaction of the purified CSAT antigen with these cytoskeletal components, and we demonstrate an interaction specifically between the CSAT antigen and talin.

Animals↗