Vinculin in relation to stress fibers in spread platelets

Nachmias, Vivianne T.; Golla, Rajasree

doi:10.1002/cm.970200303

Cited by 57 publications

(54 citation statements)

References 34 publications

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“…This also appears to occur in platelets during activation [5,25]. These different actin structures are known to be enriched in different actin-binding proteins.…”

Section: Introductionmentioning

confidence: 96%

“…Platelets are extremely rich in actin (estimated to be 20% of the soluble protein) and contain a wide range of actin-binding proteins [2]. During activation, these proteins interact with actin and each other to reorganize the actin filament structures into functional units that then mediate crucial events, such as: adherence to extracellular matrix and to other cells [3][4][5]; reorganization of the membrane skeleton [6]; extension of filopodia and lamellipodia to bring the activated platelet in contact with more distant cells [7]; contraction of the inner central cytoplasm facilitating the expulsion of granule contents such as growth factors and vasoactive substances [8]; and ultimately contractions of the whole cell which constrict the clot into a tightly adherent complex structure called a "scab" in lay terms. Each of these activities must involve the participation of different structures whose common element is filamentous actin.…”

Section: Introductionmentioning

confidence: 99%

“…None of these studies have looked comprehensively at the coordinated formation of these structures, nor have the actin filaments thought to be present in the lamellipodium that surround the platelet been visualized. Changes in distribution of individual proteins such as talin [4] and vinculin [5] have been elegantly described, but for many actin-binding proteins identified biochemically in platelets, distributions remain unknown. As a by-product of this investigation it was found that during the first 15 min of activation on glass, platelets elaborate four different actin-based structures: filopodia, a contractile ring, lamellipodia, filopodia, and stress fibers.…”

Section: Introductionmentioning

confidence: 99%

“…As a by-product of this investigation it was found that during the first 15 min of activation on glass, platelets elaborate four different actin-based structures: filopodia, a contractile ring, lamellipodia, filopodia, and stress fibers. By staining with phalloidin during fixation, these structures could be more clearly delineated than has been possible in the past [5,25]. Each of these four structures is directly related to a physiologic event in the homeostatic role of the platelet: shape change (filopodia, lamellipodia), adhesion (lamellipodia, adhesion plaques), degranulation (contractile ring) and contraction (stress-like fibers, filopodia).…”

Section: Introductionmentioning

confidence: 99%

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Cytoskeletal domains in the activated platelet

Bearer

1995

Cell Motil. Cytoskeleton

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Platelets circulate in the blood as discoid cells which, when activated, change shape by polymerizing actin into various structures, such as filopodia and stress fibers. In order to understand this process, it is necessary to determine how many other proteins are involved. As a first step in defining the full complement of actin-binding proteins in platelets, filamentous (F)-actin affinity chromatography was used. This approach identified >30 different proteins from ADP-activated human blood platelets which represented 4% of soluble protein. Although a number of these proteins are previously identified platelet actin-binding proteins, many others appeared to be novel. Fourteen different polyclonal antibodies were raised against these apparently novel proteins and used to sort them into nine categories based on their molecular weights and on their location in the sarcomere of striated muscle, in fibroblasts and in spreading platelets. Ninetythree percent of these proteins (13 of 14 proteins tested) were found to be associated with actinrich structures in vivo.Four distinct actin filament structures were found to form during the initial 15 min of activation on glass: filopodia, lamellipodia, a contractile ring encircling degranulating granules, and thick bundles of filaments resembling stress fibers. Actin-binding proteins not localized in the discoid cell became highly concentrated in one or another of these actin-based structures during spreading, such that each structure contains a different complement of proteins. These results present crucial information about the complexity of the platelet cytoskeleton, demonstrating that four different actin-based structures form during the first 15 min of surface activation, and that there remain many as yet uncharacterized proteins awaiting further investigation that are differentially involved in this process.

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“…This also appears to occur in platelets during activation [5,25]. These different actin structures are known to be enriched in different actin-binding proteins.…”

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cytoskeletal domains in the activated platelet

Bearer

1995

Cell Motil. Cytoskeleton

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“…, which forms a 1:1 complex with actin monomers, is believed to be involved in preventing actin polymerization (1)(2)(3)(4)(5)(6). Dissociation constants of the actin⅐T␤4 complex were found to be in the range of 0.4 -0.7 M for platelet actin and 0.7-2.0 M for muscle actin (4,7).…”

mentioning

confidence: 99%

Identification of Contact Sites in the Actin-Thymosin β4 Complex by Distance-dependent Thiol Cross-linking

Reichert

Heintz

Echner

et al. 1996

Journal of Biological Chemistry

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Binding sites of actin and thymosin ␤4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys 10 , Cys 374 , or to the sulfur atom of the ATP analog adenosine 5-O-(thiotriphosphate) (ATP␥S), the actin derivatives were reacted with synthetic thymosin ␤4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate crosslinking as followed by UV spectroscopy was found for Cys 374 of actin and Cys 6 of thymosin ␤4, indicating that the N terminus of thymosin ␤4 is in close proximity ( 9.2 Å) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin ␤4 analogs when the cross-linkers were anchored at Cys 10 of actin. A second contact site was identified by cross-linking of Cys 17 and Cys 28 in thymosin ␤4 with the ATP␥S derivative bound to actin, indicating that the hexamotif of thymosin ␤4 (positions 17-22) is in close proximity ( 9.2 Å) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin ␤4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.The physiological conditions within nonmuscle cells favor the assembly of actin monomers. Therefore, the pool of monomeric actin has to be maintained by complexation with small G-actin binding proteins. Particularly thymosin ␤4 (T␤4) 1 , which forms a 1:1 complex with actin monomers, is believed to be involved in preventing actin polymerization (1-6). Dissociation constants of the actin⅐T␤4 complex were found to be in the range of 0.4 -0.7 M for platelet actin and 0.7-2.0 M for muscle actin (4, 7). While complexed with T␤4, the exchange of the bound nucleotide in actin is retarded (8). In a detailed study, Vancompernolle et al. (9) have shown that binding of T␤4 to actin is mainly mediated by the hexamotif LKKTET (17-22), since loss of this sequence is paralleled by an almost complete loss of inhibitory activity. Alterations in the N-terminal part (1-16) of the peptide strongly influence the inhibitory activity of T␤4, whereas alterations in the C-terminal part (31-43) seem to be of minor importance (9). As shown by 1 H NMR spectroscopy (10, 11) T␤4 does not contain an ordered conformation in aqueous solution but tends to form an ␣-helical conformation between residues 5 and 16 (11). It has been proposed that T␤4 is likely to adopt a unique conformation upon binding actin (12).One of the binding sites of T␤4 on the actin molecule seems to be located in subdomain 1 as suggested by cross-linking studies (13,14). In order to gain more knowledge about contact sites in the actin⅐T␤4 complex, we performed a structural analysis using bifunctional thiol-specific reagents of the type alkylene-bis-[5-dithio-(2-nitrobenzoic acid)] for intermolecular crosslinki...

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