RESUMO
Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders such as cardiac failure and pulmonary injury. The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances, implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of ß-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with ß-integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation. Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching, we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases ß-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.
Assuntos
Actinas/metabolismo , Proteínas Contráteis/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Cadeias beta de Integrinas/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Animais , Sítios de Ligação , Filaminas , Fluorescência , Humanos , Ligantes , Miosina Tipo II/metabolismo , Ligação Proteica , CoelhosRESUMO
BACKGROUND: Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. METHODS: We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. RESULTS: Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera. CONCLUSION: We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.
Assuntos
Monócitos/imunologia , Western Blotting , Linhagem Celular , Citometria de Fluxo , Humanos , Microscopia Eletrônica , Tamanho da Partícula , ProteômicaRESUMO
The cellular and molecular basis of the intricate process by which megakaryocytes (MKs) form and release platelets remains poorly understood. Work has shown that proplatelets, long cytoplasmic extensions made by mature MKs, are essential intermediates in platelet biogenesis. Microtubules are the main structural component of proplatelets and it is microtubule sliding, driven by dynein motors within cortical bundles, which elongates and thins proplatelets. Kinesin motors carry their cargo of platelet-specific granules and organelles into the proplatelets using the microtubule bundles as tracks. Extension of proplatelets is associated with repeated actin-dependent bending and bifurcation, which results in considerable amplification of free proplatelet ends. Large proplatelets, dissociated from the residual MK cell body, have the capacity to mature platelets. Only the ends of proplatelets form marginal microtubule coils similar to that observed in mature platelets, demonstrating that platelet formation completes primarily at proplatelet ends. Understanding the molecular basis of platelet formation requires detailed knowledge of how the MK microtubule machinery interacts to generate proplatelets and release platelets.
Assuntos
Plaquetas/fisiologia , Citoplasma/fisiologia , Dineínas/fisiologia , Humanos , Microtúbulos/fisiologiaRESUMO
We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditions is a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas Contráteis/química , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Animais , Filaminas , Humanos , Ligação Proteica , CoelhosRESUMO
The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex-specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex-mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Proteínas Contráteis/metabolismo , Proteínas do Citoesqueleto , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Movimento Celular/fisiologia , Filaminas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Melanoma , Microscopia Eletrônica/métodos , Células Tumorais CultivadasRESUMO
The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Desenvolvimento Embrionário e Fetal , Proteínas do Tecido Nervoso/fisiologia , Animais , Linhagem Celular , Linhagem Celular Transformada , Fibroblastos , Marcação de Genes , Listeria/fisiologia , Camundongos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Recombinação Genética , Shigella flexneri/fisiologia , Vaccinia virus/fisiologia , Proteína Neuronal da Síndrome de Wiskott-AldrichRESUMO
Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP(2) binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca(2+)] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5-20 microm peptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of polyphosphoinositides in vivo.
Assuntos
Peptídeos/química , Fosfatos de Fosfatidilinositol/metabolismo , Células 3T3 , Actinas/metabolismo , Animais , Plaquetas/metabolismo , Cálcio/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Humanos , Immunoblotting , Camundongos , Microscopia de Fluorescência , Neutrófilos/metabolismo , Octoxinol/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Transdução de Sinais , Espectrometria de Fluorescência , Fatores de Tempo , Células Tumorais CultivadasRESUMO
BACKGROUND: Mammalian megakaryocytes release blood platelets through a remarkable process of cytoplasmic fragmentation and de novo assembly of a marginal microtubule band. Cell-specific components of this process include the divergent beta-tubulin isoform beta1 that is expressed exclusively, and is the predominant isoform, in platelets and megakaryocytes. The functional significance of this restricted expression, and indeed of the surprisingly large repertoire of metazoan tubulin genes, is unclear. Fungal tubulin isoforms appear to be functionally redundant, and all mammalian beta-tubulins can assemble in a variety of microtubules, whereas selected fly and worm beta-tubulins are essential in spermatogenesis and neurogenesis. To address the essential role of beta1-tubulin in its natural context, we generated mice with targeted gene disruption. RESULTS: beta1-tubulin(-/-) mice have thrombocytopenia resulting from a defect in generating proplatelets, the immediate precursors of blood platelets. Circulating platelets lack the characteristic discoid shape and have defective marginal bands with reduced microtubule coilings. beta1-tubulin(-/-) mice also have a prolonged bleeding time, and their platelets show an attenuated response to thrombin. Two alternative tubulin isoforms, beta2 and beta5, are overexpressed, and the total beta-tubulin content of beta1-tubulin(-/-) megakaryocytes is normal. However, these isoforms assemble much less efficiently into platelet microtubules and are thus unable to compensate completely for the absence of beta1-tubulin. CONCLUSIONS: This is the first genetic study to address the essential functions of a mammalian tubulin isoform in vivo. The results establish a specialized role for beta1-tubulin in platelet synthesis, structure, and function.
Assuntos
Plaquetas/fisiologia , Tubulina (Proteína)/fisiologia , Animais , Plaquetas/citologia , Linhagem da Célula , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Trombocitopenia/etiologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto , Proteínas do Tecido Nervoso/metabolismo , Pseudópodes/metabolismo , Células 3T3 , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Animais , Western Blotting , Bradicinina/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Glutationa Transferase/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Shigella/metabolismo , Transdução de Sinais , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Various agonists but also chilling cause blood platelets to increase cytosolic calcium, polymerize actin, and change shape. We report that cold increases barbed end nucleation sites in octyl glucoside-permeabilized platelets by 3-fold, enabling analysis of the intermediates of this response. Although chilling does not change polyphosphoinositide (ppI) levels, a ppI-binding peptide completely inhibits cold-induced nucleation. The C terminus of N-WASp, which inhibits the Arp2/3 complex, blocks nucleation by 40%; GDPbetaS, N17Rac and N17Cdc42 have no effects. Some gelsolin translocates to the detergent-insoluble cytoskeleton after cooling. Chilled platelets from gelsolin-deficient mice have approximately 50% fewer new actin nuclei compared with platelets from wild-type mice. EGTA completely inhibits gelsolin translocation into the cytoskeleton, and the small amount of gelsolin initially there becomes soluble. Chilling releases adducin from the detergent-resistant cytoskeleton. We conclude that platelet actin filament assembly induced by cooling involves ppI-mediated actin filament barbed end uncapping and de novo nucleation independently of surface receptors or downstream signaling intermediates besides calcium. The actin-related changes occur in platelets at temperatures below 37 degrees C, suggesting that the platelet may be more activable at temperatures at the body surface than at core temperature, thereby favoring superficial hemostasis over internal thrombosis.
Assuntos
Actinas/metabolismo , Plaquetas/ultraestrutura , Temperatura Baixa , Proteínas do Citoesqueleto , Guanosina Difosfato/análogos & derivados , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Actinas/ultraestrutura , Animais , Plaquetas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Gelsolina/metabolismo , Glucosídeos , Guanosina Difosfato/metabolismo , Hemostasia , Humanos , Camundongos , Proteínas do Tecido Nervoso/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Polímeros , Coelhos , Tionucleotídeos/metabolismo , Proteína Neuronal da Síndrome de Wiskott-AldrichRESUMO
The neurofibromatosis 2 protein product merlin, named for its relatedness to the ezrin, radixin and moesin (ERM) family of proteins, is a tumour suppressor whose absence results in the occurrence of multiple tumours of the nervous system, particularly schwannomas and meningiomas. Merlin's similarity to ERMs suggests that it might share functions, acting as a link between cytoskeletal components and the cell membrane. The N-terminus of merlin has strong sequence identity to the N-terminal actin-binding region of ezrin; here we describe in detail the merlin-actin interaction. Employing standard actin co-sedimentation assays, we have determined that merlin isoform 2 binds F-actin with an apparent binding constant of 3.6 microM and a stoichiometry of 1 mol of merlin per 11.5 mol of actin in filaments at saturation. Further, solid-phase binding assays reveal that merlin isoforms 1 and 2 bind actin filaments differentially, suggesting that the intramolecular interactions in isoform 1 might hinder its ability to bind actin. However, merlin does not bind G-actin. Studies of actin filament dynamics show that merlin slows filament disassembly with no influence on the assembly rate, indicating that merlin binds along actin filament lengths. This conclusion is supported by electron microscopy, which demonstrates that merlin binds periodically along cytoskeletal actin filaments. Comparison of these findings with those reported for ERM proteins reveal a distinct role for merlin in actin filament dynamics.
Assuntos
Actinas/metabolismo , Proteínas de Membrana/metabolismo , Neurofibromatose 2/metabolismo , Animais , Citoesqueleto/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Membrana/ultraestrutura , Meningioma/metabolismo , Meningioma/ultraestrutura , Microscopia Eletrônica , Neurofibromina 2 , Ligação Proteica , Isoformas de Proteínas/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais CultivadasRESUMO
Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.
Assuntos
Proteínas Contráteis/fisiologia , Proteínas dos Microfilamentos/fisiologia , Transdução de Sinais/fisiologia , Membrana Celular/fisiologia , FilaminasRESUMO
To understand the microscopic mechanical properties of actin networks, we monitor the motion of embedded particles with controlled surface properties. The highly resolved Brownian motions of these particles reveal the viscoelastic character of the microenvironments around them. In both non-cross-linked and highly cross-linked actin networks, particles that bind F-actin report viscoelastic moduli comparable to those determined by macroscopic rheology experiments. By contrast, particles modified to prevent actin binding have weak microenvironments that are surprisingly insensitive to the introduction of filament cross-links. Even when adjacent in the same cross-linked gel, actin-binding and nonbinding particles report viscoelastic moduli that differ by two orders of magnitude at low frequencies (0.5-1.5 rad/s) but converge at high frequencies (> 10(4) rad/s). For all particle chemistries, electron and light microscopies show no F-actin recruitment or depletion, so F-actin microheterogeneities cannot explain the deep penetration (approximately 100 nm) of nonbinding particles. Instead, we hypothesize that a local depletion of cross-linking around nonbinding particles explains the phenomena. With implications for organelle mobility in cells, our results show that actin binding is required for microenvironments to reflect macroscopic properties, and conversely, releasing actin enhances particle mobility beyond the effects of mere biochemical untethering.
Assuntos
Actinas/química , Actinas/ultraestrutura , Adsorção , Animais , Biotinilação , Reagentes de Ligações Cruzadas , Microscopia Eletrônica , Músculo Esquelético , Polilisina , Poliestirenos , Ligação Proteica , Coelhos , Soroalbumina Bovina , Propriedades de SuperfícieRESUMO
How platelet shape change initiated by a collagen-related peptide (CRP) specific for the GPVI/FcR gamma-chain complex (GPVI/FcR gamma-chain) is coupled to SLP-76, phosphoinositide (PI) 3-kinase, and gelsolin is reported. As shown by video microscopy, platelets rapidly round and grow dynamic filopodial projections that rotate around the periphery of the cell after they contact a CRP-coated surface. Lamellae subsequently spread between the projections. All the actin-driven shape changes require SLP-76 expression. SLP-76 is essential for the Ca(++) mobilization induced by CRP, whereas PI 3-kinase only modulates it. The extension of lamellae requires net actin assembly and an exposure of actin filament barbed ends downstream of PI 3-kinase. Gelsolin expression is also required for the extension of lamellae, but not for the formation of filopodia. Altogether, the data describe the role of SLP-76 in the platelet activation initiated by GPVI/FcR gamma-chain and the roles of PI 3-kinase and gelsolin in lamellae spreading. (Blood. 2000;96:3786-3792)
Assuntos
Plaquetas/efeitos dos fármacos , Gelsolina/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores de IgG/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/efeitos dos fármacos , Actinas/metabolismo , Actinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Plaquetas/citologia , Plaquetas/ultraestrutura , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Colágeno/química , Colágeno/metabolismo , Colágeno/farmacologia , Gelsolina/farmacologia , Humanos , Integrinas/metabolismo , Integrinas/fisiologia , Camundongos , Camundongos Mutantes , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Colágeno , Receptores de IgG/metabolismo , Receptores de Trombina/fisiologia , Transdução de SinaisAssuntos
Guanosina Trifosfato/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Plaquetas/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo , Receptor PAR-1 , Receptores de Trombina/metabolismoRESUMO
Cycling of actin subunits between monomeric and filamentous phases is essential for cell crawling behavior. We investigated actin filament turnover rates, length, number, barbed end exposure, and binding of cofilin in bovine arterial endothelial cells moving at different speeds depending on their position in a confluent monolayer. Fast-translocating cells near the wound edge have short filament lifetimes compared with turnover values that proportionately increase in slower moving cells situated at increasing distances from the wound border. Contrasted with slow cells exhibiting slow actin filament turnover speeds, fast cells have less polymerized actin, shorter actin filaments, more free barbed ends, and less actin-associated cofilin. Cultured primary fibroblasts manifest identical relationships between speed and actin turnover as the endothelial cells, and fast fibroblasts expressing gelsolin have higher actin turnover rates than slow fibroblasts that lack this actin-severing protein. These results implicate actin filament severing as an important control mechanism for actin cycling in cells.
Assuntos
Actinas/metabolismo , Fatores de Despolimerização de Actina , Difosfato de Adenosina/análise , Animais , Bovinos , Células Cultivadas , Fluorescência , Proteínas dos Microfilamentos/análiseRESUMO
The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin-immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin-bound ligands were eluted with ethylenediaminetetraacetic acid. An approximately 210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti-human PSGL-1 antibody and could be blotted with P-selectin-IgG. Under reducing conditions, both the predicted PSGL-1 approximately 210-kD dimer and the approximately 120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25-100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti-PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.
Assuntos
Plaquetas/metabolismo , Glicoproteínas de Membrana/sangue , Selectina-P/sangue , Animais , Anticorpos Monoclonais , Sequência de Bases , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Primers do DNA/genética , Endotélio Vascular/fisiologia , Expressão Gênica , Humanos , Leucócitos/metabolismo , Ligantes , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Ativação Plaquetária , RNA Mensageiro/sangue , RNA Mensageiro/genéticaRESUMO
Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.
Assuntos
Actinas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Plaquetas/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Trombina/metabolismo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1-mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.