scholarly journals Cytoskeletal reorganization of human platelets induced by the protein phosphatase 1/2 A inhibitors okadaic acid and calyculin A

1995 ◽  
Vol 307 (2) ◽  
pp. 439-449 ◽  
Author(s):  
Y Yano ◽  
M Sakon ◽  
J Kambayashi ◽  
T Kawasaki ◽  
T Senda ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Human Platelets ◽  
Calyculin A ◽  
Cytoskeletal Reorganization ◽  
Microtubule Organization ◽  
Electron Microscopic ◽  
Activated Platelets

Okadaic acid (OA) and calyculin A (CLA), which are potent and specific inhibitors of serine/threonine protein phosphatases type 1 and 2A, have been shown to induce drastic changes in platelet morphology. The aim of this study was to analyse the molecular mechanisms of OA- or CLA-induced cytoskeletal reorganization, with a specific focus on microtubules and actin filaments. Confocal fluorescence microscopy revealed that OA or CLA altered the distribution of microtubules from marginal band arrangements to homogeneous patterns, consistent with the transmission-electron-microscopic finding that microtubules were fragmented and redistributed into pseudopod-like processes. In thrombin-activated platelets, OA or CLA induced extremely long pseudopods containing an array of microtubules and actin filaments, and a condensed mass of actin filaments in the centre of platelets. OA or CLA induced the constriction of actin filaments without an increase in filamentous (F)-actin, and also rather significantly inhibited actin polymerization in thrombin-activated platelets. Furthermore, neither OA or CLA enhanced phosphorylation of myosin light chain (MLC). By immunoprecipitation of platelet lysate with anti-alpha-tubulin antibody, a 90 kDa protein was co-precipitated with tubulin and was predominantly phosphorylated in the presence of OA. As the time-dependent phosphorylation of 90 kDa protein correlated well with the reorganization of microtubules, these data suggest that phosphorylation and dephosphorylation of this protein might play a role in the regulation of microtubule organization. These findings indicate that OA or CLA induces reorganization of microtubules and actin filaments via the phosphorylation of a microtubule-associated 90 kDa protein and an MLC-phosphorylation-independent mechanism. mechanism.

scholarly journals Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique.

1987 ◽  
Vol 105 (4) ◽  
pp. 1771-1780 ◽  
Author(s):  
T Nakata ◽  
N Hirokawa
Keyword(s):  
Actin Filaments ◽  
Plasma Membranes ◽  
Human Platelets ◽  
Electron Microscopic Level ◽  
Immunocytochemical Study ◽  
Cytoskeletal Reorganization ◽  
Electron Microscopic ◽  
Activated Platelets ◽  
Mixed Polarity ◽  
Cross Bridges

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.

scholarly journals Cytochalasin D and E: effects on fibrinogen receptor movement and cytoskeletal reorganization in fully spread, surface-activated platelets: a correlative light and electron microscopic investigation

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 99-109 ◽  
Author(s):  
OE Olorundare ◽  
SR Simmons ◽  
RM Albrecht
Keyword(s):  
Low Voltage ◽  
Electron Microscopic Examination ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Cytochalasin D ◽  
Cytoskeletal Reorganization ◽  
Electron Microscopic ◽  
Directed Movement ◽  
Fibrinogen Receptor ◽  
Activated Platelets

Abstract This study investigates the involvement of actin microfilaments in fibrinogen receptor redistribution and cytoskeletal reorganization that takes place in fully spread, surface-activated platelets. Colloidal gold-labeled fibrinogen (Fgn-Au label) in conjunction with video- enhanced differential interference contrast light microscopy (VDIC) was used to identify fibrinogen binding sites, glycoprotein IIb/IIIb (GPIIb/IIIa), on fully spread platelets. Platelets were treated with cytochalasins D and E (5 x 10(-5) mol/L to 5 x 10(-8) mol/L) for 10 minutes, before or after incubation with Fgn-Au label. Results observed with VDIC were subsequently confirmed by high-voltage transmission and low voltage-high resolution scanning electron microscopic examination of the specimens. Preincubation of activated platelets with cytochalasin D or E (5 x 10(-5) and 5 x 10(-6) mol/L) inhibited fibrinogen receptor redistribution and abolished cytoskeletal reorganization in fully spread platelets. After surface-activated platelets were incubated with Fgn-Au label, treatment with the above concentrations of cytochalasin D or E disrupted cytoskeletal reorganization and caused random movement of previously redistributed receptor-ligand complexes. Incubation of platelets with cytochalasin E 5 x 10(-6) mol/L prevented platelet activation and spreading. Thus, actin filaments appear necessary for platelet spreading from the discoid to the fully spread stage. The ligand-triggered, cytoskeletally directed movement of fibrinogen receptors in fully spread platelets appears to be dependent on the presence of intact, polymerized actin. This movement is distinct from the cytochalasin-insensitive accumulation of GPIIb/IIIa-ligand in the channels of the open canalicular system.

scholarly journals Airway smooth muscle tone increases actin filamentogenesis and contractile capacity

2020 ◽  
Vol 318 (2) ◽  
pp. L442-L451
Author(s):  
Morgan Gazzola ◽  
Cyndi Henry ◽  
Katherine Lortie ◽  
Fatemeh Khadangi ◽  
Chan Young Park ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Muscle Tone ◽  
Coiled Coil ◽  
Traction Force

Force adaptation of airway smooth muscle (ASM) is a process whereby the presence of tone (i.e., a sustained contraction) increases the contractile capacity. For example, tone has been shown to increase airway responsiveness in both healthy mice and humans. The goal of the present study is to elucidate the underlying molecular mechanisms. The maximal force generated by mouse tracheas was measured in response to 10−4 M of methacholine following a 30-min period with or without tone elicited by the EC30 of methacholine. To confirm the occurrence of force adaptation at the cellular level, traction force generated by cultured human ASM cells was also measured following a similar protocol. Different pharmacological inhibitors were used to investigate the role of Rho-associated coiled-coil containing protein kinase (ROCK), protein kinase C (PKC), myosin light chain kinase (MLCK), and actin polymerization in force adaptation. The phosphorylation level of the regulatory light chain (RLC) of myosin, the amount of actin filaments, and the activation level of the actin-severing protein cofilin were also quantified. Although ROCK, PKC, MLCK, and RLC phosphorylation was not implicated, force adaptation was prevented by inhibiting actin polymerization. Interestingly, the presence of tone blocked the activation of cofilin in addition to increasing the amount of actin filaments to a maximal level. We conclude that actin filamentogenesis induced by tone, resulting from both actin polymerization and the prevention of cofilin-mediated actin cleavage, is the main molecular mechanism underlying force adaptation.

IQGAP2 functions as a GTP-dependent effector protein in thrombin-induced platelet cytoskeletal reorganization

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3021-3028 ◽  
Author(s):  
Valentina A. Schmidt ◽  
Lesley Scudder ◽  
Craig E. Devoe ◽  
André Bernards ◽  
Lisa D. Cupit ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Blood Platelets ◽  
Initial Step ◽  
Human Platelets ◽  
Effector Proteins ◽  
Effector Protein ◽  
Protease Activated Receptors ◽  
Gene Encoding ◽  
Downstream Effector ◽  
Activated Platelets

Abstract Human blood platelets are anucleate cells whose response to extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of hemostasis. The serine protease α-thrombin, known to activate platelets by cleavage of a family of protease-activated receptors (PARs), is the most potent physiologic activator of human platelets, though downstream effector proteins uniquely linked to platelet cytoskeletal actin polymerization remain largely uncharacterized. The gene encoding the putative rac1/cdc42 effector protein IQGAP2 was identified within the PAR gene cluster at 5q13, flanked telomeric byPAR1 and encompassing PAR3. Immunofluorescence microscopy demonstrated IQGAP2 expression in filopodial extensions of activated platelets and colocalized with F-actin in lamellipodia and filopodia of IQGAP2-transfected COS1 cells. Platelet activation by α-thrombin, but not saturating concentrations of fibrillar collagen or adenosine 5′-diphosphate, uniquely assemble an IQGAP2/arp2/3–actin cytoplasmic complex, an association regulated by guanosine triphosphate rac1 ([GTP]rac1) but not by [GTP]cdc42. Likewise, only thrombin-activated platelets resulted in rapid translocation of IQGAP2 to the platelet cytoskeleton. These observations identify a physiologic scaffolding function for IQGAP2 and establish the presence of a functional genomic unit in humans uniquely evolved to regulate thrombin-induced platelet cytoskeletal actin reorganization.

scholarly journals Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase.

1995 ◽  
Vol 129 (3) ◽  
pp. 831-842 ◽  
Author(s):  
C Guinebault ◽  
B Payrastre ◽  
C Racaud-Sultan ◽  
H Mazarguil ◽  
M Breton ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Subcellular Fraction ◽  
Critical Role ◽  
Physical Interaction ◽  
Activated Platelets ◽  
Phosphoinositide 3 Kinase

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

scholarly journals Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study.

1989 ◽  
Vol 109 (4) ◽  
pp. 1581-1595 ◽  
Author(s):  
S Okabe ◽  
N Hirokawa
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Stress Fibers ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Actin Bundles ◽  
Actin Networks ◽  
Focal Contacts ◽  
Microfilament System ◽  
Fibroblastic Cells

We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers. By 60 min after injection, stress fibers were labeled uniformly. We also analyzed the actin incorporation into polygonal nets of actin bundles. Circular dense foci, where actin bundles radiate, were stable structures, and actin filaments around the foci incorporated biotin-actin the slowest among the actin-containing structures within the injected cells. These results indicate that the rate and pattern of actin subunit incorporation differ in different regions of the cytoplasm and suggest the possible role of rapid actin polymerization at the leading margin on the protrusive movement of fibroblastic cells.

scholarly journals Arginylation Regulates Intracellular Actin Polymer Level by Modulating Actin Properties and Binding of Capping and Severing Proteins

2010 ◽  
Vol 21 (8) ◽  
pp. 1350-1361 ◽  
Author(s):  
Sougata Saha ◽  
Maureen M. Mundia ◽  
Fangliang Zhang ◽  
Ryan W. Demers ◽  
Farida Korobova ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Biochemical Properties ◽  
Cytoskeletal Organization ◽  
Actin Networks ◽  
Severe Impairment ◽  
Associated Proteins

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. To understand how arginylation affects the actin cytoskeleton, we investigated the biochemical properties and the structural organization of actin filaments in wild-type and arginyltransferase (Ate1) knockout cells. We found that Ate1 knockout results in a dramatic reduction of the actin polymer levels in vivo accompanied by a corresponding increase in the monomer level. Purified nonarginylated actin has altered polymerization properties, and actin filaments from Ate1 knockout cells show altered interactions with several associated proteins. Ate1 knockout cells have severe impairment of cytoskeletal organization throughout the cell. Thus, arginylation regulates the ability of actin to form filaments in the whole cell rather than preventing the collapse of preformed actin networks at the cell leading edge as proposed in our previous model. This regulation is achieved through interconnected mechanisms that involve actin polymerization per se and through binding of actin-associated proteins.

scholarly journals Direct visualization of how Actin Depolymerizing Factor’s filament severing and depolymerization synergizes with Capping Protein's "monomer funneling" to promote rapid polarized growth of actin filaments

2017 ◽  
Author(s):  
Shashank Shekhar ◽  
Marie-France Carlier
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Bulk Solution ◽  
Direct Visualization ◽  
Actin Networks ◽  
Capping Protein ◽  
Visual Evidence ◽  
Pointed End

AbstractA living cell’s ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like ADF/cofilin and filament cappers like Capping Protein (CP) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. While filament fragmentation by ADF/cofilin has long been demonstrated by TIRF, filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. We have also built a conceptually novel assay to directly visualize ADF’s effect on a filament population. We demonstrate that ADF’s enhanced pointed-end depolymerization leads to an increase in polymerizable actin monomers co-existing with filaments, thus promoting faster barbed-end growth. We further reveal how ADF-enhanced filament depolymerization synergizes with CP’s long-predicted “monomer funneling” and leads to skyrocketing of filament growth rates, close to estimated rates in the lamellipodia. The “Funneling model” hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few noncapped filaments transiently grow much faster, an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling by CP at the scale of individual filaments. We believe that these results enlighten our understanding of the turnover of cellular actin networks.

scholarly journals Arp2/3 complex is required for actin polymerization during platelet shape change

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4466-4474 ◽  
Author(s):  
Zhi Li ◽  
Eric S. Kim ◽  
Elaine L. Bearer
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Shape Change ◽  
Thrombin Receptor ◽  
Fab Fragments ◽  
Sequential Formation ◽  
Platelet Shape

Platelets undergo a series of actin-dependent morphologic changes when activated by thrombin receptor activating peptide (TRAP) or when spreading on glass. Polymerization of actin results in the sequential formation of filopodia, lamellipodia, and stress fibers, but the molecular mechanisms regulating this polymerization are unknown. The Arp2/3 complex nucleates actin polymerization in vitro and could perform this function inside cells as well. To test whether Arp2/3 regulated platelet actin polymerization, we used recombinant Arp2 protein (rArp2) to generate Arp2-specific antibodies (αArp2). Intact and Fab fragments of αArp2 inhibited TRAP-stimulated actin-polymerizing activity in platelet extracts as measured by the pyrene assay. Inhibition was reversed by the addition of rArp2 protein. To test the effect of Arp2/3 inhibition on the formation of specific actin structures, we designed a new method to permeabilize resting platelets while preserving their ability to adhere and to form filopodia and lamellipodia on exposure to glass. Inhibition of Arp2/3 froze platelets at the rounded, early stage of activation, before the formation of filopodia and lamellipodia. By morphometric analysis, the proportion of platelets in the rounded stage rose from 2.85% in untreated to 63% after treatment with αArp2. This effect was also seen with Fab fragments and was reversed by the addition of rArp2 protein. By immunofluorescence of platelets at various stages of spreading, the Arp2/3 complex was found in filopodia and lamellipodia. These results suggest that activation of the Arp2/3 complex at the cortex by TRAP stimulation initiates an explosive polymerization of actin filaments that is required for all subsequent actin-dependent events.

INTERACTIONS OF HUMAN PLATELETS WITH COAGULATION FACTORS IX AND IXa

1987 ◽  
Author(s):  
S S Ahmad ◽  
R Rawala ◽  
P N Walsh
Keyword(s):  
Binding Sites ◽  
Molecular Mechanisms ◽  
Dissociation Constants ◽  
Coagulation Factors ◽  
Human Platelets ◽  
Factor Ix ◽  
Binding Studies ◽  
Activated Platelets ◽  
Ca Ions ◽  
A Site

To elucidate the molecular mechanisms underlying the platelet contribution to the activation of factor IX (FIX) and FX, we have previously reported the presence of specific, high-affinity binding sites on activated platelets for FIX and FIXa (Blood 66:300a, 1985; Circulation, 74:11-238, 1986). Since both FIX and FIXa bind to a common site on thrombin-activated platelets in the presence of Ca++ ions, it is important to determine whether saturation of platelet binding sites with FIX would prevent F-IXa binding. We have therefore made direct comparisons of F-IX and F-IXa binding and examined the functional consequences of F-IXa binding in F-X activation. Gel-filtered platelets (GFP) were incubated with 125I-FIX or 125I-FlXa and centrifuged through silicone barriers to separate GFP from unbound proteins. Optimal binding of both FIX and FIXa occurred in the presence of CaCl2 (5 mM) and thrombin (0.1 U/ml), with maximal binding in 10-15 min at 37°C. Binding of both proteins was specific since a 130-fold excess of FIX or FIXa inhibited FIX binding >85% and excess FIXa inhibited FIXa binding >85%, whereas excess FIX inhibited FIXa binding to a lesser extent, and excess high Mr kininogen, FXI, FX, prothrombin, and prekallikrein inhibited binding <30%. Binding of both FIX and FIXa was rapidly reversible and saturable. The number of FIXa binding sites (551 + 48 per platelet) was significantly (p < 0.001) higher than for FIX (306 + 57). Dissociation constants (Kd) were 2.57 +0.14 nM for FIXa and 2.68 + 0.25 nM for FIX. However, the concentration of FIXa required for half-maximal rates of FX activation in the presence of FVIIIa and thrombin-activated GFP was approximately 0.1 nM. This suggests that the FIXa receptors functionally active in FX activation may bind FIXa >20-fold more tightly than indicated by equilibrium binding studies, and that FIXa binding studies should be carried out in the presence of large molar excesses of FIX. We conclude that FIX and FIXa bind to a coupon site on thrombin-activated platelets in the presence of Ca ions, and that FIXa also binds to a site distinct from that for FIX. FIXa binding to platelets appears to contribute to F-X activation.

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