Role for phosphoinositide 3-kinase in FcγRIIA-induced platelet shape change

2003 ◽  
Vol 285 (4) ◽  
pp. C797-C805 ◽  
Author(s):  
Kurt L. Barkalow ◽  
Hervé Falet ◽  
Joseph E. Italiano ◽  
Andrew van Vugt ◽  
Christopher L. Carpenter ◽  
...  
Keyword(s):  
Actin Filament ◽  
Kinase Activity ◽  
Shape Change ◽  
Small Gtpases ◽  
Pi3 Kinase ◽  
Cross Linking ◽  
Actin Assembly ◽  
Filament Assembly ◽  
Tenfold Increase ◽  
Phosphoinositide 3 Kinase

Platelets transform from disks to irregular spheres, grow filopodia, form ruffles, and spread on surfaces coated with anti-FcγRIIA antibody. FcγRIIA cross-linking leads to a tenfold increase in actin filament barbed end exposure and robust actin assembly. Activation of the small GTPases Rac and Cdc42 follows FcγRIIA cross-linking. Shape change, actin filament barbed end exposure, and quantifiable actin assembly require phosphoinositide 3-kinase (PI3-kinase) activity and a rise in intracellular calcium. PI3-kinase inhibition blocks activation of Rac, but not of Cdc42, and diminishes the association of Arp2/3 complex and CapZ with polymerized actin. Furthermore, addition of constitutively active D-3 phosphorylated polyphosphoinositides or recombinant PI3-kinase subunits to octylglucoside-permeabilized platelets elicits actin filament barbed end exposure by releasing gelsolin and CapZ from the cytoskeleton. Our findings place PI3-kinase activity upstream of Rac, gelsolin, and Arp2/3 complex activation induced by FcγRIIA and clearly distinguish the FcγRIIA signaling pathway to actin filament assembly from the thrombin receptor protease-activated receptor (PAR)-1 pathway.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

Roles of SLP-76, phosphoinositide 3-kinase, and gelsolin in the platelet shape changes initiated by the collagen receptor GPVI/FcRγ-chain complex

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3786-3792 ◽  
Author(s):  
Hervé Falet ◽  
Kurt L. Barkalow ◽  
Vadim I. Pivniouk ◽  
Michael J. Barnes ◽  
Raif S. Geha ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Shape Change ◽  
Chain Complex ◽  
Actin Assembly ◽  
Coated Surface ◽  
Phosphoinositide 3 Kinase ◽  
Collagen Receptor ◽  
Shape Changes ◽  
Platelet Shape

Abstract How platelet shape change initiated by a collagen-related peptide (CRP) specific for the GPVI/FcRγ-chain complex (GPVI/FcRγ-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/FcRγ-chain and the roles of PI 3-kinase and gelsolin in lamellae spreading.

scholarly journals Cdc42 and Phosphoinositide 3-Kinase Drive Rac-Mediated Actin Polymerization Downstream of c-Met in Distinct and Common Pathways

2007 ◽  
Vol 27 (19) ◽  
pp. 6615-6628 ◽  
Author(s):  
Tanja Bosse ◽  
Julia Ehinger ◽  
Aleksandra Czuchra ◽  
Stefanie Benesch ◽  
Anika Steffen ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Pi3 Kinase ◽  
Actin Assembly ◽  
Actin Reorganization ◽  
Membrane Ruffling ◽  
Internalin B ◽  
Phosphoinositide 3 Kinase ◽  
Hepatocyte Growth ◽  
Wave Complex

ABSTRACT Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.

scholarly journals The actin released from profilin--actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes.

1990 ◽  
Vol 110 (6) ◽  
pp. 1965-1973 ◽  
Author(s):  
F S Southwick ◽  
C L Young
Keyword(s):  
Actin Filament ◽  
Polymorphonuclear Leukocytes ◽  
Critical Concentration ◽  
Relative Concentration ◽  
Insoluble Fraction ◽  
Membrane Receptors ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Total Change ◽  
Filament Assembly

Chemoattractant stimulation of polymorphonuclear leukocytes is associated with a nearly two-fold rise in actin filament content. We examined the role of the actin monomer sequestering protein, profilin, in the regulation of PMN actin filament assembly during chemoattractant stimulation using a Triton extraction method. Poly-L-proline-conjugated Sepharose beads were used to assess the relative concentration of actin bound to profilin with high enough affinity to withstand dilution (profilin-actin complex) and DNase I-conjugated beads to measure the relative concentration of actin in the Triton-soluble fraction not bound to profilin. Actin associated with the Triton-insoluble fraction (F-actin) was also measured. In unstimulated PMN, the relative concentration of actin bound to profilin was maximum. After FMLP stimulation, profilin released actin monomers within 10 s, with the profilin-actin complex concentration reaching a nadir by 40 s and remaining low as long as the cells were exposed to chemoattractant (up to 30 min). If FMLP was dissociated from PMN membrane receptors using t-BOC, actin reassociated with profilin within 20 s. Quantitative analysis of these reactions, however, revealed that profilin release of and rebinding to actin could account for only a small percentage of the total change in F-actin content. Determination of the total profilin and actin concentrations in PMN revealed that the molar ratio of profilin to actin was 1 to 5.2. When purified actin was polymerized in PMN Triton extract containing EGTA, removal of profilin from the extract minimally affected (12% reduction) the high apparent critical concentration at which actin began to assemble. Although profilin released actin at the appropriate time to stimulate actin assembly during exposure to chemoattractants, the concentration of profilin in PMN was insufficient to explain the high unpolymerized actin content in unstimulated PMN and the quantity of actin released from profilin too small to account for the large shifts from unpolymerized to polymerized actin associated with maximal chemoattractant stimulation.

Actin Assembly Takes Off Like a Rocket!

2013 ◽  
Vol 21 (2) ◽  
pp. 8-9
Author(s):  
Stephen W. Carmichael ◽  
Jeffrey L. Salisbury
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cells ◽  
Actin Assembly ◽  
Elongation Factors ◽  
Delicate Balance ◽  
Filament Assembly ◽  
Two Factors

Actin filament assembly occurs in all eukaryotic cells and involves a delicate balance between factors that promote assembly and factors that inhibit assembly. Filament assembly begins with a process of nucleation and then proceeds via elongation. Filament assembly in vivo requires nucleation and elongation factors to overcome barriers that could either bind actin monomers to inhibit nucleation or “cap” the ends of elongating filaments. The formation of most cellular actin structures depends on two or more such factors, which may interact directly. The interaction between two factors that initiate nucleation and promote assembly has recently been demonstrated by Dennis Breitsprecher, Richa Jaiswal, Jeffrey Bombardier, Christopher Gould, Jeff Gelles, and Bruce Goode. Interestingly, the model of these factors in action (Figure 1) resembles a rocket launcher!

An Aplysia cell adhesion molecule associated with site-directed actin filament assembly in neuronal growth cones

1996 ◽  
Vol 109 (12) ◽  
pp. 2843-2854 ◽  
Author(s):  
C. Thompson ◽  
C.H. Lin ◽  
P. Forscher
Keyword(s):  
Cell Adhesion ◽  
Membrane Protein ◽  
Actin Filament ◽  
De Novo ◽  
Lateral Diffusion ◽  
Actin Assembly ◽  
Neuronal Growth ◽  
Cytoskeletal Remodeling ◽  
Interaction Sites ◽  
Filament Assembly

During neuronal growth cone-target interactions, a programmed sequence of cytoskeletal remodeling has been described, involving increased actin assembly at the target site and directed microtubule extension into it. The cell adhesion protein apCAM rapidly accumulates at such interaction sites, suggesting a possible role in regulating cytoskeletal remodeling. To test this hypothesis we crosslinked apCAM to varying degrees with antibodies. Secondary immunocomplexes exhibited a classical patching and capping response; in contrast, high density crosslinking of apCAM by antibody coated beads triggered localized actin assembly accompanied by formation of tail-like actin structures referred to as inductopodia. When beads were derivatized with increasing amounts of anti-apCAM they displayed three sequential dose-dependent kinetic states after binding: (1) lateral diffusion in the plane of the membrane; (2) restricted diffusion due to coupling with underlying F-actin; and (3) translocation in the plane of the membrane driven by de novo actin filament assembly local to bead binding sites, i.e. inductopodia formation. In contrast, lectin coated beads were far less efficient in triggering inductopodia formation despite demonstrated membrane protein binding. This work provides evidence that crosslinking of a diffusable membrane protein, apCAM, to threshold levels, can trigger highly localized actin filament assembly and rapid remodeling of neuronal cytoarchitecture.

The relationship between CR3 deficiency and neutrophil actin assembly

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1973-1979
Author(s):  
FS Southwick ◽  
TH Howard ◽  
T Holbrook ◽  
DC Anderson ◽  
TP Stossel ◽  
...  
Keyword(s):  
Actin Filament ◽  
Polymorphonuclear Leukocytes ◽  
Family Members ◽  
Surface Expression ◽  
Actin Assembly ◽  
Normal Surface ◽  
Filament Assembly ◽  
Type 3 ◽  
The Relationship ◽  
Total Concentrations

Polymorphonuclear leukocytes (PMN) with a deficiency of the complement receptor type 3 (CR3) membrane glycoprotein family have impairments in the ability to adhere to surfaces as well as chemotactic and phagocytic defects, processes that require a functional contractile apparatus. PMN from the patient with neutrophil actin dysfunction (NAD) displayed similar functional characteristics to those with CR3 deficiency suggesting the two disorders may be the same disease. In order to evaluate the relationship between CR3 deficiency and actin assembly, actin filament assembly was measured in PMN from six previously reported homozygotes (two severe and four moderate CR3-deficient patients) as well as five heterozygotes for CR3 deficiency. PMN from all patients had normal unstimulated concentrations of F-actin and after exposure to the chemotactic peptide FMLP (5 x 10(-7) mol/L for 5 to 40 seconds at 25 degrees C) assembled actin normally. Pretreatment of normal PMN with concentrations of monoclonal anti-alpha CR3 antibody, capable of blocking PMN adherence, also failed to impair FMLP- induced actin filament assembly. CR3 glycoprotein expression was measured in PMNs from the mother, father, and older sister of the NAD patient (N Engl J Med 291:1093, 1974). Actin filament assembly was recently shown to be defective in PMNs from all three family members. The total concentrations of the alpha and beta CR3 subunits were below normal in PMN detergent extracts from the mother (25% of simultaneous controls) and older sister (56% of control). PMN surface expression of these two subunits was also found to be depressed (mother, 50%; older sister, 63% of control). These findings suggest these two NAD family members are heterozygote carriers for CR3 deficiency as well as NAD. Simultaneous studies of the father, however, demonstrated normal total concentrations of both the alpha and beta CR3 subunits (126% of controls) as well as normal surface expression of both subunits after phorbol myristate acetate stimulation and incubation at 37 degrees C (mean, 112% of controls) but slightly lower than normal levels after FMLP stimulation (mean, 83%). These findings indicate that CR3 deficiency generally is not associated with defective actin filament assembly and support the conclusion that NAD represents a unique kindred in which PMN actin function differs from previously reported genotypes of CR3 deficiency.

The Mechanism of Actin-Filament Assembly and Cross-Linking

1982 ◽  
pp. 15-44 ◽  
Author(s):  
Thomas D. Pollard ◽  
Ueli Aebi ◽  
John A. Cooper ◽  
Marshall Elzinga ◽  
Walter E. Fowler ◽  
...  
Keyword(s):  
Actin Filament ◽  
Cross Linking ◽  
Filament Assembly

Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 959-964 ◽  
Author(s):  
A. C. Azim ◽  
K. Barkalow ◽  
J. Chou ◽  
J. H. Hartwig
Keyword(s):  
Plasma Membranes ◽  
Kinase Activity ◽  
Small Gtpases ◽  
Cytoskeletal Proteins ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cell Interior ◽  
Activated Platelets ◽  
Octyl Glucoside ◽  
Triton X

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.

The phosphoinositide 3-kinase pathway and cancer

2005 ◽  
Vol 7 (10) ◽  
pp. 1-22 ◽  
Author(s):  
David Stokoe
Keyword(s):  
Signal Transduction ◽  
Kinase Activity ◽  
Malignant Potential ◽  
Pi3 Kinase ◽  
Signal Transduction Pathways ◽  
Constitutive Activation ◽  
And Migration ◽  
Phosphoinositide 3 Kinase ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway

Human tumours emerge as the result of multiple genetic and epigenetic aberrations that allow the proto-cancer cell to escape normal social control. Many signal transduction pathways become constitutively active during this process, and one whose importance is increasingly being appreciated involves phosphoinositide 3-kinase (PI3-kinase). This pathway normally regulates important cell decisions such as growth, division, survival and migration, and when deregulated it can confer malignant potential to the ensuing tumour. However, constitutive activation of the PI3-kinase pathway might provide attractive therapeutic targets for the design of small-molecule inhibitors. This review discusses events upstream and downstream of PI3-kinase activity in the PI3-kinase signalling pathway, how PI3-kinase is deregulated in human tumourigenesis, and how this is being targeted clinically.

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