Summary Neuronal growth cone filopodia contain guidance receptors and contribute to axon guidance, however the mechanism by which the guidance cue netrin increases filopodia density is unknown. Here we demonstrate that TRIM9, an E3 ubiquitin ligase that localizes to filopodia tips and binds the netrin receptor DCC, interacts with and ubiquitinates the barbed-end polymerase VASP to modulate filopodial stability during netrin-dependent axon guidance. Studies with murine TRIM9+/+ and TRIM9−/− cortical neurons, along with a non-ubiquitinatable VASP mutant, demonstrate that TRIM9-mediated ubiquitination of VASP reduces VASP filopodial tip localization, VASP dynamics at tips, and filopodial stability. Upon netrin treatment, VASP is deubiquitinated, which promotes VASP tip localization and filopodial stability. TRIM9 deletion induces axon guidance defects in vitro and in vivo, whereas a gradient of deubiquitinase inhibition promotes axon turning in vitro. We conclude that a gradient of TRIM9-mediated ubiquitination of VASP creates a filopodial stability gradient during axon turning.

Despite the fact that lysophosphatidic acid (LPA) has been identified as a main platelet-activating lipid of mildly oxidized low-density lipoprotein (LDL) and human atherosclerotic lesions, it remains unknown whether it is capable of activating platelets in blood. We found that LPA at concentrations slightly above plasma levels induces platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. 1-alkyl-LPA (16:0 fatty acid) was almost 20-fold more potent than 1-acyl-LPA (16:0). LPA directly induced platelet shape change in blood and platelet-rich plasma obtained from all blood donors. However, LPA-stimulated platelet aggregation in blood was donor dependent. It could be completely blocked by apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors P2Y 1 and P2Y 12 . These substances also inhibited LPA-induced aggregation of platelet-rich plasma and aggregation and serotonin secretion of washed platelets. These results indicate a central role for ADP-mediated P2Y 1 and P2Y 12 receptor activation in supporting LPA-induced platelet aggregation. Platelet aggregation and platelet-monocyte aggregate formation stimulated by LPA was insensitive to inhibition by aspirin. We conclude that LPA at concentrations approaching those found in vivo can induce platelet shape change, aggregation, and platelet-monocyte aggregate formation in whole blood and suggest that antagonists of platelet P2Y 1 and P2Y 12 receptors might be useful preventing LPA-elicited thrombus formation in patients with cardiovascular

In platelets stimulated by thrombin to secrete and aggregate, cofilin is rapidly dephosphorylated leading to its activation. Cofilin by severing existing actin filaments and stimulating F-actin polymerization on newly created barbed ends dynamizes the actin cytoskeleton. We previously found that cofilin dephosphorylation is Ca 2؉ -dependent and occurs upstream of degranulation in stimulated platelets. We report now in thrombinstimulated platelets that Rac1 and class II PAKs (PAK4/5/6) were rapidly (within 5 seconds) activated, whereas PAK1/2 (class I PAKs) phosphorylation was slower. The Rac1-specific inhibitor NSC23766 blocked phosphorylation of class II PAKs, but not PAK1/2. Moreover, inhibition of the Ca 2؉ /calmodulindependent phosphatase calcineurin inhibited Rac1 activation and class II PAKs phosphorylation. Prevention of Rac1 activation by calcineurin inhibition or NSC23766 also blocked cofilin dephosphorylation and platelet granule secretion indicating that a calcineurin/Rac1/ class II PAKs pathway regulates cofilin dephosphorylation leading to secretion.We further found that PI3-kinases were activated downstream of Rac1, but were not involved in regulating cofilin dephosphorylation and secretion in thrombinstimulated platelets. Our study unravels a Ca 2؉ -dependent pathway of secretion in stimulated platelets as a signaling pathway linking Rac1 activation to actin dynamics: calcineurin3Rac13class II PAKs3cofilin activation. We further demonstrate that this pathway is separate and independent of the protein kinase C (PKC) pathway mediating secretion. (Blood. 2009;114:415-424)

Cofilin is a regulator of actin filament dynamics. We studied whether during platelet activation Rho kinase stimulates LIM kinase (LIMK) leading to subsequent phosphorylation and inactivation of cofilin. Platelet shape change and aggregation/secretion were induced by low and high concentrations of thrombin, respectively. We found that during these platelet responses Rho kinase activation was responsible for mediating rapid Thr508 phosphorylation and activation of LIMK-1 and for the F-actin increase during shape change and, in part, during secretion. Surprisingly, during shape change cofilin phosphorylation was unaltered, and during aggregation/secretion cofilin was first rapidly dephosphorylated by an okadaic acid-insensitive phosphatase and then slowly rephosphorylated by LIMK-1. LIMK-1 phosphorylation and cofilin dephosphorylation and rephosphorylation during aggregation were independent of integrin ␣ IIb ␤ 3 engagement. Cofilin phosphorylation did not regulate cofilin association with F-actin and was unrelated to the F-actin increase in thrombin-activated platelets. Our study identifies LIMK-1 as being activated by Rho kinase in thrombinstimulated platelets. Two counteracting pathways, a cofilin phosphatase and LIMK-1, are activated during platelet aggregation/secretion regulating cofilin phosphorylation sequentially and independently of integrin ␣ IIb ␤ 3 engagement. Rho kinasemediated F-actin increase during platelet shape change and secretion involves a mechanism other than LIMK-1-mediated cofilin phosphorylation, raising the possibility of another LIMK substrate regulating platelet actin assembly. IntroductionDynamic remodeling of actin structures underlies the different morphologic and functional platelet responses such as shape change, spreading, secretion, and aggregation. 1,2 The remodeling of actin is mediated by factors that regulate actin polymerization and depolymerization, disassembly of existing filaments, formation of new filaments, crosslinking of filaments to networks, and bundling of actin filaments. [3][4][5] They include signaling proteins that regulate actin dynamics as well as proteins that bind directly to actin and modulate the diverse actin structures. A key protein regulating actin remodeling is cofilin, 6,7 an essential, ubiquitously expressed and highly conserved actin-binding protein. Binding of cofilin to actin filaments stabilizes a twisted form of F-actin, thereby weakening lateral subunit interactions and promoting filament severing and depolymerization. 8,9 However, filament severing by cofilin also results in the generation of free barbed ends, which in turn is crucial for efficient enhancement of actin polymerization. [10][11][12] Cofilin is therefore an actin dynamizing protein, which favors depolymerization or polymerization of actin, depending on the cellular content of actin filaments relative to actin monomers and free barbed ends. 13 In unstimulated cells cofilin is present both in a phosphorylated and a nonphosphorylated form. 14,15 The depolymerizing activity of co...