Abstract
Platelets undergo profound shape changes upon adhesion to damaged blood vessel walls that are mediated by reorganisation of the actin cytoskeleton in response to receptor-mediated signalling cascades. The highly conserved 56 kDa multidomain cyclase associated protein 1 (CAP1) works in concert with cofilin and profilin to modulate actin filament turnover by facilitating cofilin-mediated actin filament severing and depolymerisation and catalysing profilin-mediated regeneration of actin monomers for reutilisation in growing filaments. CAP1 is abundant in platelets but its roles remain unexplored. We report that in suspended platelets CAP1 localises predominantly at the cell cortex whereas in spread platelets it is uniformly distributed in the cytoplasm, with enrichment at the cell cortex and the periphery of actin nodules. Upon subcellular fractionation most CAP1 was found cytosolic but part associated to the membrane fraction in an actin-independent manner. Interestingly, upon stimulation with thrombin a significant proportion of the membrane-associated CAP1 translocates to the cytosol. This relocalisation was prevented by prior treatment with PGI2 or the nitric oxide donor GSNO, or by inhibition of GSK3. Our results place CAP1 at a crossroad of signalling pathways that control platelet activation by contributing to actin remodelling at the cell cortex and actin nodules during platelet spreading.
Highlights
Platelets in circulation adopt a discoid shape that is maintained thanks to a highly organised cytoskeleton that includes a peripheral microtubule coil, a network of cross-linked actin filaments that fill the cytoplasmic space and, connected to this, a cortical spectrin-based skeleton associated to the plasma membrane[1]
The antibody we used throughout this study proved specific for human cyclase associated protein 1 (CAP1) but we have demonstrated the presence of CAP1 in murine platelets using a different antibody
Since CAP1 is an actin-binding protein, we further investigated whether this membrane association is mediated by actin
Summary
Platelets in circulation adopt a discoid shape that is maintained thanks to a highly organised cytoskeleton that includes a peripheral microtubule coil, a network of cross-linked actin filaments that fill the cytoplasmic space and, connected to this, a cortical spectrin-based skeleton associated to the plasma membrane[1]. Platelets undergo profound shape changes when they adhere to the damaged blood vessel wall, first becoming spherical, and producing filopodia and actin nodules that progress to lamellipodia and stress fibres as the cell spreads and flattens[2]. These changes in platelet morphology are mediated by reorganisation of the actin cytoskeleton in response to multiple receptor-mediated signalling cascades[3]. CAP1 is a predominantly cytosolic protein, a small fraction has been found associated to membranes[12] It usually shows a diffuse cytoplasmic localisation and accumulates in dynamic actin structures at the cell cortex where it co-localises with cofilin[13,14,15]. Our study paves the way for further studies towards establishing the role of CAP1 in platelet actin dynamics and platelet function
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