WASH coats mature lysosomes and is required for exocytosis of indigestible material.

SummaryThe actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for a5b1 integrin recycling. WASH regulates the trafficking of endosomal a5b1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.

• Murine and human megakaryocytes assemble podosomes.• Megakaryocyte podosomes remodel matrix.Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane. (Blood. 2013;121(13):2542-2552

Cells use various actin-based motile structures to allow them to move across and through matrix of varying density and composition. Podosomes are actin cytoskeletal structures that form in motile cells and that mediate adhesion to substrate, migration, and other specialized functions such as transmigration through cell and matrix barriers. The podosome is a unique and interesting entity, which appears in the light microscope as an individual punctum, but is linked to other podosomes like a node on a network of the underlying cytoskeleton. Here, we discuss the signals that control podosome assembly and dynamics in different cell types and the actin organising proteins that regulate both the inner actin core and integrin-rich surrounding ring structures. We review the structure and composition of podosomes and also their functions in various cell types of both myeloid and endothelial lineage. We also discuss the emerging idea that podosomes can sense matrix stiffness and enable cells to respond to their environment. V C 2013 Wiley Periodicals, Inc.

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P 2 , PtdIns(3,5)P 2 , PtdIns(4,5)P 2 ], and phosphatidylinositol trisphosphate [PtdIns(3,4,5) ]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP 3 and PtdInsP 2 regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS 3 . Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples. As our understanding of lipids as highly dynamic structures with both structural and signaling roles matures, one group stands out. These are the phosphoinositides, which regulate a host of cellular events, such as membrane trafficking, secretion, adhesion, migration, cell survival, and replication (1-4). Many of these outcomes are regulated by interactions between phosphoinositides and effector proteins bearing specific phosphoinositide binding domains (e.g., ENTH, FYVE, PH, PX) (5). Within this lipid family, the best known are phosphatidylinositol (PtdIns), the parent of the higher phosphorylated forms, which itself has no known signaling role, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ]. PtdIns(4,5)P 2 is the precursor to diacylglycerol and inositol 1,4,5-trisphosphate in receptor signaling and also directly regulates cytoskeletal reorganizations and the activities of enzymes such as phospholipase D. PtdIns(3,4,5)P 3 regulates cell movement, apoptosis, metabolism, and proliferation via the activation of phosphoinositide dependent kinase 1, protein kinase B, and other PH and PX domain proteins. However, it is now also apparent that other PtdInsP 2 regioisomers, such as phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P 2 ], together with the phosphatidylinositol monophosphate regioisomers phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 5-phosphate (PtdIns5P), have their own specific functions. PtdIns3P and PtdIns4P regulate membrane trafficking, whereas PtdIns5P has been reported to bind to the PHD domain i...

Summary. Background: MyosinIIs are adenosine triphosphate-driven molecular motors that form part of a cellÕs contractile machinery. They are activated by phosphorylation of their light chains, by either activation of myosin light chain (MLC) kinase or inhibition of MLC phosphatase via Rho kinase (ROCK). MyosinIIa phosphorylation underlies platelet rounding and stress fiber formation. Objective: To identify the functional significance of myosinIIa in platelet spreading and thrombus formation on collagen using inhibitors of ROCK (Y27632) and myosinII (blebbistatin). Results: Stress fiber formation on collagen is inhibited by both Y27632 and blebbistatin. A substantial proportion of spread platelets generate internal holes or splits on collagen, presumably because of a reduction in contractile strength. Platelet integrity, however, is maintained. In an in vitro model, thrombus embolization on collagen is increased in the presence of Y27632 and blebbistatin at intermediate shear, leading to a reduction in platelet aggregate growth. Moreover, Y27632 causes a marked reduction in thrombus formation in an in vivo laser-injury model. Conclusions: MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

To cite this article: Calaminus SDJ, Thomas S, McCarty OJT, Machesky LM, Watson SP. Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading. J Thromb Haemost 2008; 6: 1944-52.Summary. Background: During platelet spreading, the actin cytoskeleton undergoes marked changes, forming filopodia, lamellipodia and stress fibres. In the present study, we report the identification of a novel actin-rich structure, termed an actin nodule, which appears prior to lamellipodia and stress fibre formation. Methods: Platelet spreading was monitored using human platelets and mouse GFP-actin platelets using real-time and end-point DIC, and fluorescent and electron microscopy (EM). Results: We identified a small, novel actin structure, the actin nodule, in the early stages of adhesion and spreading, which we hypothesize to be a precursor of lamellipodia and stress fibres. Nodule formation shows an inverse correlation to Rho kinase and myosin-II activity, is independent of PI3-kinase, but dependent on Src kinase activity. Actin nodules contain multiple proteins, including Arp2/3, Fyn, Rac, and b1-and b3-integrins, but not Src. EM analysis revealed that actin filaments extend in all directions from the nodules. Actin nodules are present on multiple matrices, including fibrinogen, laminin and VWF + botrocetin. Conclusion: This work identifies a novel platelet actin structure, which we propose is a precursor to both lamellipodia and stress fibres and acts to drive platelet spreading.

Summary. Background: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flowdependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin a IIb b 3 . Objective: To evaluate the ability of GPIb and a IIb b 3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. Results: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A 2 (TxA 2 ), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of a IIb b 3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon a IIb b 3 and PI3-kinase. Furthermore, a IIb b 3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA 2 . The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an a IIb b 3 -independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an a IIb b 3 -dependent manner. Conclusions: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.