Simon D. J. Calaminus

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

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

Analysis of intact phosphoinositides in biological samples

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P3], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [32P]inorganic phosphate or [3H]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 PtdInsP3 and PtdInsP2 regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS3. Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.

The Arp2/3 activator WASH regulates α5β1-integrin-mediated invasive migration

The 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 α5β1 integrin recycling. WASH regulates the trafficking of endosomal α5β1 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.

Podosomes in adhesion, migration, mechanosensing and matrix remodeling

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.

MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability

Summary.  Background: MyosinIIs are adenosine triphosphate‐driven molecular motors that form part of a cell’s 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.

Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane

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.

von Willebrand factor mediates platelet spreading through glycoprotein Ib and αIIbβ3 in the presence of botrocetin and ristocetin, respectively

Summary.  Background: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow‐dependent 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 αIIbβ3. Objective: To evaluate the ability of GPIb and αIIbβ3 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 A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb‐dependent mechanism that is independent of αIIbβ3 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 αIIbβ3 and PI3‐kinase. Furthermore, αIIbβ3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. 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 αIIbβ3‐independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an αIIbβ3‐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.

Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcRγ‐chain, which has an immunoreceptor tyrosine‐containing activation motif (ITAM). Objective: To determine why GPVI–FcRγ signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI‐reactive snake venom toxin convulxin. Methods and results: Using a nuclear factor of activated T‐cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen‐induced GPVI–FcRγ signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte‐associated immunoglobulin‐like receptor‐1 (LAIR‐1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. Conclusion: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR‐1. In platelets, we propose that GPVI–FcRγ has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.

Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading.

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 β1‐ and β3‐ 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.

Vav family proteins are required for optimal regulation of PLCγ2 by integrin αIIbβ3

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCγ (phospholipase Cγ) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin αIIbβ3, which has recently been shown to regulate PLCγ2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced αIIbβ3-mediated PLCγ2 tyrosine phosphorylation and reduced Ca2+ mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCγ2 by integrin αIIbβ3, but that their requirement is by-passed upon G-protein receptor activation.