Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3

Background: Phosphatidylinositol 4,5-bisphosphate (PIP2) has been implicate d in the regulation of the actin cytoskeleton and Vesicle trafficking. It s timulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein c omplex Arp2/3. Other studies show that actin polymerizes from cholesterol-s phingolipid-rich membrane microdomains called 'rafts', in a manner dependen t on tyrosine phosphorylation. Although actin has been implicated in vesicl e trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in r afts, or if it is linked to vesicle movements. Results: Overexpression of type I phosphatidylinositol phosphate 5-kinase ( PIP5KI), which synthesizes PIP2, promoted actin polymerization from membran e-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexp ression. PV increased PIP2 levels, suggesting that it induces comets by cha nging PIP2 homeostasis and by increasing tyrosine phosphorylation. Platelet -derived growth factor (PDGF) enhanced PV-induced comet formation, and thes e stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2 /3 involvement was established using dominant-negative WASP constructs, End ocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with meth yl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. Conclusions: Sphingolipid-cholesterol rafts are preferred platforms for mem brane-linked actin polymerization. This is mediated by in situ PIP2 synthes is and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin com ets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.