The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P-2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P-2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P-2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P-2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P-2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P-2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P-2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca2+-dependent exocytosis.