The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters.
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 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)P2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P2 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)P2 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)P2 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)P2 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)P2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca2+-dependent exocytosis.
Aoyagi
Kyota
K
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro, Tokyo 153-8902, Japan.
Sugaya
Tsukiko
T
Umeda
Masato
M
Yamamoto
Seiji
S
Terakawa
Susumu
S
Takahashi
Masami
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis; D015151:Immunoblotting; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000502:physiology; D011188:Potassium / Q000737:chemistry; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D051381:Rats
D008051:Lipid Bilayers; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D050765:Qa-SNARE Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C116930:Pikfyve protein, mouse; C501303:PIP5KI protein, rat; D005033:Ethylmaleimide; D011188:Potassium