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First published online May 10, 2006
doi: 10.1242/10.1242/jcs.02912

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Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells

Gerald R. V. Hammond^1,*,, Stephen K. Dove², Alastair Nicol³, Jef A. Pinxteren⁴, Daniel Zicha³ and Giampietro Schiavo^1,

¹ Molecular Neuropathobiology, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, UK
² School of Bioscience, University of Birmingham, Birmingham, B15 2TT, UK
³ Light Microscopy Laboratories, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, UK
⁴ Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research, Albert Baertsoenkaai 3, 9000 Ghent, Belgium

Authors for correspondence (e-mail: gerald.hammond{at}cancer.org.uk; giampietro.schiavo{at}cancer.org.uk)

Accepted 1 February 2006

The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P₂ in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis. Using a novel quantitative immunofluorescence technique, we report that plasma membrane PtdIns(4,5)P₂ becomes transiently depleted upon activation of exocytosis, and is not detected on the membranes of fusing granules. Depletion is caused by phospholipase C activity, and is mandatory for exocytosis. Although phospholipase C is required for Ca²⁺ release from internal stores, the majority of the requirement for PtdIns(4,5)P₂ hydrolysis occurs downstream of Ca²⁺ signalling - as shown in permeabilised cells, where the inositol (1,4,5)-trisphosphate-Ca²⁺ pathway is bypassed. Neither generation of the PtdIns(4,5)P₂ metabolite, diacylglycerol (DAG) or simple removal and/or sequestration of PtdIns(4,5)P₂ are sufficient for exocytosis to occur. However, treatment of permeabilised cells with DAG induces a small potentiation of exocytosis, indicating that it may be required. We propose that a cycle of PtdIns(4,5)P₂ synthesis and breakdown is crucial for exocytosis to occur in mast cells, and may have a more general role in all professional secretory cells.

Key words: Diacylglycerol, Exocytosis, Neomycin, Phosphoinositide, Phospholipase C

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JCS 2006 119: 1001. [Full Text]  

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