Availability of PIP3 at the Plasma Membrane Regulates Secretion in Pc12 Cells

Abstract

The phosphoinositide PIP2 has long been implicated in secretion; however despite the demonstration of high affinity binding between synaptotagmin and PIP3, the role of the latter in secretion has received little attention. We have investigated this question using two complementary approaches; expression of PIP2 or PIP3 targeting Pleckstrin Homology (PH) domains, and up or down regulation of the PIP3 dephosphorylating enzyme PTEN (Phosphatase and TENsin homolog). The PH-domain of GRP1 (General receptor for phosphoinositides), which we show to have high specificity for PIP3, is equally as effective at inhibiting secretion as the PH domain of phospholipase C delta 1, which is generally accepted as binding to PIP2. Using super-resolution Stochastic Optical Reconstruction Microscopy we have shown that PIP2 and PIP3 clusters do not colocalize, and find that PIP3 clusters, but not PIP2 clusters, are reduced in number by over expression of PTEN (Phosphatase and TENsin homolog). Analysis of single vesicle rates of secretion with TIRF (Total Internal Reflection Fluorescence) microscopy shows a correlation between secretion rates and cellular PTEN content. These results implicate PIP3 in the secretory machinery, and may illuminate some of the neurological impacts of PTEN mutations, such as epilepsy and Autism Spectrum Disorders.