Unusual Features of Chick VSP and its Potential Role in Regulation of Cell Structure

Abstract

Voltage-sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the PTEN-like region with phosphoinositide-phosphatase activities. VSP is conserved from sea squirt to human, but little is known about its physiological roles. Here we characterize the molecular properties of chick ortholog of VSP (designated as Gg-VSP). Gg-VSP shares basic properties with other VSP orthologs, including voltage sensing by the transmembrane segments and phosphoinositide phsophatase activities of the cytoplasmic region. Imaging of PI(3,4)P2-sensitive fluorescent protein in Xenopus oocyte showed that membrane depolarization triggers dephosphorylation of PI(3,4,5)P3 into PI(3,4)P2. Notably, about 10% of cell populations of DF-1 cells transiently expressing Gg-VSP exhibited morphological feature of cell process outgrowths. This was accompanied by the decrease of actin filaments as detected by phalloidin-staining. Transfection of the voltage sensor mutant, Gg-VSPR153Q, showing shift of voltage dependence to a lower voltage, facilitated cell process outgrowths. Coexpression of chick PTEN cDNA suppressed this effect of Gg-VSP. Visualization of PI(3,4)P2 with the Ds-Red fused with the FAPP1-derived pleckstrin homology (PH) domain showed more fluorescent clusters than luciferase-transfected control cells, suggesting that cell process outgrowths paralleled with the increase of PI(3,4)P2. Whole mount in situ hybridization and immunostaining of chicken brain showed that Gg-VSP is expressed in neurons. These findings suggest that one of the VSP’s functions is to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile.