Fixation-induced cell blebbing on spread cells inversely correlates with phosphatidylinositol 4,5-bisphosphate level in the plasma membrane

Siyuan Zhao; Huanhuan Liao; Meiying Ao; Li Wu; Xiaojun Zhang; Yong Chen

doi:10.1016/j.fob.2014.02.003

Fixation-induced cell blebbing on spread cells inversely correlates with phosphatidylinositol 4,5-bisphosphate level in the plasma membrane

FEBS Open Bio. 2014 Feb 14:4:190-9. doi: 10.1016/j.fob.2014.02.003. eCollection 2014.

Authors

Siyuan Zhao¹, Huanhuan Liao², Meiying Ao³, Li Wu², Xiaojun Zhang², Yong Chen²

Affiliations

¹ Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China.
² Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China ; School of Life Sciences and Food Engineering, Nanchang University, Nanchang, Jiangxi 330031, China.
³ School of Life Sciences and Food Engineering, Nanchang University, Nanchang, Jiangxi 330031, China ; Department of Pharmacy, Science and Technology College, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330025, China.

Abstract

While most attention has been focused on physiologically generated blebs, the molecular mechanisms for fixation-induced cell blebbing are less investigated. We show that protein-fixing (e.g. aldehydes and picric acid) but not lipid-stabilizing (e.g. OsO4 and KMnO4) fixatives induce blebbing on spread cells. We also show that aldehyde fixation may induce the loss or delocalization of phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane and that the asymmetric distribution of fixation-induced blebs on spread/migrating cells coincides with that of PIP2 on the cells prefixed by lipid-stabilizing fixatives (e.g., OsO4). Moreover, fixation induces blebbing less readily on PIP2-elevated spread cells but more readily on PIP2-lowered or lipid raft-disrupted spread cells. Our data suggest that fixation-induced lowering of PIP2 level at cytoskeleton-attaching membrane sites causes bleb formation via local breakdown of the membrane-cytoskeleton coupling.

Keywords: Cell blebbing; Cell fixation; DAG, 1,2-diacylglycerol; DIC, differential interference contrast; HUVECs, human umbilical vein endothelial cells; Human umbilical vein endothelial cells (HUVECs); IP3, inositol 1,4,5-trisphosphate; Lipid rafts; MβCD, methyl-β-cyclodextrin; PI3K, phosphoinositide-3 kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PLC, phospholipase C; Phosphatidylinositol 4,5-bisphosphate (PIP2); TBS, Tris-buffered saline; THP-1-derived macrophages.