title: Fixation-induced cell blebbing on spread cells inversely correlates with phosphatidylinositol 4,5-bisphosphate level in the plasma membrane.
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.
authors.affiliation: Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Zhao
authors.affiliation: 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.
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Liao
authors.affiliation: 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.
authors.forename: Meiying
authors.initials: M
authors.lastname: Ao
authors.affiliation: 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.
authors.forename: Li
authors.initials: L
authors.lastname: Wu
authors.affiliation: 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.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
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