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.

Keywords

• Cell fixation
• Cell blebbing
• Phosphatidylinositol 4,5-bisphosphate (PIP2)
• Human umbilical vein endothelial cells (HUVECs)
• Lipid rafts
• THP-1-derived macrophages