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

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. OsO(4) and KMnO(4)) fixatives induce blebbing on spread cells. We also show that aldehyde fixation may induce the loss or delocalization of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the plasma membrane and that the asymmetric distribution of fixation-induced blebs on spread/migrating cells coincides with that of PIP(2) on the cells prefixed by lipid-stabilizing fixatives (e.g., OsO(4)). Moreover, fixation induces blebbing less readily on PIP(2)-elevated spread cells but more readily on PIP(2)-lowered or lipid raft-disrupted spread cells. Our data suggest that fixation-induced lowering of PIP(2) level at cytoskeleton-attaching membrane sites causes bleb formation via local breakdown of the membrane–cytoskeleton coupling.