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

•Protein- but not lipid-stabilizing fixatives induce cell blebbing of spread cells.•Asymmetric distribution of fixation-induced blebs coincides with that of PIP2.•Fixation less readily induces blebbing on spread cells with elevated PIP2 levels.•Fixation more readily induces blebbing on spread cells...

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Published in:FEBS open bio 2014-01, Vol.4 (1), p.190-199
Main Authors: Zhao, Siyuan, Liao, Huanhuan, Ao, Meiying, Wu, Li, Zhang, Xiaojun, Chen, Yong
Format: Article
Language:English
Subjects:
Aldehydes
Cell blebbing
Cell fixation
Cytoskeleton
Experiments
Fixatives
Human umbilical vein endothelial cells (HUVECs)
Lipid rafts
Lipids
Molecular modelling
Penicillin
Phosphatidylinositol
Phosphatidylinositol 4,5-bisphosphate (PIP2)
Phosphatidylinositol 4,5-diphosphate
Physiology
Picric acid
plasma membrane
Potassium permanganate
Proteins
THP-1-derived macrophages
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Summary:•Protein- but not lipid-stabilizing fixatives induce cell blebbing of spread cells.•Asymmetric distribution of fixation-induced blebs coincides with that of PIP2.•Fixation less readily induces blebbing on spread cells with elevated PIP2 levels.•Fixation more readily induces blebbing on spread cells with lower PIP2 levels.•Disruption of lipid rafts enhances fixation-induced blebbing of spread cells. 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.
ISSN:2211-5463
2211-5463
DOI:10.1016/j.fob.2014.02.003