Loss of CIP4 affects membrane fluidity. (A) Decreased membrane fluidity in CIP4-deficient cells by fluorescence anisotropy study with PMA treatment. Fluorescent anisotropy values (r) are in response to stimulation by PMA. Results are given as percentage of time zero values. A lower r value means higher membrane fluidity. Cells with CIP4 knockdown have impaired response in terms of plasma membrane fluidity, when compared with control cells (P < .05 when comparing control and shRNA CIP4 cells for 20, 40, and 50 minutes). (B) Decreased membrane fluidity in cells deficient for CIP4 treated with fibronectin. Fluorescent anisotropy value was in response to integrin stimulation by fibronectin. Results are percentage of control (BSA coating) conditions. After labeling, CHRF cells were incubated on a fibronectin- or BSA-coated plate for 2 hours. CIP4 knockdown cells display decreased membrane fluidity compared with control. (P = .002 for shCIP4 compared, P = .10 for shRNA WASP). (C-G). Simulated NMR deuterium order parameters calculations confirm decreased fluidity in the presence of CIP4 protein. The deuterium order parameters, Scd, were plotted (C) for each carbon in the unsaturated POPC acyl chain (D) and show a 2.34 ± 0.02% decrease between the lipids near the protein (E) and the protein-free system. The order parameter is a function of the angle formed by a carbon-deuterium bond on the lipid acyl chain and the vector perpendicular to the membrane surface (F-G) and a decrease in order parameter is associated with an increase in disorder of the acyl chain.
