Profilin Diffusion in Live Cell Membranes

Abstract

<p>Profilin is a ubiquitously expressed protein involved in the regulation of cell proliferation, apoptosis and motility. In vitro, it is essential in the coordination of actin filament assembly and disassembly. However, the dynamics of profilin in live cells is still largely unknown. The epithelial breast cancer cell line MDA-MB-231 was transfected with wild type GFP-profilin, or GFP-profilin mutants R88A, R88E/R136D, H119E or H133S. These mutations affect the binding of profilin to phosphoinositide lipids, phosphoinositide lipids and actin, actin, or poly-proline rich domains respectively.</p> <p>Image stacks of the cell membrane were acquired by TIRF microscopy and analysed using image mean square displacement (iMSD) analysis to determine if the diffusion modes (isotropic, confined or transiently confined) and diffusion rates were altered by the perturbation of profilin interactions by the different mutations. The cells were further perturbed by treatment by the actin filament disrupting drugs Cytochalasin D and Latrunculin A, by removal of cholesterol in membrane by methyl-cyclodextrin or by serum starvation.</p> <p>The diffusion of wild type profilin and the profilin mutations respond in different ways to treatments, giving information not only on profilin dynamics in the cell membrane but also on the structure and organisation of the membrane itself.</p>