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Davey, Rhonda
- PublicationQuantitative image mean squared displacement (iMSD) analysis of the dynamics of profilin 1 at the membrane of live cellsImage mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories.
- PublicationProfilin Diffusion in Live Cell Membranes(University of New England, 2019-06-07)
; ; ; 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.
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.
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.