Actin filament assembly and mechanics are crucial for muscle contraction, maintenance of cell structure, motility, and division. Actin filament assembly occurs in a crowded intracellular environment consisting of various types of molecules, including small organic molecules known as osmolytes. Ample evidence highlights the protective functions of osmolytes such as trimethylamine-N-oxide (TMAO), including their effects on protein stability and their ability to counteract cellular osmotic stress. Recently, TMAO has been shown to counteract the denaturing effects of urea on actin filament assembly based on bulk fluorescence assays. Yet, how TMAO affects individual actin filament assembly dynamics and mechanics is not well understood. We hypothesize that, owing to its protective nature, TMAO will enhance filament assembly kinetics and stiffen actin filaments due to increased stability. In this study, we investigate osmolyte-dependent actin filament assembly kinetics and bending mechanics by measuring filament elongation rates and bending persistence lengths in the presence of TMAO using total internal reflection fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays. We demonstrate that TMAO enhances increases the elongation rates of individual filaments as well as steady-state average filament lengths and enhances filament bending stiffness. Taken together, our results show that the physiochemical properties of the intracellular environment can regulate actin assembly dynamics and mechanics. This study will help identify molecular mechanisms of how small organic osmolytes modulate cytoskeletal protein filament assembly and mechanics in living cells.
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Master of Science (M.S.)
College of Graduate Studies
Nanoscience Technology Center
Length of Campus-only Access
Masters Thesis (Open Access)
Kravchuk, Pavlo, "How Small Organic Osmolytes Modulate Actin Filament Assembly Kinetics and Mechanics" (2022). Electronic Theses and Dissertations, 2020-. 1238.