The assembly, mechanics, and structure of actin cytoskeleton are critical for eukaryotic cellular processes such as structural support, cellular motility, and intracellular transport. Actin assembly occurs in crowded intracellular environments containing many ions and macromolecules. While the individual roles of cation and crowding on actin assembly and mechanics are well established, how the combined cation and crowding effects modulate the formation, mechanics and structural transitioning of actin filaments and bundles are not fully understood. Here, we investigate the impacts of various cation and crowded environments on the bending stiffness, length, organization, secondary structure, and kinetics of actin filaments and bundles. Fluorescence microscopy and biophysical analysis of filaments in crowding indicates an increase to filament bending stiffness and reductions in length, while molecular dynamics (MD) simulations demonstrate crowding alters filament conformations and inter-subunit contacts. Direct visualization and analysis of bundle stiffness and geometry by fluorescence imaging and atomic force microscopy (AFM) show changes to bundle longitudinal and transverse mechanics as well as bundle packing, displaying a dissimilar dependence on either cation or crowding conditions. Furthermore, spectroscopy results illustrate bundles experience changes to secondary structure and hydrogen bonding with shifts from a-helices to ß-sheets supporting observed bundle stiffness in cation and crowded environments. In addition, bundle assembly kinetics demonstrate that bundle formation is sensitive to variations in electrostatic and crowding conditions, potentially influencing bundle mechanics and structure. These investigations suggest that cation and crowding effects may work counteractively leading to alterations in bundle formation, mechanics, and structure.
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Doctor of Philosophy (Ph.D.)
College of Medicine
Burnett School of Biomedical Sciences
Length of Campus-only Access
Doctoral Dissertation (Open Access)
College of Medicine
Castaneda, Nicholas, "Regulation of Actin Assembly, Mechanics, and Structure by Intracellular Environmental Factors" (2021). Electronic Theses and Dissertations, 2020-. 661.