Hydrodynamic and Polyelectrolyte Properties of Single Actin Filaments and Actin Bundling Formation

Actin filaments, bundles, and other higher-order structures have been extensively studied due to their capability to change their polyelectrolyte and hydrodynamic properties to external and internal alterations within the cytoskeleton to achieve specific biological functions. For instance, branched and crosslinked networks in a quasi-two-dimensional sheet make up the lamellipodium at the front of the cell. They are the main engine of cell movement since they push the cell membrane by polymerizing against it. Aligned bundles underlie filopodia, the fingerlike structures at the front of the cell, essential for the directional response of the cell. A thin layer of actin mesh (a mix of bundled straight and branched filaments), called the cell cortex, coats the plasma membrane at the back and sides of the cell, which is essential for cell shape maintenance and changes. Nevertheless, the underlying biophysical principles and molecular mechanisms that support actin's polyelectrolyte and hydrodynamic nature remain elusive. Thus, the central objective of this research is to gain a fundamental understanding of these properties and their biological functions under different intracellular environments and protein concentrations. In the first analysis, we focused the investigation on actin filaments at low protein concentrations where inter-filament interactions can be neglected. Unlike conventional approaches, we characterized hydrodynamics properties of polydisperse, semiflexible charged actin filaments without (dynamic light scattering, DLS) and with (electrophoresis light scattering, ELS) an external electric field perturbation using the same sample and experimental conditions. In the DLS experiments, we measured the longitudinal diffusion coefficient, which characterizes the Brownian motion of the filaments in a dispersion solution. In the ELS experiments, we measured the electrophoretic mobility, which represents the movement of the charged actin filaments relative to the liquid under the influence of an applied electric field. We also developed novel protocols based on bio-statistical tools to minimize errors and ensure the reproducibility of our results. Additionally, we characterized the longitudinal diffusion coefficient and electrophoretic mobility properties using an extended semiflexible wormlike chain model and asymmetric polymer length distribution theory. This characterization is novel since it is obtained from non-invasive experiments in hydrodynamic conditions. Finally, we considered several g-actin and polymerization buffers to elucidate the impact of their chemical composition, reducing agents, pH values, and ionic strengths on the filament translational diffusion coefficient, electrophoretic mobility, structure factor, asymmetric length distribution, effective filament diameter, electric charge, zeta potential, and semi-flexibility. In the second and final analysis, we investigated the polyelectrolyte and hydrodynamic properties of actin bundles arising from inter-filament interactions at increasing protein and divalent ionic concentrations. We extended the previous approach to characterize these structures under various pH solution levels, and calcium and magnesium concentrations, which usually appear in different cellular compartments and locations at physiological conditions. We determined the effective charge, inter-filament distance, semi-flexibility, length distribution, and diameter of actin bundles and their relationship with the number of actin filaments, their charge, semi-flexibility, and diameter characterized at low protein concentrations. This comparison revealed how the biological environment and inter-filament interactions form and stabilize actin bundles. The outcomes of this research project will provide the bases and theoretical framework to characterize more complex cytoskeleton structures, particularly those formed by crosslinkers and binding proteins governing other vital biological functions in eukaryotic cells under physiological and pathological conditions. While other highly anionic semiflexible polyelectrolytes, including ARNs, DNAs, microtubules, and tobacco viruses, have different biomolecular sizes and lengths, charge, and semi-flexibility, their mechanical and polyelectrolyte properties resemble those of actin filaments. Thus, suitable modifications of this approach might also be applicable to characterize other biomolecules relevant to biophysics and neuroscience.