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Abstract

Cells need to move, divide, change shape, transport cargo, and explore their environment. To perform this diverse array of functions, cells dynamically construct and rearrange actin filaments into distinct networks through interactions between actin and unique combinations of actin-binding proteins (ABPs). These proteins constitute the actin cytoskeleton. The protein-protein interactions, which typically occur on the scale of ångströms to nanometers, lead to profound changes in the emergent actin filament networks at the micron scale. By understanding the mechanisms that govern the behavior at the molecular level, one can gain a deeper understanding of the higher-order structures that emerge. This appreciation is key to identifying how healthy cells carry out important cytoskeletal functions and how unhealthy cells, some with only one amino acid mutated, fail to do so. In recent years, research has demonstrated that the behavior of a wide range of ABPs can be modulated by mechanical forces. The effect of this is that cells can utilize the actin cytoskeleton in ways that are tailored to particular mechanical stimuli. Interestingly, different forms of mechanoregulation have been reported. Some systems respond when forces are applied to the ABP, as in the case of the actin nucleation and elongation factor, formin. For other systems, the behavior of the ABP changes when force is applied to the actin filaments themselves, as in the case of mechanosensitive LIM domains. Additionally, the effect of the force can either enhance ABP binding or activity, as in the case of LIM and certain isoforms of formin, respectively, or limit protein activity, as in one isoform of formin. However, many of these interesting phenomena have not been explained in mechanistic detail. Here, I present a body of work that explains the molecular origins of important behaviors and interactions that govern the actin cytoskeleton. First, I use classical molecular dynamics simulations to elucidate why one end of a bare actin filament has much faster polymerization kinetics than the other, and report that the conformational change between actin monomers in solution and actin subunits in filaments occurs gradually as additional subunits are added. In the second project, I apply forces to actin filaments using steered molecular dynamics. This reveals that a distinct residue switch governs at which interface the actin filament fragments. However, before fragmentation occurs, a metastable ‘cracked’ state presents a unique strain-induced binding surface. I find that mechanosensitive LIM domains bind the crack, and these binding poses offer natural explanations for what conveys mechanosensitivity to LIM domains. Third, I use a coarse grained model to simulate the process of cytokinetic ring assembly in a fission yeast cell. These results demonstrate how the mechanoregulation of formin impacts the higher-order emergent phenomenon of ring formation. Fourth, I set up and use a microfluidics apparatus to exert forces onto individual formin molecules anchored to the coverslip. This reveals that a single formin dimer isoform can be either enhanced or inhibited when subjected to tension. Lastly, I report on ongoing projects aimed at understanding the mechanism of spontaneous actin filament nucleation and inorganic phosphate release, and suggest future directions.

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