Abstract
Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy–based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.
Highlights
Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements
To investigate the basal F-actin severing activity of the gelsolin preparations used in the present study under standardized conditions, we first checked the disassembly of actin filaments in solution in the presence of EGTA (1 mM) or CaCl2 (100 μM) (Fig. 1)
In a first set of experiments, we found that the rate of spontaneous actin depolymerization is very low in the presence of EGTA, whereas addition of CaCl2 slightly favors the monomeric form of actin even in the absence of gelsolin (Fig. 1A)
Summary
Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Myosin motor activity fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surfaceadsorbed heavy meromyosin (HMM) motor fragments. We used an in vitro motility assay, where actin filaments are propelled by surfaceadsorbed heavy meromyosin (HMM) motor fragments This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. To the best of our knowledge, these issues have not been previously studied
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