Abstract
Drebrin E is a regulatory protein of intracellular force produced by actomyosin complexes, that is, myosin molecular motors interacting with actin filaments. The expression level of drebrin E in nerve cells decreases as the animal grows, suggesting its pivotal but unclarified role in neuronal development. Here, by applying the microscopic heat pulse method to actomyosin motility assay, the regulatory mechanism is examined from the room temperature up to 37 °C without a thermal denaturing of proteins. We show that the inhibition of actomyosin motility by drebrin E is eliminated immediately and reversibly during heating and depends on drebrin E concentration. The direct observation of quantum dot-labeled drebrin E implies its stable binding to actin filaments during the heat-induced sliding. Our results suggest that drebrin E allosterically modifies the actin filament structure to regulate cooperatively the actomyosin activity at the maintained in vivo body temperature.
Highlights
Drebrin E is a regulatory protein of intracellular force produced by actomyosin complexes, that is, myosin molecular motors interacting with actin filaments
On the basis of our results, we propose that the inhibition of actomyosin activity by drebrin E is not a simple competitive binding to actin filaments with myosin but involves a cooperative modification of actin filament structure
The current study investigated the drebrin E-mediated modulation of actomyosin activity and its thermal sensitivity using the optically controlled microscopic heat pulse method
Summary
E concentration, IC50, Hill coefficient, and baseline, respectively. The change of the sliding velocity curve shape from decay-type to sigmoidal-type with the temperature increase is determined as (i) the nonlinear decrease in sliding velocity with drebrin E concentration and (ii) the higher concentration of drebrin E required at a higher temperature to gain an inhibitory effect. The data obtained in the presence of 31 nM Qdot-labeled drebrin E as in Figure 3 were combined with those in Figure 4B,C (open small circles). The change of trend with temperature (Figure 4C) was described quantitatively by the half-maximum inhibitory concentration of drebrin (IC50) and Hill coefficients shown in Figure 4D and Table S1. The IC50 value that corresponded to half of the maximum velocity gradually increased from a few nanomolar up to over 30 nM of drebrin concentration as the temperature rose. The Hill coefficient, which reflects the degree of cooperativity in the given reaction, was kept slightly above 1.0 at a temperature below ∼34 °C, whereas it abruptly increased to 4.0 at 36.5−37.5 °C
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