Histamine causes adjacent endothelial cells to retract from each another. We examined phosphorylation of the 20-kD myosin light chain (MLC20) in human umbilical vein endothelial cells (HUVECs) exposed to histamine to determine if we could find evidence to support the hypothesis that retraction of these cells in response to histamine represents an actomyosininitiated contraction of the endothelial cytoskeleton. We found that MLC20 in HUVECs was constitutively phosphorylated

Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (P i ) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining P i in its active site, by either elevating P i in solution or by introducing a mutation in myosin's active site (S217A) to slow P irelease from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s À1 ) and without a detectable delay, both in the absence and presence of 30 mM P i . The elevated levels of P i did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that P i rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower P i release rate. These findings provide direct evidence that myosin Va generates a powerstroke with P i still in its active site.

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force generating lever arm in myosin V. We directly compared the FRET results with single molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), while our simulations suggest the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and non-muscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosin’s mechanics and unloaded kinetics are not affected by thin filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin filament regulatory proteins do not affect either the identity or magnitude of myosin’s primary load-dependent transition. Interestingly, at low ATP concentrations, thin filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for both disease modeling and computational models of muscle contraction.Significance StatementHuman heart contraction is powered by the molecular motor β-cardiac myosin, which pulls on thin filaments consisting of actin and the regulatory proteins troponin and tropomyosin. In some muscle and non-muscle systems, these regulatory proteins tune the kinetics, mechanics, and load dependence of the myosin working stroke. Despite having a central role in health and disease, it is not well understood whether the mechanics or kinetics of β-cardiac myosin are affected by regulatory proteins. We show that regulatory proteins do not affect the mechanics or load-dependent kinetics of the working stroke at physiologically relevant ATP concentrations; however, they can affect the kinetics at low ATP concentrations, suggesting a mechanism beyond simple steric blocking. This has important implications for modeling of cardiac physiology and diseases.

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