Model for processive movement of myosin V and myosin VI

Myosin V and myosin VI are two classes of two-headed molecular motors of the myosin superfamily that move processively along helical actin filaments in opposite directions. Here we present a hand-over-hand model for their processive movements. In the model, the moving direction of a dimeric molecular motor is automatically determined by the relative orientation between its two heads at free state and its head's binding orientation on track filament. This determines that myosin V moves toward the barbed end and myosin VI moves toward the pointed end of actin. During the moving period in one step, one head remains bound to actin for myosin V whereas two heads are detached for myosin VI: The moving manner is determined by the length of neck domain. This naturally explains the similar dynamic behaviors but opposite moving directions of myosin VI and mutant myosin V (the neck of which is truncated to only one-sixth of the native length). Because of different moving manners, myosin VI and mutant myosin V exhibit significantly broader step-size distribution than native myosin V. However, all three motors give the same mean step size of 36 nm (the pseudo-repeat of actin helix). Using the model we study the dynamics of myosin V quantitatively, with theoretical results in agreement with previous experimental ones.Comment: 18 pages, 7 figure

Myosin II filament dynamics in actin networks revealed with interferometric scattering microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect, and image individual membrane attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network

The coagulation of myosin in muscle

The coagulation of myosin is one of the few changes in the proteins of muscle known to take place during contraction and rigor. Although under certain conditions as much as one-third of the total protein of muscle may become insoluble, the significance of this change for the shortening of muscle is not understood. And yet the recognition of a definite transformation in the substance of muscle should be of value in investigating the mechanism of contraction, especially when one recalls that nearly all of those abortive theories of contraction that have been formulated since the time of Descartes have been based on knowledge of systems supposed to be analogous to muscle, rather than on a knowledge of the properties of living muscle itself. As a step towards an understanding of the chemical properties of the "living machinery" of muscle (as distinguished from the metabolic transformations in muscle) I have, accordingly, investigated the coagulation of myosin. I have already shown how the coagulation of myosin in muscle is related to the denaturation and coagulation of isolated myosin (Mirsky, 1935-36 and 1936-37). By measurements of protein sulfhydryl groups it was found that the coagulation of myosin in muscle differs from the coagulation of myosin and other proteins brought about by the usual denaturing agents (such as heat and acid) but resembles the coagulation of myosin caused by dehydration. At this point it is important to recall that when myosin is said to coagulate in muscle it is not supposed that myosin actually precipitates from solution. In muscle probably only a very small part of the myosin present is dissolved (Smith, 1934). That there is a change in myosin in muscle is inferred from the fact that at one time the protein can be dissolved in certain media in which at another time it cannot be dissolved; the myosin is said to have coagulated. Apparently myosin in muscle can pass from one gel state to another. How these states differ will be considered in this paper

Myosin VI Lever Arm Rotation: Fixed or Variable?

Two recent articles addressed the power-stroke of myosin VI molecules during stepping. Although both groups measured the angles of fluorescent probes attached on the myosin VI molecule lever arm using polarized fluorescence techniques, they differ about whether the myosin VI lever arm rotation is fixed1 or variable2. Here we discuss the causes of the discrepancy between the two studies and the implications for myosin VI processive motility

A Branched Kinetic Scheme Describes the Mechanochemical Coupling of Myosin Va Processivity in Response to Substrate

Myosin Va is a double-headed cargo-carrying molecular motor that moves processively along cellular actin filaments. Long processive runs are achieved through mechanical coordination between the two heads of myosin Va, which keeps their ATPase cycles out of phase, preventing both heads detaching from actin simultaneously. The biochemical kinetics underlying processivity are still uncertain. Here we attempt to define the biochemical pathways populated by myosin Va by examining the velocity, processive run-length, and individual steps of a Qdot-labeled myosin Va in various substrate conditions (i.e., changes in ATP, ADP, and Pi) under zero load in the single-molecule total internal reflection fluorescence microscopy assay. These data were used to globally constrain a branched kinetic scheme that was necessary to fit the dependences of velocity and run-length on substrate conditions. Based on this model, myosin Va can be biased along a given pathway by changes in substrate concentrations. This has uncovered states not normally sampled by the motor, and suggests that every transition involving substrate binding and release may be strain-dependent. © 2012 Biophysical Society

Stratification relieves constraints from steric hindrance in the generation of compact acto-myosin asters at the membrane cortex

Recent in-vivo studies have revealed that several membrane proteins are driven to form nanoclusters by active contractile flows arising from F-actin and myosin at the cortex. The mechanism of clustering was shown to be arising from the dynamic patterning of transient contractile platforms (asters) generated by actin and myosin. Myosin-II, which assemble as minifilaments consisting of tens of myosin heads, are rather bulky structures and hence a concern could be that steric considerations might obstruct the emergence of nanoclustering. Here, using coarse-grained, agent-based simulations that respect the size of constituents, we find that in the presence of steric hindrance, the patterns exhibited by actomyosin in two dimensions, do not resemble the steady state patterns observed in our in-vitro reconstitution of actomyosin on a supported bilayer. We then perform simulations in a thin rectangular slab, allowing the separation of a layer of actin filaments from those of myosin-II minifilaments. This recapitulates the observed features of in-vitro patterning. Using super resolution microscopy, we find direct evidence for stratification in our in-vitro system. Our study suggests the possibility that molecular stratification may be an important organising feature of the cortical cytoskeleton in-vivo