James A. Spudich

Myosin-V is a processive actin-based motor

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport perhaps working with microtubule-based motors such as kinesin,. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor, like kinesin,. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.

Optimized localization-analysis for single-molecule tracking and super-resolution microscopy

We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fishers information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.

Quantitative measurements of force and displacement using an optical trap

We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.

Myosin VI is a processive motor with a large step size

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.

Assays for actin sliding movement over myosin-coated surfaces.

Publisher Summary One important result from in vitro studies of the interaction of the major proteins of muscle, actin and myosin, has been the growing recognition that nearly any aspect of muscle mechanics can be studied in a model system consisting of purified proteins. This chapter is a compilation of techniques for purified in vitro motility assays for actin sliding movement over myosin. Several forms of myosin, including filaments, monomers, and soluble proteolytic fragments, have been found to work well in aetin sliding movement assays. The focus is limited to studies using skeletal muscle proteins, but only slight modification of these protocols may be necessary for proteins derived from smooth muscle and nonmuscle sources. The properties of the protein preparations used are critical to reproducibility of actin sliding movement assays. The methods presented in the chapter are trustworthy preparations but are not singularly successful. However, in particular it should be noted that myosin subfragment preparations that work well in solution experiments might not be optimal for use in movement assays.

Myosin step size: Estimation from slow sliding movement of actin over low densities of heavy meromyosin☆

We have estimated the step size of the myosin cross-bridge (d, displacement of an actin filament per one ATP hydrolysis) in an in vitro motility assay system by measuring the velocity of slowly moving actin filaments over low densities of heavy meromyosin on a nitrocellulose surface. In previous studies, only filaments greater than a minimum length were observed to undergo continuous sliding movement. These filaments moved at the maximum speed (Vo), while shorter filaments dissociated from the surface. We have now modified the assay system by including 0.8% methylcellulose in the ATP solution. Under these conditions, filaments shorter than the previous minimum length move, but significantly slower than Vo, as they are propelled by a limited number of myosin heads. These data are consistent with a model that predicts that the sliding velocity (v) of slowly moving filaments is determined by the product of vo and the fraction of time when at least one myosin head is propelling the filament, that is, v = vo [1-(1-ts/tc)N], where ts is the time the head is strongly bound to actin, tc is the cycle time of ATP hydrolysis, and N is the average number of myosin heads that can interact with the filament. Using this equation, the optimum value of ts/tc to fit the measured relationship between v and N was calculated to be 0.050. Assuming d = vots, the step size was then calculated to be between 10nm and 28 nm per ATP hydrolyzed, the latter value representing the upper limit. This range is within that of geometric constraint for conformational change imposed by the size of the myosin head, and therefore is not inconsistent with the swinging cross-bridge model tightly coupled with ATP hydrolysis.

Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo.

Three threonine residues in the tail region of Dictyostelium myosin II heavy chain have been implicated previously in control of myosin filament formation. Here we report the in vitro and in vivo consequences of converting these sites to alanine residues, which eliminates phosphorylation at these positions, or to aspartate residues, which mimics the negative charge state of the phosphorylated molecule. Alanine substitution allows in vitro assembly and in vivo contractile activity, although this myosin shows substantial over-assembly in vivo. Aspartate substitution eliminates filament assembly in vitro and renders the myosin unable to drive any tested contractile event in vivo. These results demonstrate that heavy chain phosphorylation plays a key modulatory role in controlling myosin function in vivo.

Gene replacement in Dictyostelium: generation of myosin null mutants.

The eukaryotic slime mold Dictyostelium discoideum has a single conventional myosin heavy chain gene (mhcA). The elimination of the mhcA gene was achieved by homologous recombination. Two gene replacement plasmids were constructed, each carrying the G418 resistance gene as a selective marker and flanked by either 0.7 kb of 5′ coding sequence and 0.9 kb of 3′ coding sequence or 1.5 kb of 5′ flanking sequence and 1.1 kb of 3′ flanking sequence. Myosin null mutants (mhcA‐ cells) were obtained after transformation with either of these plasmids. The mhcA‐ cells are genetically stable and are capable of a variety of motile processes. Our results provide genetic proof that in Dictyostelium the conventional myosin gene is required for growth in suspension, normal cell division and sporogenesis, and illustrate how gene targeting can be used as a tool in Dictyostelium.

Chapter 18 Purification of Muscle Actin

Publisher Summary This chapter focuses on the purification methods of muscle actin. With the advent of polyacrylamide gel electrophoresis as a highly resolving analytical tool for ascertaining protein purity, it became evident that muscle actin isolated by classic procedure contained significant amounts of actomyosin-associated muscle proteins such as tropomyosin and α-actinin. SDS-polyacrylamide gels have met with widespread use as a general method for obtaining muscle actin. A problem in establishing methods for actin purification resides in the level of purity. Emerging experimentation in cell biology and, specifically, cytoskeletal biochemistry requires probing sensitive properties of actin itself and actin associations with other cell proteins. The chapter explores some of the pitfalls associated with actin purification and clarifies in some detail the correct usage and expected result from each step of the widely used muscle actin purification. Additional steps to eliminate trace contaminants are also described.

The Mechanism of Myosin VI Translocation and Its Load-Induced Anchoring

Myosin VI is thought to function as both a transporter and an anchor. While in vitro studies suggest possible mechanisms for processive stepping, a biochemical basis for anchoring has not been demonstrated. Using optical trapping, we observed myosin VI stepping against applied forces. Step size is not strongly affected by such loads. At saturating ATP, myosin VI kinetics shows little dependence on load until, at forces near stall, its stepping slows dramatically as load increases. At subsaturating ATP or in the presence of ADP, stepping kinetics is significantly inhibited by load. From our results, we propose a mechanism of myosin VI stepping that predicts a regulation through load of the motors roles as transporter and anchor.