David M. Warshaw

Cargo transport: molecular motors navigate a complex cytoskeleton.

Intracellular cargo transport requires microtubule-based motors, kinesin and cytoplasmic dynein, and the actin-based myosin motors to maneuver through the challenges presented by the filamentous meshwork that comprises the cytoskeleton. Recent in vitro single molecule biophysical studies have begun to explore this process by characterizing what occurs as these tiny molecular motors happen upon an intersection between two cytoskeletal filaments. These studies, in combination with in vivo work, define the mechanism by which molecular motors exchange cargo while traveling between filamentous tracks and deliver it to its destination when going from the cell center to the periphery and back again.

Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.

Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.

Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro.

SummaryDifferences in the mechanical properties of mammalian smooth, skeletal, and cardiac muscle have led to the proposal that the myosin isozymes expressed by these tissues may differ in their molecular mechanics. To test this hypothesis, mixtures of fast skeletal, V1 cardiac, V3 cardiac and smooth muscle (phosphorylated and unphosphorylated) myosin were studied in an in vitro motility assay in which fluorescently-labelled actin filaments are observed moving over a myosin coated surface.Pure populations of each myosin produced actin filament velocities proportional to their actin-activated ATPase rates. Mixtures of two myosin species produced actin filament velocities between those of the faster and slower myosin alone. However, the shapes of the myosin mixture curves depended upon the types of myosins present. Analysis of myosin mixtures data suggest that: (1) the two myosins in the mixture interact mechanically and (2) the same force-velocity relationship describes a myosins ability to operate over both positive and negative forces. These data also allow us to rank order the myosins by their average force per cross-bridge and ability to resist motion (phosphorylated smooth > skeletal = V3 cardiac > V1 cardiac). The results of our study may reflect the mechanical consequence of multiple myosin isozyme expression in a single muscle cell.

Single-Molecule Mechanics of R403Q Cardiac Myosin Isolated From the Mouse Model of Familial Hypertrophic Cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is an inherited cardiac disease that can result in sudden death in the absence of any overt symptoms. Many of the cases documented to date have been linked with missense mutations in the beta-myosin heavy chain gene. Here we present data detailing the functional impact of one of the most deadly mutations, R403Q, on myosin motor function. Experiments were performed on whole cardiac myosin purified from a mouse model of FHC to eliminate potential uncertainties associated with protein expression systems. The R403Q mutant myosin demonstrated 2.3-fold higher actin-activated ATPase activity, 2.2-fold greater average force generation, and 1.6-fold faster actin filament sliding in the motility assay. The force- and displacement-generating capacities of both the normal and mutant myosin were also characterized at the single molecule level in the laser trap assay. Both control and mutant generated similar unitary forces ( approximately 1 pN) and displacements ( approximately 7 nm) without any differences in event durations. On the basis of the distribution of mean unitary displacements, this mutation may possibly perturb the mechanical coordination between the 2 heads of cardiac myosin. Any of these observations could, alone or possibly in combination, result in abnormal power output and potentially a stimulus for the hypertrophic response.

Cardiac V1 And V3 Myosins Differ in Their Hydrolytic and Mechanical Activities In Vitro

The two mammalian cardiac myosin heavy chain isoforms, alpha and beta, have 93% amino acid homology, but hearts expressing these myosins exhibit marked differences in their mechanical activities. To further understand the function of these cardiac myosins as molecular motors, we compared the ability of these myosins to hydrolyze ATP and to both translocate actin filaments and generate force in an in vitro motility assay. V1 myosin has twice the actin-activated ATPase activity and three times the actin filament sliding velocity when compared with V3 myosin. In contrast, the force-generating ability of these myosins is quite different when the total force produced by a small population of myosin molecules (> 50) is examined. V1 myosin produces only one half the average cross-bridge force of V3 myosin. With discrete areas of primary structural heterogeneity known to exist between alpha and beta heavy chains, the differences we report in the hydrolytic and mechanical activities of the motors are explored in the context of potential structural and kinetic differences between the V1 and V3 myosins.

A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap.

Two smooth muscle myosin heavy chain isoforms differ by a 7-amino- acid insert in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the inserted isoform is mainly found in phasic muscle. The inserted isoform has twice the actin-activated ATPase activity and actin filament velocity in the in vitro motility assay as the non-inserted isoform. We used the laser trap to characterize the molecular mechanics and kinetics of the inserted isoform ((+)insert) and of a mutant lacking the insert ((−)insert), analogous to the isoform found in tonic muscle. The constructs were expressed as heavy meromyosin using the baculovirus/insect cell system. Unitary displacement (d) was similar for both constructs (∼10nm) but the attachment time (ton for the (−)insert was twice as long as for the (+)insert regardless of the [MgATP]. Both the relative average isometric force (Favg(−insert)/Favg(+insert))=1.1±0.2 (mean±se) using the in vitro motility mixture assay, and the unitary force (F∼1pN) using the laser trap, showed no difference between the two constructs. However, as under unloaded conditions, ton under loaded conditions was longer for the (−)insert compared with the (+)insert construct at limiting [MgATP]. These data suggest that the insert in this surface loop does not affect the mechanics but rather the kinetics of the cross-bridge cycle. Through comparisons of ton from d measurements at various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and MgATP binding.

Actin filament mechanics in the laser trap

Numerous biological processes, including muscular contraction, depend upon the mechanical properties of actin filaments. One such property is resistance to bending (flexural rigidity, EI). To estimate EI, we attached the ends of fluorescently labelled actin filaments to two microsphere‘handles’ captured in independent laser traps. The positions of the traps were manipulated to apply a range of tensions (0--8 pN)to the filaments via the microsphere handles. With increasing filament tension, the displacement of the microspheres was inconsistent with a microsphere-filament system that is rigid. We maintain that this inconsistency is due to the microspheres rotating in the trap and the filaments bending near their attachments to accommodate this rotation. Fitting the experimental data to a simple model of this phenomena, we estimate actins EI to be ×15 × 103 pN nm2, a value within the range of previously reported results, albeit using a novel method. These results both: support the idea that actin filaments are more compliant than historically assumed; and, indicate that without appropriately pretensioning the actin filament in similar laser traps, measurements of unitary molecular events (e.g. myosin displacement) may be significantly underestimated

R403Q and L908V mutant β-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level

Familial hypertrophic cardiomyopathy (FHC) is a disease of the sarcomere. In the β-myosin heavy chain gene, which codes for the mechanical enzyme myosin, greater than 40 point mutations have been found that are causal for this disease. We have studied the effect of two mutations, the R403Q and L908V, on myosin molecular mechanics. In the in vitro motility assay, the mutant myosins produced a 30% greater velocity of actin filament movement (νactin). At the single molecule level, νactin ∼d/ton, where d is the myosin unitary step displacement and ton is the step duration. Laser trap studies were performed at 10 μM MgATP to estimate d and ton for the normal and mutant myosin molecules. The increase in νactin can be explained by a significant decrease in the average tons in both the R403Q and L908V mutants (∼30 ms) compared to controls (∼40 ms), while d was not different for all myosins tested (∼7 nm). Thus the mutations affect the kinetics of the cross-bridge cycle without any effect on myosins inherent motion and force generating capacity. Based on these studies, the primary signal for the hypertrophic response appears to be an apparent gain in function of the individual mutant myosin molecules.

Myosin V and Kinesin act as tethers to enhance each others' processivity

Organelle transport to the periphery of the cell involves coordinated transport between the processive motors kinesin and myosin V. Long-range transport takes place on microtubule tracks, whereas final delivery involves shorter actin-based movements. The concept that motors only function on their appropriate track required further investigation with the recent observation that myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of myosin Vs diffusion on microtubules is a significant enhancement of the processive run length of kinesin when both motors are present on the same cargo. The degree of run length enhancement correlated with the net positive charge in loop 2 of myosin V. On actin, myosin V also undergoes longer processive runs when kinesin is present on the same cargo. The process that causes run length enhancement on both cytoskeletal tracks is electrostatic. We propose that one motor acts as a tether for the other and prevents its diffusion away from the track, thus allowing more steps to be taken before dissociation. The resulting run length enhancement likely contributes to the successful delivery of cargo in the cell.

Collaborative Dynamic DNA Scanning by Nucleotide Excision Repair Proteins Investigated by Single- Molecule Imaging of Quantum-Dot-Labeled Proteins

How DNA repair proteins sort through a genome for damage is one of the fundamental unanswered questions in this field. To address this problem, we uniquely labeled bacterial UvrA and UvrB with differently colored quantum dots and visualized how they interacted with DNA individually or together using oblique-angle fluorescence microscopy. UvrA was observed to utilize a three-dimensional search mechanism, binding transiently to the DNA for short periods (7 s). UvrA also was observed jumping from one DNA molecule to another over approximately 1 microm distances. Two UvrBs can bind to a UvrA dimer and collapse the search dimensionality of UvrA from three to one dimension by inducing a substantial number of UvrAB complexes to slide along the DNA. Three types of sliding motion were characterized: random diffusion, paused motion, and directed motion. This UvrB-induced change in mode of searching permits more rapid and efficient scanning of the genome for damage.