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.

SUMMARY 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 ~1 μm 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.

Myosin V, a double-headed molecular motor, transports organelles within cells by walking processively along actin, a process that requires coordination between the heads. To understand the mechanism underlying this coordination, processive runs of single myosin V molecules were perturbed by varying nucleotide content. Contrary to current views, our results show that the two heads of a myosin V molecule communicate, not through any one mechanism but through an elaborate system of cooperative mechanisms involving multiple kinetic pathways. These mechanisms introduce redundancy and safeguards that ensure robust processivity under differing physiologic demands.A s a processive motor, a myosin V molecule carries its intracellular cargo for long distances along an actin track, taking multiple steps before detaching and deriving the energy for each step from the hydrolysis of ATP (1-3). Determining the mechanism of this processive movement is essential for explaining diseases such as Griscelli syndrome in which a mutation to human myosin Va leads to hypopigmentation and severe neurological impairment (4, 5). Each of the two heads of myosin V catalyzes the hydrolysis of ATP and after release of hydrolysis products (P i and ADP) generates motion with a rotation of its long lever arm (6-8). When both heads function together, the myosin V molecule walks along actin in a hand-over-hand fashion, taking 36-nm strides (7,(9)(10)(11)(12). One of the most critical and intriguing questions, however, remains unanswered. How do the two heads coordinate their biochemical and mechanical cycles to maintain processive movement? Solution kinetic studies to date have focused only on single-headed myosin V constructs, from which coordination between the heads can only be inferred (13-15). Therefore, we expressed double-headed, heavy meromyosin V molecules with a C-terminal yellow fluorescent protein (YFP-HMM M5 ) such that processive movement of individual myosin V molecules could be visualized by total internal reflectance fluorescence (TIRF) microscopy (16) and then described in terms of run length and velocity (17). By perturbing the biochemical cycle through addition of ATP, ADP, or P i , we assessed the impact of these ligands on myosin V processivity, identified the myosin state from which a processive run most likely terminates, and showed that the two heads of myosin V are coordinated to generate processive movement through an elaborate system of cooperative mechanisms. Methods YFP-HMMM5 Expression. YFP was cloned onto the C terminus of HMM M5 . The final construct contains the first 1,098 amino acids (G1098) of murine myosin V HMM, followed by a linker region coding for the amino acids VTGS, followed by YFP and a FLAG epitope for purification. Sf9 cells were coinfected with two recombinant viruses, one encoding for the myosin V heavy chain and one for calmodulin. The calmodulin was a mutant deficient in calcium binding (CaM⌬all) to ensure complete occupancy of all the IQ motifs (18). After 72 h of infection, cells were pelleted a...

Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery.cytoskeleton ͉ molecular motor ͉ motility ͉ processivity C oordinated organelle transport along certain secretory pathways starts with kinesin-powered movement on microtubules and finishes with vertebrate myosin Va (myoVa)-based motility on actin (1-4). This trip requires that both motors be present when a microtubule-actin intersection is encountered, but how these two motors find one another or the cargo they share is still unknown. Another challenge is to understand how cargo is delivered to its destination by way of the complex cytoskeletal network. Vertebrate myoVa is a processive motor that can travel long distances along its actin track (5). However, the cytoskeletal intersections that myoVa must encounter along its journey may present a physical barrier to forward motion or an alternate path to its final destination.To investigate this issue from myoVa's point of view, we observed single myoVa molecules labeled with highly photostable quantum dots (Qdots) in an objective-type total internal reflectance microscope (6) as they maneuvered through a model of cytoskeletal intersections created on a microscope coverslip coated with either actin filaments (Fig. 1A) or actin filaments and microtubules (Fig. 1B). In this model system, actin-actin intersections could inhibit or alter myoVa's direction of travel while actin-microtubule intersections should act as a hurdle to myoVa's processive movement. Given myoVa's ability to take 72-nm steps as it walks processively along actin filaments in a handover-hand fashion (6-9) and its inherent flexibility (10, 11), we have observed that myoVa can easily maneuver past actin filament intersections. In contrast, when encountering a microtubule, myoVa cannot step over this physical barrier, but surprisingly can step onto the microtubule and begin a onedimensional diffusive search. Thus, myoVa has evolved to handle the challenges of the cytoskeletal network and through its interaction with the microtubule can effectively scan for its transport partner, kinesin, and/or its cargo. Results and Discussion MyoVa Dynamics at Actin Filament Intersections.To determine what happens when myoVa encounters an overlapping actin filament, we adhered Alexa Fluor 660 phalloidin-labeled actin filaments to the coverslip, followed by TRITC...

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.