Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development, and promoting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility from the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting MyBP-H may be functionally silent. However, our results suggest an active role. Small angle x-ray diffraction of intact larval tails revealed MyBP-H contributes to the compression of the myofilament lattice accompanying stretch or contraction, while in vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake". These results provide new insights and raise questions about the role of the C-zone during muscle development.

Imaging ATP Consumption in Resting Skeletal Muscle: One Molecule at a Time.

Striated muscle contraction is the result of sarcomeres, the basic contractile unit, shortening because of hydrolysis of adenosine triphosphate (ATP) by myosin molecular motors. In noncontracting, "relaxed" muscle, myosin still hydrolyzes ATP slowly, contributing to the muscle's overall resting metabolic rate. Furthermore, when relaxed, myosin partition into two kinetically distinct subpopulations: a faster-hydrolyzing "relaxed" population, and a slower-hydrolyzing "super relaxed" (SRX) population. How these two myosin subpopulations are spatially arranged in the sarcomere is unclear, although it has been proposed that myosin-binding protein C (MyBP-C) may stabilize the SRX state. Because MyBP-C is found only in a distinct region of the sarcomere, i.e., the C-zone, are SRX myosin similarly colocalized in the C-zone? Here, we imaged the binding lifetime and location (38-nm resolution) of single, fluorescently labeled boron-dipyrromethene-labeled ATP molecules in relaxed skeletal muscle sarcomeres from rat soleus. The lifetime distribution of fluorescent ATP-binding events was well fitted as an admixture of two subpopulations with time constants of 26 ± 2 and 146 ± 16 s, with the longer-lived population being 28 ± 4% of the total. These values agree with reported kinetics from bulk studies of skeletal muscle for the relaxed and SRX subpopulations, respectively. Subsarcomeric localization of these events revealed that SRX-nucleotide-binding events are fivefold more frequent in the C-zone (where MyBP-C exists) than in flanking regions devoid of MyBP-C. Treatment with the small molecule myosin inhibitor, mavacamten, caused no change in SRX event frequency in the C-zone but increased their frequency fivefold outside the C-zone, indicating that all myosin are in a dynamic equilibrium between the relaxed and SRX states. With SRX myosin found predominantly in the C-zone, these data suggest that MyBP-C may stabilize and possibly regulate the SRX state.

Mechanical Characteristics of Ultrafast Zebrafish Larval Swimming Muscles.

Zebrafish (Danio rerio) swim within days of fertilization, powered by muscles of the axial myotomes. Forces generated by these muscles can be measured rapidly in whole, intact larval tails by adapting protocols developed for ex vivo muscle mechanics. But it is not known how well these measurements reflect the function of the underlying muscle fibers and sarcomeres. Here, we consider the anatomy of the 5-day-old, wild-type larval tail, and implement technical modifications to measuring muscle physiology in intact tails. Specifically, we quantify fundamental relationships between force, length, and shortening velocity, and capture the extreme contractile speeds required to swim with tail-beat frequencies of 80-100 Hz. Therefore, we analyze 1000 frames/s videos to track the movement of structures, visible in the transparent tail, which correlate with sarcomere length. We also characterize the passive viscoelastic properties of the preparation to isolate forces contributed by nonmuscle structures within the tail. Myotomal muscles generate more than 95% of their maximal isometric stress (76 ± 3 mN/mm2) over the range of muscle lengths used in vivo. They have rapid twitch kinetics (full width at half-maximal stress: 11 ± 1 ms) and a high twitch/tetanus ratio (0.91 ± 0.05), indicating adaptations for fast excitation-contraction coupling. Although contractile stress is relatively low, myotomal muscles develop high net power (134 ± 20 W/kg at 80 Hz) in cyclical work loop experiments designed to simulate the in vivo dynamics of muscle fibers during swimming. When shortening at a constant speed of 7 ± 1 muscle lengths/s, muscles develop 86 ± 2% of isometric stress, whereas peak instantaneous power during 100 Hz work loops occurs at 18 ± 2 muscle lengths/s. These approaches can improve the usefulness of zebrafish as a model system for muscle research by providing a rapid and sensitive functional readout for experimental interventions.

Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity.

The cell's dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.

Myosin Va molecular motors manoeuvre liposome cargo through suspended actin filament intersections in vitro.

Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell's three-dimensional (3D) highway of actin filaments. At actin filament intersections, the intersecting filament is a structural barrier to and an alternate track for directed cargo transport. Here we use 3D super-resolution fluorescence imaging to determine the directional outcome (that is, continues straight, turns or terminates) for an ∼10 motor ensemble transporting a 350 nm lipid-bound cargo that encounters a suspended 3D actin filament intersection in vitro. Motor-cargo complexes that interact with the intersecting filament go straight through the intersection 62% of the time, nearly twice that for turning. To explain this, we develop an in silico model, supported by optical trapping data, suggesting that the motors' diffusive movements on the vesicle surface and the extent of their engagement with the two intersecting actin tracks biases the motor-cargo complex on average to go straight through the intersection.

Flexural Stiffness of Myosin Va Subdomains as Measured from Tethered Particle Motion.

Myosin Va (MyoVa) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton. The motor's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor's structure. Specifically, changes in calcium may regulate motor processivity by altering the motor's lever arm stiffness and thus its interhead communication. In order to measure the flexural stiffness of MyoVa subdomains, we use tethered particle microscopy, which relates the Brownian motion of fluorescent quantum dots, which are attached to various single- and double-headed MyoVa constructs bound to actin in rigor, to the motor's flexural stiffness. Based on these measurements, the MyoVa lever arm and coiled-coil rod domain have comparable flexural stiffness (0.034 pN/nm). Upon addition of calcium, the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm. In addition, the flexural stiffness of the full-length MyoVa construct is an order of magnitude less stiff than both a single lever arm and the coiled-coil rod. This suggests that the MyoVa lever arm-rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network.

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport.

Myosin Va (myoV) and myosin VI (myoVI) are processive molecular motors that transport cargo in opposite directions on actin tracks. Because these motors may bind to the same cargo in vivo, we developed an in vitro "tug of war" to characterize the stepping dynamics of single quantum-dot-labeled myoV and myoVI motors linked to a common cargo. MyoV dominates its myoVI partner 79% of the time. Regardless of which motor wins, its stepping rate slows due to the resistive load of the losing motor (myoV, 2.1 pN; myoVI, 1.4 pN). Interestingly, the losing motor steps backward in synchrony with the winning motor. With ADP present, myoVI acts as an anchor to prevent myoV from stepping forward. This model system emphasizes the physical communication between opposing motors bound to a common cargo and highlights the potential for modulating this interaction by changes in the cell's ionic milieu.

Simultaneous observation of tail and head movements of myosin V during processive motion.

Processive stepping of myosin Va (myoV) has been tracked by monitoring either the tail position (center of mass) or the position of one or both heads. Here, we combine these two approaches by attaching a quantum dot to one of the motor domains and a bead to the tail. Using laser trapping and total internal reflection microscopy, the position of one head and the tail are observed simultaneously as myoV moves processively on an actin filament bundle against the resistive load of the laser trap. The head moves one step (73 ± 10 nm) for every two steps of the tail (35 ± 9 nm). One tail step occurs concurrently with quantum dot-labeled head movement, whereas the other occurs with movement of the unlabeled head, consistent with a hand-over-hand model. Load increases the probability of the motor taking a back step. The back step is triggered by the motor taking a shorter forward step (head step, 68 ± 11 nm; tail step, 32 ± 10 nm), likely one actin monomer short of its preferred binding site. During a back step, the motor reverses its hand-over-hand motion, with the leading head detaching and reattaching to one of multiple actin sites behind the trailing head. After a back step, the motor can correct its mistake and step processively forward at resistive loads <0.7 piconewton or stall or detach at higher loads. Back stepping may provide a mechanism to ensure efficient cargo delivery even when myoV encounters obstacles within the actin cytoskeletal meshwork or when other motors are attached to the same cargo.

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.

Biopanning Phage-Display Libraries on Small Tissue Sections Captured by Laser Capture Microdissection.

Phage-display technology has been widely used for developing tumor-targeting agents. Laser capture microdissection (LCM) has proven to be an accurate method to select specific cells from histological sections. Our goal was to develop a method to combine phage-display with LCM to obtain phage-displayed ligands that bind to selected cells in human solid tumors. Two panning strategies were evaluated and optimized. The first strategy was to pan on patient tissue mounted to LCM slides before LCM occurred. The poor panning output showed that phage did not tolerate the drying conditions during LCM. The second strategy was to pan on tumor cells from the patient tumor tissue that were isolated by LCM. The catapulted tumor cells were transferred to a filter unit which retained cells but allowed rinsing of unbound phage. Six commercially available filter units were evaluated and the one with the lowest nonspecific binding to phage was selected for the panning steps. The smallest number of cells (500) in which panning could be successfully accomplished was also determined. A micropipette system was developed to further decrease background by removing catapulted cells from the filter unit after panning was complete. This left behind nearly all background binding phage in the filter unit. This strategy led to the selection of individual phage antibody clones (5 out of 79 tested) specific for tumor cells of the patient's cancer tissue. Immunofluorescence staining on tumor tissues from the same patient showed that these clones have selective signals on tumor island cells, while the scFv library only showed low nonspecific signals on tumor tissues. We established a method of panning on a small number of LCM-captured solid tumor specimens. The quick identification of specific phage-displayed antibodies in the cancer tissue of human patients will greatly enhance the therapy and diagnosis of cancer.

Myosin Va maneuvers through actin intersections and diffuses along microtubules.

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.

Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity.

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.

Myosin V processivity: multiple kinetic pathways for head-to-head coordination.

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 mutant heterodimeric myosin with one inactive head generates maximal displacement.

Each of the heads of the motor protein myosin II is capable of supporting motion. A previous report showed that double-headed myosin generates twice the displacement of single-headed myosin (Tyska, M.J., D.E. Dupuis, W.H. Guilford, J.B. Patlak, G.S. Waller, K.M. Trybus, D.M. Warshaw, and S. Lowey. 1999. Proc. Natl. Acad. Sci. USA. 96:4402-4407). To determine the role of the second head, we expressed a smooth muscle heterodimeric heavy meromyosin (HMM) with one wild-type head, and the other locked in a weak actin-binding state by introducing a point mutation in switch II (E470A). Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP. Optical trap measurements revealed that the heterodimer generated unitary displacements of 10.4 nm, strikingly similar to wild-type HMM (10.2 nm) and approximately twice that of single-headed subfragment-1 (4.4 nm). These data show that a double-headed molecule can achieve a working stroke of approximately 10 nm with only one active head and an inactive weak-binding partner. We propose that the second head optimizes the orientation and/or stabilizes the structure of the motion-generating head, thereby resulting in maximum displacement.