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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.