M. Yusuf Ali

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.

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.

Random Walk of Processive, Quantum Dot-Labeled Myosin Va Molecules within the Actin Cortex of COS-7 Cells

Myosin Va (myoVa) is an actin-based intracellular cargo transporter. In vitro experiments have established that a single myoVa moves processively along actin tracks, but less is known about how this motor operates within cells. Here we track the movement of a quantum dot (Qdot)-labeled myoVa HMM in COS-7 cells using total internal reflectance fluorescence microscopy. This labeling approach is unique in that it allows myoVa, instead of its cargo, to be tracked. Single-particle analysis showed short periods (</=0.5 s) of ATP-sensitive linear motion. The mean velocity of these trajectories was 604 nm/s and independent of the number of myoVa molecules attached to the Qdot. With high time (16.6 ms) and spatial (15 nm) resolution imaging, Qdot-labeled myoVa moved with sequential 75 nm steps per head, at a rate of 16 s(-1), similarly to myoVa in vitro. Monte Carlo modeling suggests that the random nature of the trajectories represents processive myoVa motors undergoing a random walk through the dense and randomly oriented cortical actin network.

Diffusive Movement of Processive Kinesin-1 on Microtubules

The processive motor kinesin‐1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence microscopy of kinesin bound to a carboxylated quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti‐HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well established that kinesin undergoes a salt‐dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin‐ΔC) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles.

Myosin VI Must Dimerize and Deploy Its Unusual Lever Arm in Order to Perform Its Cellular Roles

It is unclear whether the reverse-direction myosin (myosin VI) functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single-α-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions.

Myosin VI has a one track mind versus myosin Va when moving on actin bundles or at an intersection

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin‐actin) or mixed polarity (α‐actinin‐actin) bundles. The stepping dynamics of individual Qdot‐labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVIs preference to stay on track, which may be critical to its role as an intracellular anchor.

Quantum dot labeling strategies to characterize single-molecular motors.

Recent advances in single-molecule labeling and detection techniques allow high-resolution imaging of the motion of single molecules. Molecular motors are biological machines that convert chemical energy into mechanical work. Myosin Va (MyoVa) is a well-characterized processive molecular motor, essential for cargo transport in living organisms. Quantum dots (Qdots) are fluorescent semiconductor nanocrystals that are extremely useful for single-molecule studies in biological sciences. High-resolution video microscopy and single-particle tracking of a Qdot-labeled MyoVa motor molecule allow the detection of individual steps in vitro and in live cells.

Small teams of myosin Vc motors coordinate their stepping for efficient cargo transport on actin bundles

Myosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to move continuously on single actin filaments. Single molecules of myoVc cannot take multiple hand-over-hand steps from one actin-binding site to the next without dissociating, in stark contrast to the well studied myosin Va (myoVa) isoform. At low salt, single myoVc motors can, however, move processively on actin bundles, and at physiologic ionic strength, even teams of myoVc motors require actin bundles to sustain continuous motion. Here, we linked defined numbers of myoVc or myoVa molecules to DNA nanostructures as synthetic cargos. Using total internal reflectance fluorescence microscopy, we compared the stepping behavior of myoVc versus myoVa ensembles and myoVc stepping patterns on single actin filaments versus actin bundles. Run lengths of both myoVc and myoVa teams increased with motor number, but only multiple myoVc motors showed a run-length enhancement on actin bundles compared with actin filaments. By resolving the stepping behavior of individual myoVc motors with a quantum dot bound to the motor domain, we found that coupling of two myoVc motors significantly decreased the futile back and side steps that were frequently observed for single myoVc motors. Changes in the inter-motor distance between two coupled myoVc motors affected stepping dynamics, suggesting that mechanical tension coordinates the stepping behavior of two myoVc motors for efficient directional motion. Our study provides a molecular basis to explain how teams of myoVc motors are suited to transport cargos such as zymogen granules on actin bundles.

A Branched Kinetic Scheme Describes the Mechanochemical Coupling of Myosin Va Processivity in Response to Substrate

Myosin Va is a double-headed cargo-carrying molecular motor that moves processively along cellular actin filaments. Long processive runs are achieved through mechanical coordination between the two heads of myosin Va, which keeps their ATPase cycles out of phase, preventing both heads detaching from actin simultaneously. The biochemical kinetics underlying processivity are still uncertain. Here we attempt to define the biochemical pathways populated by myosin Va by examining the velocity, processive run-length, and individual steps of a Qdot-labeled myosin Va in various substrate conditions (i.e., changes in ATP, ADP, and P(i)) under zero load in the single-molecule total internal reflection fluorescence microscopy assay. These data were used to globally constrain a branched kinetic scheme that was necessary to fit the dependences of velocity and run-length on substrate conditions. Based on this model, myosin Va can be biased along a given pathway by changes in substrate concentrations. This has uncovered states not normally sampled by the motor, and suggests that every transition involving substrate binding and release may be strain-dependent.

Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex

We investigated the role of full-length Drosophila Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and K10 mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one K10 mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.