Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va

Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems—the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32–125 motors per μm²), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track.Myosin Va is a processive, actin-based molecular motor critical for transport, morphology, and positioning of a wide variety of intracellular cargoes and organelles (1, 2). Although a single myosin Va can transport cargo in vitro, intracellular membrane-bound vesicular cargoes have a high surface density of motor proteins (3–5). Ensembles of motors that transport cargo in vitro have been shown to demonstrate enhanced run lengths (6–9) and slower movement, relative to the behavior of a single motor (6, 8–13). Slower velocities are generally attributed to negative interference between motors that arise when rigidly coupled, high duty ratio motors step asynchronously (12). The stepping kinetics of myosin Va are load sensitive, whereby resistive loads slow the stepping rate, whereas assistive loads produce only a modest acceleration (14–16). This asymmetric load dependence causes an overall slowing for a motor ensemble. Indicative of the complexity of the intracellular environment, Efremov et al. (17) have shown that physiological vesicle transport (whether microtubule- or actin-based) does not directly mirror the behavior of the responsible molecular motor but is sensitive to aspects of the cargo itself. In some microtubule-based transport systems, velocities faster than the capacity of the transporting motor have been observed in cells (18, 19). However, motion of the cytoskeletal track may contribute to this enhanced motion (20).Most studies of in vitro cargo transport have a motor protein ensemble coupled to rigid cargoes such as quantum dots (Qdots) (8, 21), silica beads (12), DNA scaffolds (9, 22, 23), or directly to glass substrates (24). However, intracellular vesicles have fluid membranes that would be expected to allow the vesicle-attached motors (or coupled groups of motors) to diffuse within the membrane, so that motor ensemble transport of lipid-bound vesicles may be distinct from that observed with rigid cargo. Thus, more physiologically relevant studies have used liposomes (25) or isolated vesicles (26) with their in vivo complement of motors (26–28), or even intracellular organelles artificially coupled to a known motor type (17). Here, we attached recombinant myosin Va heavy meromyosin (myoVa) molecules at varying motor density to 100- to 650-nm lipid vesicles with membrane characteristics that were either fluid- or gel-like. When vesicles composed of fluid membranes are transported by an ensemble of myoVa molecules, they demonstrate velocities exceeding both those of identical vesicles transported by a single motor and those of individual, unloaded motors. Gel-like vesicles demonstrate velocities equal to or slower than a single motor. We developed a simulation of vesicular transport that supports at least one simple model wherein reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas enhanced transport velocities result from the concerted effects of myoVa’s stepping and the preferential detachment of the trailing motor in the ensemble from the actin track. This proposed bias in motor detachment and subsequent recentering of the vesicle above the remaining motors would generate an additional forward-directed displacement of the vesicle, providing the additional vesicular velocity. Therefore, we propose that the physical properties of the cargo itself contribute to the emergent transport behavior.