Abstract
Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.
Highlights
Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors
Myosin-5B is a processive motor that moves in 36 nm steps
The movement of quantum dot (QD)-labeled myosin-5B heavy meromyosin on actin filaments was recorded on a custom-built multicolor total internal reflection fluorescence (TIRF) microscope (Fig. 1a, Methods section)[41]
Summary
Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Single-molecule studies uniformly indicate that myosin-5A is a high duty ratio motor (i.e., each head is strongly bound to actin for the majority of the actomyosin ATPase cycle) that coordinates its catalytic motor domains to move processively in 36 nm steps on actin, a prerequisite for efficient cargo transport[19,26,27,28]. We show that Ca2+ finely regulates myosin-5B motility in vitro by uncoupling the mechanical and enzymatic activity of the motor, giving insight into its transport function in neuronal cells. Our study provides mechanistic insight into the molecular basis underlying myosin-5B-based transport and offers a detailed model of its trafficking function in the crowded actin cytoskeleton of mammalian cells
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