Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level

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.

Unconventional Myosins: How Regulation Meets Function

“…Similarly, Ca 2+ -induced relief of auto-inhibition and the resulting ATPase activation and association with cargo has been observed for myosin Vb in vivo and in vitro [117]. Interestingly, although Ca 2+ does not directly affect the activity of the motor of myosin V [21,118], it significantly impairs its processivity in vitro by inducing dissociation of CaM from one or more of the IQ motifs, and therefore compromising the stiffness of the neck [118][119][120]. Therefore, although Ca 2+ activates myosin V through release of the auto-inhibition, it also results in a mechanically compromised myosin.…”

“…As described above, Ca 2+ has been shown to activate myosin Va in vitro [21,112], by releasing this auto-inhibition [113,114]. However, the in vivo relevance of such activation is still debated [117][118][119][120]. Instead, the interaction of myosin V with its cargo adaptors has been accepted as the main physiological mechanism of activation.…”

Processivity of molecular motors under vectorial loads

“…The present model can be extended to study the effects of the vectorial character of the applied load on the mechanics of dynein and myosin motors, since the load-velocity and -detachment rate behaviours of kinesins, dyneins, and myosins are similar [14][15][16][17]. This can help to understand the mechanics of collective function of different molecular motors, owing to the force-dependent interactions of the motors.…”

“…They measured the vertical load component F z , where the horizontal component F x was resisting (<0, applied against the stepping direction). More recently, single-molecule optical trapping experiments [14][15][16][17] have studied the function of molecular motors under assisting loads F x (>0, applied in the stepping direction) as well. They have shown that the motors exhibit similar responses to the applied load direction: i) their velocity decreases with resisting loads, but changes slightly when the load is assisting, and ii) motors favor faster detachment rate under assisting loads than resisting ones.…”

α-catenin switches between a slip and an asymmetric catch bond with F-actin to cooperatively regulate cell junction fluidity

“…A single a-catenin unfolds to bear force on actin Here, we used ultrafast force-clamp spectroscopy, a technique with sub-millisecond and sub-nanometer resolution based on laser tweezers 9,[15][16][17] , to dissect the mechanosensitivity of single mammalian a-catenin homodimers and a-b-catenin heterodimers. We first analyzed single purified recombinant a-catenin homodimers (named as a-catenin below).…”