Information flow, Gating, and Energetics in dimeric molecular motors

Molecular motors belonging to the kinesin and myosin super family hydrolyze ATP by cycling through a sequence of chemical states. These cytoplasmic motors are dimers made up of two linked identical monomeric globular proteins. Fueled by the free energy generated by ATP hydrolysis, the motors walk on polar tracks (microtubule or filamentous actin) processively, which means that only one head detaches and executes a mechanical step while the other stays bound to the track. Thus, the one motor head must regulate chemical state of the other, referred to as "gating", a concept that is not fully understood. Inspired by experiments, showing that only a fraction of the energy from ATP hydrolysis is used to advance the kinesin motors against load, we demonstrate that additional energy is used for coordinating the chemical cycles of the two heads in the dimer - a feature that characterizes gating. To this end, we develop a general framework based on information theory and stochastic thermodynamics, and establish that gating could be quantified in terms of information flow between the motor heads. Applications of the theory to kinesin-1 and Myosin V show that information flow occurs, with positive cooperativity, at external resistive loads that are less than a critical value, Fc. When force exceeds Fc, effective information flow ceases. Interestingly, Fc, which is independent of the input energy generated through ATP hydrolysis, coincides with force at which the probability of backward steps starts to increase. Our findings suggest that transport efficiency is optimal only at forces less than Fc, which implies that these motors must operate at low loads under in vivo conditions.

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin.

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin. The unprecedented number of high-resolution structures of a single myosin finally enabled us to assemble a nearly complete structural model of the myosin-V motor cycle and describe the molecular principles of force production.

Autoregulation and Dual Stepping Mode of MYA2, an Arabidopsis Myosin XI Responsible for Cytoplasmic Streaming

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD), When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining TIRF microscopy and the FIONA method, revealed that the MYA2 processively moved on actin with three different step sizes: −28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes, hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2 − 3 pN). These results indicated that MYA2 is more flexible than the myosin V responsible for vesicle transport in animal cells. Such flexibility may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.

The Role of Calmodulin vs. Synaptotagmin in Exocytosis

Exocytosis is a Ca2+-regulated process that requires the participation of Ca2+ sensors. In the 1980s, two classes of Ca2+-binding proteins were proposed as putative Ca2+ sensors: EF-hand protein calmodulin, and the C2 domain protein synaptotagmin. In the next few decades, numerous studies determined that in the final stage of membrane fusion triggered by a micromolar boost in the level of Ca2+, the low affinity Ca2+-binding protein synaptotagmin, especially synaptotagmin 1 and 2, acts as the primary Ca2+ sensor, whereas calmodulin is unlikely to be functional due to its high Ca2+ affinity. However, in the meantime emerging evidence has revealed that calmodulin is involved in the earlier exocytotic steps prior to fusion, such as vesicle trafficking, docking and priming by acting as a high affinity Ca2+ sensor activated at submicromolar level of Ca2+. Calmodulin directly interacts with multiple regulatory proteins involved in the regulation of exocytosis, including VAMP, myosin V, Munc13, synapsin, GAP43 and Rab3, and switches on key kinases, such as type II Ca2+/calmodulin-dependent protein kinase, to phosphorylate a series of exocytosis regulators, including syntaxin, synapsin, RIM and Ca2+ channels. Moreover, calmodulin interacts with synaptotagmin through either direct binding or indirect phosphorylation. In summary, calmodulin and synaptotagmin are Ca2+ sensors that play complementary roles throughout the process of exocytosis. In this review, we discuss the complementary roles that calmodulin and synaptotagmin play as Ca2+ sensors during exocytosis.

Myosin V Regulates Spatial Localization of Different Forms of Neurotransmitter Release in Central Synapses

Synaptic active zone (AZ) contains multiple specialized release sites for vesicle fusion. The utilization of release sites is regulated to determine spatiotemporal organization of the two main forms of synchronous release, uni-vesicular (UVR) and multi-vesicular (MVR). We previously found that the vesicle-associated molecular motor myosin V regulates temporal utilization of release sites by controlling vesicle anchoring at release sites in an activity-dependent manner. Here we show that acute inhibition of myosin V shifts preferential location of vesicle docking away from AZ center toward periphery, and results in a corresponding spatial shift in utilization of release sites during UVR. Similarly, inhibition of myosin V also reduces preferential utilization of central release sites during MVR, leading to more spatially distributed and temporally uniform MVR that occurs farther away from the AZ center. Using a modeling approach, we provide a conceptual framework that unites spatial and temporal functions of myosin V in vesicle release by controlling the gradient of release site release probability across the AZ, which in turn determines the spatiotemporal organization of both UVR and MVR. Thus myosin V regulates both temporal and spatial utilization of release sites during two main forms of synchronous release.

Microtubule-independent movement of the fission yeast nucleus

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.