Molecular function of Myosin-l

University Of Pennsylvania, Philadelphia PA

Investigators

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Abstract

Abstract The goal of this R37-supported research program is to obtain a fundamental understanding of how myosin motors function and interact with proteins, lipid membranes, and other biomolecules to power structural arrangements and motile events that are crucial for eukaryotic life. Our strategy has been to define the physical properties of the myosin family to better model and test function. To this end, we made fundamental discoveries into the mechanisms of chemomechanical coupling of all myosins and provided new insights into the mechanical attachments of membrane interactions. We will continue along the lines of the original specific aims, and we will build from our discoveries to address new and exciting questions related to these Aims. Aim 1: Determine the structural origin of myosin force sensing. Our focus is to determine how myosins sense and respond to mechanical load. Recent progress has given us an unprecedented look into the myosin-I structural states that span the force-sensing transition that control exit from force-bearing states. Using our new structural "know-how," we engineered Myo1b tension-sensing properties into mechanically divergent Myo1c, and we were able to engineer a low duty ratio myo1b into a high duty ratio motor. Our newest work has resulted in specific predictions regarding the roles of key residues conserved in most members of the myosin superfamily in tuning tension sensitivity. Chemomechanical tuning via these allosteric connections will be tested, which will allow us to (1) understand the basic molecular biophysics of energy transduction (the holy grail of myosin biophysics), (2) understand the effect of disease causing mutations that are on this allosteric path that impact tension sensing, and (3) design, engineer, and express myosins of altered mechanosensitivity for cell biological experiments to probe the molecular functions of myosin. Our development of a high-speed optical trapping system (µs time resolution, Woody et al, 2018), will allow us to probe the entry into the force-bearing states, including the phosphate-release step. Mechano-diversity in this transition is controversial and virtually unexplored. We will continue our cryo-EM work to determine the structure of Myo1c, a motor with highly divergent tension sensing properties. We will also use cryo-EM to determine the structure of mechanically strained myosin, which will be facilitated by the novel cryo-EM analysis techniques developed in our recent paper (Mentes et al. (2018)), and the generation of engineered Myo1b dimers that, when actin-bound, have a positive and negative mechanical strains that substantially affect ADP release. Investigation of myosin-I function in living cells, under working conditions, has been extraordinarily difficult, as identifying the population of proteins that are actively generating force has not been possible. Thus, we are very excited about the development of FRET-force-sensors for exploring myosin function. Correlation of in vitro calibration studies with cellular experiments is novel and powerful, and these tools will be continued to be developed and applied during the next funding period. Aim 2: Biochemical and Mechanical Properties of Myosin-I Binding Proteins and Membranes. The most impactful outcome of our Aim-2 research is the discovery that the myosin-I motor domain plays a defining role in development of left-right (L/R) asymmetry in Drosophila, through planar cell polarity mechanisms (Lebreton et al., 2018). The molecular roles of myosin in this process are unknown, as are the mechanisms by which a subset of myosin-I isoforms power leftward turning of actin filaments. Nevertheless, a key feature of the mechanism is that once myosins are bound to the membrane, specific isoforms can establish left from right. Strong evidence points to myosin-I isoforms binding to sites of cell-cell adhesion via direct interaction of the tail domains with cadherins at sites of Arp2/3-mediated actin growth. Thus, we will undertake a series of biophysical and biochemical investigations to gain a better understanding the fundamental properties of Myo1C and Myo1D individually and in complex protein ensembles. Already underway are cryo-EM experiments with the two isoforms to examine actin binding sites and lever arm geometries to determine the origin of isoform-specific chirality. We will also investigate the biochemical and mechanical interactions (e.g., affinities, attachment durations, adhesion forces) of Drosophila myosin-I with E-cadherin using the methods outlined in our original proposal and our prior publications. It is possible that that myosin drives L/R asymmetry by directing the geometry of the actin network, as suggested for formins (Tee et al., (2015) Nat Cell Bio 17:445-57). Thus, we will study the effect of myosin-I on actin network polymerization and geometry using micro-patterned coverslips to grow Arp2/3-nucleated actin in a spatially controlled fashion as a function of myosin-I content. Aim 3: Determine the mechanisms of tropomyosin regulation of myosin-1. We will explore the diversity of regulation in the myosin-1 family by examining the effect of tropomyosin on one isoform from each of the phylogenetic subgroups (Myo1b, Myo1c, Myo1, Myo1g), and we will determine the detailed biochemical mechanism for modulation of myosin-I function. A eukaryotic expression system have provided us with tag-free, N-terminal acetylated tropomyosin isoforms which we will use for biochemical and motility experiments.