Project 1: LIM domains as cytoskeletal strain sensors Lucas Axiotakis, Jr. Collaborator: Clare Waterman (NHLBI) Proteomic studies have demonstrated that LIM domains, common but poorly characterized protein-protein interaction motifs, are enriched in the sequences of proteins which localize to the actin-adhesion system in response to contractility. This has lead to the proposal that LIM domains could act as mechanosensors by unknown mechansims. The 3 LIM domains of the cytoskeletal protein zyxin confer rapid localization of this molecule to sites of acute mechanical strain in the actin cytoskeleton, leading us to hypothesize that other LIM proteins would mechanosense by an analogous mechanism. In preliminary studies, we undertook a live-cell imaging-based screen of the localization of 29 cytoskeletal LIM domain containing proteins at strain sites, identifying a subset (30%) that colocalize with zyxin. We have now additionally established a cell-stretching assay for localization of LIM domain proteins to remodeled stress fibers produced by mechanical insult, which are also enriched with zyxin. We find that a largely overlapping set of LIM proteins relocalize in this assay, suggesting that the same underlying signal is being sensed by LIM domains at strain sites and remodeled stress fibers. Current efforts are focused on dissecting the modularity of LIM domains for localizing effector molecules (e.g. actin repair machinery) to mechanically induced actin structures, as well as the effects of LIM domain number on mechanosensor activity. Future efforts will focus on biochemical studies to establish the molecular identity of factors recognized by LIM domains in a mechanosensitive fashion. Project 2: The structural basis of F-actin interactions with binding partners Laura Y. Kim Pinar S. Gurel Collaborators: Sharon Campbell (UNC), Ami Mankodi (NINDS), Roberto Dominguez (UPenn), Zev Bryant (Stanford), Thomas Friedman (NIDCD) Previous structural evidence suggests that F-actin is structurally polymorphic, with multiple conformations of the actin protomer being compatible with the helical lattice of the filament. We and others have hypothesized that mechanical forces impinging on a filament can influence this structural landscape, which would then modulate the interaction of a filament with actin binding partners (ABPs). An important prerequisite of this model is that ABPs prefer to bind specific conformations of F-actin (as has been established for one binding partner, cofilin). However, few F-actin-ABP interactions have been visualized due to the technical challenges of structurally analyzing this system. We have thus established a robust pipeline for obtaining high-resolution cryo-EM reconstructions of F-actin in complex with binding partners. In collaboration with the Campbell lab at UNC, we have described the interaction between the critical adhesion factor vinculin and actin (JMB 2016), and proposed a model of how force could strengthen this bond. We are now engaged in follow-up studies of disease mutants of vinculins cardiomyopathy-associated splice isoform metavinculin, which we have found to be defective in tuning the architecture of actin networks. Additionally, we have initiated structural studies of several other actin-ABP interactions, with a focus on ABPs associated with genetic diseases that can potentially be linked to mechanotransduction or mechanical homeostasis defects. These include the factors ZASP (collaboration: Mankodi lab, NINDS) and the CH-domain containing protein dystrophin (collaboration: Dominguez lab, UPenn), both of which are associated with hereditary muscular dystrophies, as well as the unconventional motors myosin VI (collaboration: Bryant lab, Stanford) and myosin XV (collaboration: Friedman lab, NIDCD), mutations in which lead to deafness due to defects in the biogenesis and maintenance of actin-rich stereocilia. In addition to helping fulfill our long-term goal of defining the actin-ABP structural landscape, these studies potentially stand to benefit human health by shedding light on the biophysical etiology of currently intractable genetic conditions. Project 3: Visualizing mechanically induced conformational changes in F-actin Pinar S. Gurel Collaborators: James Sellers (NHLBI) In addition to our efforts to visualize the structural landscape of actin-ABP interactions in the absence of exogenous forces, we are also developing methodology to directly test the hypothesis that mechanical force alters actin conformation. We have developed a novel reconstitution system to visualize actin under load that is compatible with both dynamic biophysical studies with fluorescence microscopy and high-resolution structural studies with cryo-EM. We immobilize active myosin motors onto the carbon substrate of holey EM grids, suspending motor-engaged actin filaments over holes, where we can visualize the effects of myosin activity on bare regions of the filaments. Preliminary studies demonstrate the appearance of a novel sinusoidal actin conformation under these conditions. We are currently refining our system to dissect what type (i.e. tension or compression) and what amount of force are required to elicit these conformational changes. Future efforts will focus on detailed structural studies of actin under load and the influence of these structures on ABP binding.

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Kim, Laura Y; Thompson, Peter M; Lee, Hyunna T et al. (2016) The Structural Basis of Actin Organization by Vinculin and Metavinculin. J Mol Biol 428:10-25