Cellular actin protrusions (e.g. filopodia, microvilli, and stereocilia) display a broad range of lengths and lifetimes critically related to their specific cellular function. Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Our laboratory has made significant advances towards elucidating the mechanisms that underlie the formation, regulation, renewal, and life span of stereocilia. Studies on actin turnover in stereocilia as well as the identification of several deafness-related proteins essential for proper stereocilia structure and function provide new insights into the mechanisms and molecules involved in stereocilia length regulation, long-term maintenance, and potetnial for repair following overstimulation or acoustic trauma. ? ? Myosins and their cargo have been implicated in formation and elongation of actin protrusions, but the mechanisms by which they influence F-actin elongation are diverse and not fully understood. We demonstrated that PTPRQ and Myosin VI, two proteins that have been previously associated with inherited deafness, form a molecular complex that dynamically maintains the organization of the cell surface coat at the stereocilia base and helps maintain the structure of the overall stereocilia bundle. We demonstrated that PTPRQ is intensely localized at the base of stereocilia where it forms a cell surface coat. The restricted localization of PTPRQ in wild type mice and its diffuse distribution in myosin VI-deficient mice suggest that myosin VI-based cargo transportation regulates the compartmentalization of PTPRQ. Based on these results and on computational analyses of the distribution of these two proteins we propose that PTPRQ couples the membrane to myosin VI thereby exerting a tethering force, which counteracts the membrane curvature-induced force that tends to lift the membrane away from the actin cytoskeleton at the stereocilia base. When either PTPRQ or myosin VI is missing, the curvature force lifts the membrane between the stereocilia upward resulting in fusion. Localization of PTPRQ-myosin VI at the stereocilia base provides the force that keeps the stereocilia separated and functional for normal hearing. This system demonstrates that the counter action of diffusion by molecular motors may be a general mechanism that can be used by a cell to localize certain components in actin protrusions.? ? Two other proteins implicated in inherited deafness, myosin IIIa, a plus end directed motor, and espin1, an actin bundling protein containing an actin-monomer-binding WH2 (WASP homology 2) motif, have been shown to influence the length and shape of mechanosensory stereocilia of the inner ear. Ongoing studies in our lab demonatrate that espin 1, the only isoform of espin that contains ankyrin repeats, shows a spatial and temporal pattern of localization at the tips of stereocilia similar to that described for myosin IIIa, and that the espin 1 ankyrin repeats domain (ARD) interacts with a unique conserved domain in the myosin IIIa carboxyl-terminal tail region. We show that, like myosin IIIa, espin 1 causes stereocilia elongation when overexpressed in cultured hair cells. Using a heterologous expression system we show an extraordinary elongation of filopodia resulting from the transport of espin 1 to the plus ends of filopodial F-actin by myosin IIIa, and that this elongation is dependent on espin 1 WH2 activity. This study provides the basis for understanding the role myosin IIIa and espin 1 play in regulating stereocilia length, presenting a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.? ? We also collaborated with Nir Gov from the Weizmann Institute to produce a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diusion and di-rected motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin laments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that infuence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.
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