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. In the process of investigating whether myosin IIIb, a paralog of myosin IIIa, could compensate for myosin IIIa function and explain the DFNB30 late onset hearing loss, we have discovered an entirely novel form of cargo-dependent myosin motility. Myosin IIIa is presumed to translocate along actin filaments in an inchworm fashion, where a conserved domain in the tail (THDII) provides a second actin-binding site that prevents complete dissociation of the myosin from the actin with each myosin head power-stroke. Intriguingly, myosin IIIb has been presumed to lack motile activity because it does not contain a THDII domain. We observed, however, that when myosin IIIb and espin 1 are co-transfected in COS7 cells, the behavior of myosin IIIb is nearly identical to myosin IIIa. Using Co-IP and co-transfection assays we showed that myosin IIIb binds to espin 1 through its THDI and uses the actin-binding domain of espin 1 as a crutch or replacement for the missing THDII to translocate to the tips of actin protrusions. Immunolocalization and transfection assays showed that myosin IIIb forms the same pattern of localization as myosin IIIa at the tips of stereocilia. These studies demonstrate that myosin IIIb can compensate for myosin IIIa function and provide a framework to further investigate DFNB30 late onset deafness. In collaboration with Christopher Yengo (Penn State University), we showed that intermolecular auto-phosphorylation regulates myosin IIIa activity and localization in stereocilia and other actin protrusions. Myosin IIIa has a kinase domain that is thought to auto-regulate its activity. Because myosin IIIa tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate its biochemical activity, cellular localization, and cellular function. While inactivation of the myosin IIIa kinase domain with the point mutation K50R did not alter maximal ATPase activity, phosphorylation of myosin IIIa resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of myosin IIIa autophosphorylation was unchanged by the presence of actin but found to be dependent upon myosin IIIa concentration within the range of 0.1-1.2 M, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of myosin IIIa lacking the kinase domain decreases when co-expressed with kinase-active, full-length myosin IIIa. The cellular consequence of reduced myosin IIIa tip localization was a decreased filopodial density along the cell periphery, identifying a novel cellular function for myosin IIIa in mediating the formation and stability of actin-based protrusions. These results suggest that myosin IIIa motor activity is regulated through a novel mechanism involving concentration-dependent autophosphorylation In the past year, in collaborations with Henrique von Gersdorff (OHSU) we used serial section and electron tomographic reconstructions to inventory all ribbon synapses in a model hair cell. By pairing our comprehensive ultrastructural data (precise spatial relationships, vesicle pools, and synaptic geometry) with physiology performed in Henriques lab, we explored how the design features of these ribbon synapses might allow them to convert the hair cells graded electrical potentials into multivesicular release over a wide range of stimulus intensities. By maintaining a population of docked synaptic vesicles with a gradient of release thresholds, the synapse appears able to adapt the efficacy of vesicle release to the intensity of stimulation.
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