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 advances towards elucidating the mechanisms that underlie the formation, regulation, renewal, and life span of stereocilia. Inner ear hair cell stereocilia are composed of parallel, uniformly polarized and crosslinked actin filaments. In earlier studies we have shown that in developing neonatal hair cells stereocilia actin filaments are continuously polymerizing and depolymerizing in a treadmilling process that results in a dynamic renewal process while maintaining steady-state lengths. Whether stereocilia actin filaments are dynamically turning over throughout the lifetime of the hair cell, and whether mature stereocilia have any structural plasticity has remained an important open question in the hearing research field, especially since structural plasticity in hair cell stereocilia may have implications for the development of therapeutic interventions for both inherited and acquired hearing losses. A flurry of recent papers has reported multiple myosin motor proteins involved in regulating stereocilia structures by transporting actin-regulatory cargo to the tips of stereocilia. In a recent study, we showed that two paralogous class 3 myosins Myo3a and Myo3b both transport the actin-regulatory protein Espin 1 (Esp1) to stereocilia and filopodia tips in a remarkably similar, albeit non-identical fashion. We followed up this study with experimental and computational data (in collaboration with Nir Gov, Weizmann Institute) that suggests that subtle differences between these two proteins biophysical and biochemical properties can help us understand how these myosin species target and regulate the lengths of actin protrusions. Through the combination of experimental and computational approaches, we were able to dissect the underlying molecular components of these myosin:cargo dynamics. This work demonstrates the intricate nature of the interactions between molecular motors within cellular protrusions, and the usefulness of theoretical modeling in deciphering them. Specifically, we can begin to ask and answer detailed questions about the contribution of different aspects of myosin motility, (e.g., actin-binding and motor activity), to not only their biophysical behavior, but also their physiological function. Indeed, these results strongly indicate that Myo3b can behave quite similarly to Myo3a, but they also show that Myo3b falls short of Myo3a activity in terms of elongation activity. This finding is one plausible explanation for the late-onset phenotype associated with mutations in MYO3A. Overall, these studies lend stronger support for further exploring the potential role for Myo3b in compensating for Myo3a, and how these two motor proteins interact in a physiological context. We also helped Nir Gov develop and test a reaction diffusion concept to describe the initiation of actin-based protrusions, and compared with experimental observations. Reaction-diffusion models have been used to describe pattern formation on the cellular scale, and traditionally do not include feedback between cellular shape changes and biochemical reactions. We introduce here a distinct reaction-diffusion-elasticity approach: the reaction-diffusion part describes bi-stability between two actin orientations, coupled to the elastic energy of the cell membrane deformations. This coupling supports localized patterns, even when such solutions do not exist in the self-inhibited reaction-diffusion system. We studied the stereocilia localization an role of a PDZD7, a scaffolding protein linked to the Usher syndrome protein network. Usher syndrome is the leading cause of genetic deaf-blindness. It has been reported that monoallelic mutations in PDZD7 increase the severity of Usher type II syndrome caused by mutations in USH2A and VLGR1, which respectively encode usherin and VLGR1. PDZ domain-containing 7 protein (PDZD7) is a paralog of the scaffolding proteins harmonin and whirlin, which are implicated in Usher type 1 and type 2 syndromes. While usherin and VLGR1 have been reported to form the hair cell stereocilia ankle-links, harmonin localizes to the stereocilia upper tip-link density (UTLD) and whirlin localizes to both the stereocilia tip and ankle-link regions. In collaboration with Peter Gillespie from the Oregon Hearing Research Center, we used mass spectrometry to show that PDZD7 is expressed in stereocilia at a comparable molecular abundance to VLGR1. We also show by immunofluorescence and by overexpression of tagged proteins that PDZD7 localizes to the stereocilia ankle-link region, overlapping with usherin, whirlin, and VLGR1. Finally, we show in LLC-PK1 cells that the cytosolic domains of usherin and VLGR1 can bind to both whirlin and PDZD7. These observations are consistent with PDZD7 being a modifier of the USH2 phenotype and candidate gene for USH2, and suggest that PDZD7 is a second scaffolding component of the ankle-link complex. In collaboration with Henrique von-Gersdorf from the Vollum Institute we showed that sharp calcium nanodomains beneath the ribbon promote highly synchronous multivesicular release at hair cell synapses. Hair cell ribbon synapses exhibit several distinguishing features. Structurally, a dense body, or ribbon, is anchored to the presynaptic membrane and tethers synaptic vesicles;functionally, neurotransmitter release is dominated by large EPSC events produced by seemingly synchronous multivesicular release. However, the specific role of the synaptic ribbon in promoting this form of release remains elusive. Using complete ultrastructural reconstructions and capacitance measurements of bullfrog amphibian papilla hair cells dialyzed with high concentrations of a slow calcium buffer (10 mM EGTA), we found that the number of synaptic vesicles at the base of the ribbon correlated closely to those vesicles that released most rapidly and efficiently, while the rest of the ribbon-tethered vesicles correlated to a second, slower pool of vesicles. Combined with the persistence of multivesicular release in extreme calcium buffering conditions (10 mM BAPTA), our data argue against the calcium-dependent compound fusion of ribbon-tethered vesicles at hair cell synapses. Moreover, during hair cell depolarization, our results suggest that elevated calcium levels enhance vesicle pool replenishment rates. Finally, using calcium diffusion simulations, we propose that the ribbon and its vesicles define a small cytoplasmic volume where calcium buffer is saturated, despite 10 mM BAPTA conditions. This local buffer saturation permits fast and large calcium rises near release sites beneath the synaptic ribbon that can trigger multiquantal EPSCs. We conclude that, by restricting the available presynaptic volume, the ribbon may be creating conditions for the synchronous release of a small cohort of docked vesicles. We also examined the localization of otoconin 90 and otolin, two proteins essential for the biomolecular control of mineralization of otoconia, the extracelluar biomineral structures that overlay vestibular sensory organs. We observed that partial demineralization of otoconia causes loosening of the anchoring of interotoconial fibrils which are required to attach otoconia to each other. We postulate that in humans an analogous demineralization of the otoconia subsurface layer, which causes loss of firm anchoring of the interconnecting fibrils and detachment of otoconia results in the benign positional vertigo, the most common cause of vertigo.

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Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
2012
Total Cost
$2,188,902
Indirect Cost
Name
National Institute on Deafness and Other Communication Disorders
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Ebrahim, Seham; Avenarius, Matthew R; Grati, M'hamed et al. (2016) Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like. Nat Commun 7:10833
Andrade, Leonardo R; Salles, Felipe T; Grati, M'hamed et al. (2016) Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus. J Struct Biol 194:139-46
Raval, Manmeet H; Quintero, Omar A; Weck, Meredith L et al. (2016) Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions. J Biol Chem 291:22781-22792
Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul et al. (2016) Hemi-fused structure mediates and controls fusion and fission in live cells. Nature 534:548-52
Mittal, Rahul; Grati, M'hamed; Sedlacek, Miloslav et al. (2016) Characterization of ATPase Activity of P2RX2 Cation Channel. Front Physiol 7:186
Ebrahim, Seham; Ingham, Neil J; Lewis, Morag A et al. (2016) Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia. Cell Rep 15:935-43
Kurima, Kiyoto; Ebrahim, Seham; Pan, Bifeng et al. (2015) TMC1 and TMC2 Localize at the Site of Mechanotransduction in Mammalian Inner Ear Hair Cell Stereocilia. Cell Rep 12:1606-17
Yochelis, A; Ebrahim, S; Millis, B et al. (2015) Self-organization of waves and pulse trains by molecular motors in cellular protrusions. Sci Rep 5:13521
Andrade, Leonardo R (2015) Evidence for changes in beta- and gamma-actin proportions during inner ear hair cell life. Cytoskeleton (Hoboken) 72:282-91
Francis, Shimon P; Krey, Jocelyn F; Krystofiak, Evan S et al. (2015) A short splice form of Xin-actin binding repeat containing 2 (XIRP2) lacking the Xin repeats is required for maintenance of stereocilia morphology and hearing function. J Neurosci 35:1999-2014

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