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Browsing Biology Department Theses and Dissertations by Subject "Actin"
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Item The role of ADF and cofilin in auditory sensory cell development(2020-12) McGrath, Jamis; Perrin, Benjamin; Cummins, Theodore; Belecky-Adams, Teri; Balakrishnan, Lata; Zhao, BoOur ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.Item Stereocilia Morphogenesis and Maintenance is Dependent on the Dynamics of Actin Cytoskeletal Proteins(2019-05) Roy, Pallabi; Perrin, Benjamin J.; Atkinson, Simon J.; Belecky-Adams, Teri; Hashino, Eri; Baucum II, Anthony J.Age-related hearing loss is an acute health problem affecting people worldwide, often arising due to defects in the proper functioning of sensory hair cells in the inner ear. The apical surface of sensory hair cells contains actin-based protrusions known as stereocilia, which detect sound and head movements. Since hair cells are not regenerated in mammals, it is important to maintain the functioning of stereocilia for the life of an organism to maintain hearing ability. The actin filaments within a stereocilium are extensively crosslinked by various actin crosslinking proteins, which are important for stereocilia development and maintenance. Multiple studies have shown that the stereocilia actin core is exceptionally stable whereas actin is dynamic only at the tips of stereocilia. However, whether the actin crosslinking proteins, which are nearly as abundant as actin itself, are similarly stable or can freely move in and out of the core remains unknown. Loss or mutation of crosslinkers like plastin-1, fascin-2, and XIRP2 causes progressive hearing loss along with stereocilia degeneration while loss of espin prevents stereocilia from even developing properly. Do these phenotypes stem from an unstable stereocilia core? Does crosslinking confer stability to the core? To address these questions, we generated novel transgenic reporter lines to monitor the dynamics of actin in mice carrying fascin-2R109H mutation and espin null mice and also to study the dynamics of actin crosslinkers, in vivo and ex-vivo. We established that actin crosslinkers readily exchange within the highly stable F-actin structure of the stereocilia core. In addition, we determined that stereocilia degeneration in mice carrying fascin-2R109H mutation and espin null mice could possibly occur due to a less stable actin core. These studies suggest that dynamic crosslinks stabilize the core to maintain proper stereocilia functioning. Future work warrants understanding the reason behind the importance of dynamic crosslinks within a stable stereocilia core. Actin stability not only depends on actin crosslinkers, but also on actin filament composition as evident from distinct stereocilia degeneration and progressive hearing loss patterns in hair-cell specific knockout of actin isoforms. Although beta- and gamma- actin polypeptide sequences differ by only 14 four amino acids, whether the latter determine the unique function of each cytoplasmic actin isoform was previously unknown. Here we determined that these four critical amino acids determine the unique functional importance of beta-actin isoform in sensory hair cells. Taken together, our study demonstrates that actin cytoskeletal proteins are important for the morphogenesis and maintenance of stereocilia.