Indiana University-Purdue University Indianapolis (IUPUI) / Our 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.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第12780号 / 論生博第6号 / 新制||生||38(附属図書館) / 30763 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 豊島 文子, 教授 松本 智裕 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21401号 / 生博第402号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 見学 美根子, 教授 上村 匡, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23334号 / 生博第452号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 渡邊 直樹, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Diseases of heart muscle, such as hypertrophic and dilated cardiomyopathy, are often caused by mutations in proteins of the sarcomere, including actin, troponin, tropomyosin, and myosin. The molecular mechanisms of disease-causing mutations remain unclear because the process of cardiac muscle contraction and the corresponding mutational insults are incompletely defined. To elucidate the underlying mechanisms of cardiac muscle contraction, its regulation, and the effects of disease-causing mutations, the structures of sarcomeric protein assemblies must first be solved. In this dissertation we use interdisciplinary structural biology techniques, including cryo-electron microscopy (cryo-EM), protein-protein docking, and molecular dynamics simulations to investigate the interactions between actin, tropomyosin, and myosin. This structural work is foundational in identifying the molecular effects of mutations. In the first project, we present a novel cryo-EM structure of the cardiac-isoform actomyosin-tropomyosin complex. This structure, which utilizes bovine masseter β-myosin, provides the foundation for understanding the molecular effects of cardiomyopathy-causing mutations that occur at the actomyosin interface. Furthermore, by pairing our structure with protein-protein docking methods and molecular dynamics simulations, we identify complementary and periodic electrostatic interactions between the myosin surface loop 4 and tropomyosin. We hypothesize that these interactions are essential in switching between contraction- and relaxation-mediating states. In a follow up study, we test our myosin loop 4 hypotheses by creating a human cardiac β-myosin all-glycine loop 4 mutant, which abolishes nearly all electrostatic interactions between myosin and tropomyosin. After designing the mutant, we solve the cryo-EM structures of the wild-type and mutant actin-myosin-tropomyosin complexes to high resolution. Our structures confirm that the loop 4 mutant abolishes its interaction with tropomyosin and suggests that the tropomyosin cable on mutant actomyosin filaments is shifted to a new position. Subsequent molecular dynamics simulations corroborate our cryo-EM finding that tropomyosin on mutant actomyosin is displaced from the wild type position. Interaction energy calculations derived from these simulations suggest that the mutant position is significantly less stable than the wild-type. This work provides further evidence that loop 4 interactions are key in stabilizing tropomyosin position during contraction. Finally, to extend our work on the human cardiac actomyosin-tropomyosin complex, we provide insights into the ADP release step of the cardiac β-myosin kinetic cycle. Here, we use a composite method of helical and single-particle cryo-EM reconstruction techniques to solve the structures of the human cardiac actin-myosin-tropomyosin filament in the presence and absence of ADP-Mg2+. This work elucidates the structural basis of cardiac β-myosin ADP release and provides insight into the force-sensing mechanism of the cardiac motor. Lastly, we use our structures to probe how cardiomyopathy-causing mutations potentially disrupt the ADP-to-rigor transition, leading to altered myosin contractility. Overall, the structures solved in this dissertation generate fundamental understanding about the function of the cardiac thin filament and the motor protein, myosin. Moreover, this research provides a framework that connects the initial molecular insults of mutations to the disruption of proper regulation that leads to pathological progression. / 2023-08-24T00:00:00Z

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Formins are potent regulators of actin dynamics that can remodel the actin cytoskeleton to control cell shape, cell cytokinesis, and cell morphogenesis. The defining feature of formins is the formin homology 2 (FH2) domain (Paul and Pollard, 2008), which promotes actin filament assembly while processively moving along the polymerizing filament barbed end. INFT-1 is one of six formin family members present in Caenorhabditis elegans (Hunt-Newbury et al., 2007) and is most closely related to vertebrate INF2, an inverted formin with regulatory domains in the C- rather than N-terminus. Nematode INFT-1 contains both formin homology 1 (FH1) and formin homology 2 (FH2) domains. However, it does not share the regulatory N-terminal Diaphanous Inhibitory Domain (DID) domain and C-terminal Diaphanous Autoregulatory Domain (DAD) domain found in mammalian INF2. In contrast to mammalian INF2, the sequence of INFT-1 starts immediately at FH1 domain and C-terminal region of INFT-1 shares little homology with INF2, suggesting that elegans INFT-1 is regulated by other mechanisms. We used fluorescence spectroscopy to determine the effect of INFT-1 FH1FH2 on actin assembly and total internal reflection fluorescence microscopy to investigate how INFT-1 formin homology 1 and formin homology 2 domains (FH1FH2) mediate filament nucleation and elongation. INFT-1 FH1FH2 nucleates actin filament and promote actin assembly. However, INFT-1 FH1FH2 reduces filament barbed-end elongation rates in the absence or presence of profilin. Evidences demonstrated that INFT-1 is non-processive, indicating a unique mechanism of nucleation. INFT-1 nucleation efficiency is similar to the efficiency of Arabidopsis FORMIN1 (AFH1), another non-processive formin. High phosphate affected the assembly activity of INFT-1 FH1FH2 in the absence or presence of profilin. INFT is thus the second example of a non-processive formin member and will allow a more detailed understanding of the mechanistic difference between processive and non-processive formins. / Master of Science