Regulation of bipolar myosin filaments by the giant scaffold protein obscurin

巨型支架蛋白暗蛋白对双极肌球蛋白丝的调节

• 批准号: RGPIN-2016-06793
• 负责人: Schoeck, Frieder
• 金额: $ 2.26万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=664505
• 关键词: Regulation; bipolar; myosin; filaments; giant

The overall objective of my NSERC research program is to understand the modulation and regulation of the actomyosin cytoskeleton during development. Here we propose to investigate the giant scaffold protein obscurin, a myosin-binding protein. While there are three obscurin family members in vertebrates, there is only a single obscurin gene in Drosophila. We have previously shown that obscurin localizes to the M-line of sarcomeres, the smallest functional contractile unit of muscles, and plays a crucial role in myofibril assembly, especially required for the proper anchoring of myosin thick filaments at the M-line. Here we plan to analyze genetic obscurin mutants for their role in epithelial development, to identify and characterize proteins, in particular nonmuscle myosin, interacting with obscurin, and to investigate bipolar filament architecture in nonmuscle cells. This will help us understand the cell biological function of obscurin and its various protein domains.***Aim 1: Investigate cellular defects in obscurin mutants. We have so far analyzed the function of hypomorphic obscurin mutants in myofibrils. We will now analyze these mutants in epithelial tissues and propose to generate null mutants by targeted mutagenesis using the CRISPR-Cas9 technique. We hypothesize that obscurin in addition to its muscle functions plays a role in epithelial development, because obscurin localizes to the cell cortex of epithelial cells and shows defects in morphogenesis and cell shape when depleted. We will analyze larval salivary glands, imaginal discs, and embryonic epithelia. This will identify novel nonmuscle functions common to all obscurin family members. ***Aim 2: Investigate bipolar filament architecture. We hypothesize that obscurin interacts directly with nonmuscle myosin in epithelial cells. We will purify tagged nonmuscle myosin rod domains and tagged obscurin domains to test interaction in vitro. We raised an antibody against the DH domain of obscurin, which will be used together with other antibodies to employ superresolution microscopy. This will help determine the relative localization and orientation of obscurin within actin fibers. We have also generated a transgene covering the N-terminus of obscurin. Affinity purification (AP) from adults and mass spectrometry (MS) identified two strong interactors: titin, which is known to interact with vertebrate obscurin, and tropomyosin-1, demonstrating the feasibility of this approach. We will repeat AP-MS with embryonic extracts to identify nonmuscle obscurin interactors and analyze them biochemically and genetically. This will uncover how obscurin regulates nonmuscle myosin filaments.***Conclusion: This proposal offers the unique chance to better understand the function of a crucial actomyosin-regulating protein in a model organism, providing insights into the basic cell biological and developmental functions of obscurin.

我的NSERC研究计划的总体目标是了解在发育过程中肌动球蛋白细胞骨架的调制和调节。在这里，我们建议调查巨大的支架蛋白obscurin，肌球蛋白结合蛋白。虽然在脊椎动物中有三个obscurin家族成员，但在果蝇中只有一个obscurin基因。我们以前已经表明，obscurin本地化的肌节，肌肉的最小功能收缩单位的M-线，并在肌原纤维组装，特别是需要适当的锚定肌球蛋白粗丝在M-线中起着至关重要的作用。在这里，我们计划分析遗传obscurin突变体在上皮发育中的作用，以确定和表征蛋白质，特别是非肌肉肌球蛋白，与obscurin相互作用，并调查双极丝结构在非肌肉细胞。这将有助于我们了解obscurin及其各种蛋白质结构域的细胞生物学功能。目的1：研究obscurin突变体中的细胞缺陷。到目前为止，我们已经分析了肌原纤维中的亚型obscurin突变体的功能。我们现在将分析上皮组织中的这些突变体，并提出使用CRISPR-Cas9技术通过靶向诱变产生无效突变体。我们推测，除了其肌肉功能的obscurin在上皮细胞的发育中发挥作用，因为obscurin定位于上皮细胞的细胞皮质，并显示缺陷的形态发生和细胞形状时耗尽。我们将分析幼虫的唾液腺、成虫盘和胚胎上皮。这将确定所有obscurin家族成员共同的新的非肌肉功能。* 目标2：研究双极灯丝架构。我们推测，obscurin直接与上皮细胞中的非肌肉肌球蛋白相互作用。我们将纯化标记的非肌肉肌球蛋白杆域和标记的obscurin域，以测试在体外的相互作用。我们提出了一个抗体对DH域的obscurin，这将与其他抗体一起使用超分辨率显微镜。这将有助于确定暗蛋白在肌动蛋白纤维内的相对定位和方向。我们还产生了覆盖obscurin的N-末端的转基因。亲和纯化（AP）从成人和质谱（MS）确定了两个强大的相互作用：肌联蛋白，这是已知的脊椎动物obscurin和原肌球蛋白-1相互作用，证明了这种方法的可行性。我们将重复AP-MS与胚胎提取物，以确定nonmuscle obscurin相互作用，并分析他们的生化和遗传。这将揭示obscurin如何调节非肌肉肌球蛋白丝。结论：这一提议提供了一个独特的机会，以更好地了解一个关键的肌动球蛋白调节蛋白在模式生物中的功能，提供了对obscurin的基本细胞生物学和发育功能的见解。