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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684035
• 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节和调节。在这里，我们建议研究巨支架蛋白，一种肌球蛋白结合蛋白。虽然在脊椎动物中有三个隐蛋白家族成员，但在果蝇中只有一个隐蛋白基因。我们之前的研究表明，暗蛋白定位于肌节的m线，这是肌肉最小的功能性收缩单位，在肌原纤维的组装中起着至关重要的作用，尤其是在m线上正确锚定肌球蛋白粗丝所需要的。在这里，我们计划分析遗传暗色蛋白突变体在上皮发育中的作用，识别和表征蛋白质，特别是与暗色蛋白相互作用的非肌肉肌球蛋白，并研究非肌肉细胞中的双极丝结构。这将有助于我们了解蒙昧蛋白及其各种蛋白结构域的细胞生物学功能。***目的1：研究暗色蛋白突变体的细胞缺陷。到目前为止，我们已经分析了亚形态的隐蛋白突变体在肌原纤维中的功能。我们现在将分析上皮组织中的这些突变体，并建议使用CRISPR-Cas9技术通过靶向诱变产生零突变体。我们假设，除了它的肌肉功能外，暗孔蛋白还在上皮细胞的发育中发挥作用，因为暗孔蛋白定位于上皮细胞的细胞皮层，并且在耗尽时显示出形态发生和细胞形状的缺陷。我们将分析幼虫的唾液腺、影像盘和胚胎上皮。这将确定所有暗黑蛋白家族成员共有的新的非肌肉功能。***目的2：研究双极灯丝结构。我们假设暗布林直接与上皮细胞中的非肌球蛋白相互作用。我们将纯化标记的非肌肉肌球蛋白棒结构域和标记的模糊蛋白结构域，以测试它们在体外的相互作用。我们提出了一种针对obscurin DH结构域的抗体，该抗体将与其他抗体一起用于超分辨显微镜。这将有助于确定隐蛋白在肌动蛋白纤维中的相对定位和取向。我们还生成了一个覆盖蒙皮蛋白n端的转基因。来自成人的亲和纯化（AP）和质谱（MS）鉴定出两个强相互作用物：titin（已知与脊椎动物的obscurin相互作用）和原肌球蛋白-1，证明了该方法的可行性。我们将用胚胎提取物重复AP-MS来鉴定非肌肉的暗色蛋白相互作用物，并对其进行生化和遗传学分析。这将揭示暗色蛋白如何调节非肌肉肌球蛋白细丝。***结论：这一发现为更好地理解模型生物中一个关键的肌动球蛋白调节蛋白的功能提供了独特的机会，为理解模糊蛋白的基本细胞生物学和发育功能提供了见解。