Regulation of bipolar myosin filaments by the giant scaffold protein obscurin

我的NSERC研究项目的总体目标是了解肌动蛋白细胞骨架在发育过程中的调制和调节。在这里，我们建议研究巨大的支架蛋白Oblcurin，一种肌球蛋白结合蛋白。虽然脊椎动物有三个黑暗素家族成员，但果蝇只有一个黑暗素基因。我们之前已经证明，暗蛋白定位于肌节的M线，肌节是肌肉最小的功能收缩单位，在肌原纤维的组装中起着至关重要的作用，尤其是肌球蛋白粗丝在M线上的适当锚定所必需的。在这里，我们计划分析遗传暗蛋白突变体在上皮发育中的作用，鉴定和表征与暗蛋白相互作用的蛋白质，特别是非肌肉肌球蛋白，并研究非肌肉细胞中的双极细丝结构。这将有助于我们了解Oblcurin的细胞生物学功能及其各种蛋白结构域。 目的1：研究黑素突变体的细胞缺陷。到目前为止，我们已经分析了亚型暗黑蛋白突变体在肌原纤维中的功能。我们现在将在上皮组织中分析这些突变，并建议使用CRISPR-Cas9技术通过靶向突变产生零突变。我们推测，除了肌肉功能外，黑色素还在上皮细胞的发育中发挥作用，因为黑色素定位于上皮细胞的细胞皮质，当耗尽时，会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、想象盘和胚胎上皮。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：研究双极纤维的结构。我们假设暗蛋白与上皮细胞中的非肌肉肌球蛋白直接相互作用。我们将纯化标记的非肌肉肌球蛋白杆状结构域和标记的Oblcurin结构域，以测试体外相互作用。我们提出了一种针对Oblcurin的DH域的抗体，它将与其他抗体一起用于超分辨显微镜。这将有助于确定肌动蛋白纤维中遮盖蛋白的相对定位和方向。我们还产生了一种覆盖暗蛋白N末端的转基因。成虫的亲和纯化(AP)和质谱分析(MS)确定了两个很强的相互作用分子：已知与脊椎动物黑斑蛋白相互作用的肌动蛋白和原肌球蛋白-1，证明了这种方法的可行性。我们将用胚胎提取物重复AP-MS，以确定非肌肉遮盖蛋白的相互作用，并对它们进行生化和遗传学分析。这将揭示暗蛋白是如何调节非肌肉肌球蛋白细丝的。 结论：这一提议为更好地理解一个重要的肌动球蛋白调节蛋白在模式生物中的功能提供了独特的机会，为深入了解Oblcurin的基本细胞生物学和发育功能提供了洞察力。

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.