Decoding the functions of myosin II isoforms with super-resolution microscopy

用超分辨率显微镜解码肌球蛋白 II 亚型的功能

• 批准号: 10244905
• 负责人: Dylan Tyler Burnette
• 金额: $ 39.18万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10244905
• 关键词: Address; Adhesions; Biological Models; Blood Vessels; Cardiac Myocytes; Cell division; Cells; Contractile System; Cytokinesis; Data; Developmental Process; Disease Progression; Equilibrium; Filament; Generations; Goals; Health Benefit; Interphase; Intervention; Life; Malignant Neoplasms; Microfilaments; Microscopy; Mitosis; Modeling; Molecular; Molecular Motors; Motor Activity; Muscle; Muscle Contraction; Myosin Type II; Neoplasm Metastasis; Nonmuscle Myosin Type IIA; Paper; Pathologic Processes; Play; Postdoctoral Fellow; Primary Neoplasm; Process; Protein Isoforms; Reporting; Research; Resolution; Role; Shapes; Signal Transduction; Smooth Muscle Myocytes; Structure; Testing; Vascular Diseases; Work; base; cancer cell; cell cortex; cell motility; density; design; migration; non-muscle myosin; predictive modeling; programs; rho GTP-Binding Proteins

Decoding the functions of myosin II isoforms with super-resolution microscopy 1) Background and key gaps in our understanding. Cells modify their shape and surroundings to drive processes vital for eukaryotic life, including cell division, cell migration, and muscle contraction. Forces generated by the molecular motor, myosin II, drive these processes. Thus, how myosin II assembles filaments capable of generating force inside of cells is central to our understanding of both developmental processes and the force-dependent progression of diseases such as cancer. Previous studies have provided elegant details of how a single filament of myosin II assembles. Steric hindrance limits the number of myosin II molecules that can be added to a single filament. In order to increase the scale of force generation inside of cells, myosin II filaments are organized into arrays referred to as “stacks”. While it is well documented that both muscle and non-muscle isoforms of myosin II are found within stacks, we do not know how stacks assemble. 2) Description of recent progress by the PI. At the end of his post-doctoral work, the PI showed that super-resolution microscopy could be used to resolve the structure of a non-muscle myosin IIA (NMIIA) filament and that a stack of filaments somehow grows from a single filament at the edge of a migrating cancer cell (Burnette et al, JCB 2014). The first independent paper from the Burnette lab subsequently defined the steps through which a NMIIA filament physically grows into a stack in a mechanisms we call “expansion”, and that this is regulated by the motor activity of NMIIA, the density of surrounding actin filaments, and Rho GTPase signaling (Fenix et al, MBoC 2016). Expansion occurs at the edge of migrating cells during interphase and in the contractile ring during cell division. The second paper from the Burnette lab showed a force balance between myosin II-based contractility and adhesion was controlling the shape of the cleavage furrow (Taneja et al, Scientific Reports 2016), similar to how the leading edge of a crawling cell obtains its shape (Burnette et al. JCB 2014). We now have data suggesting NMIIA and NMIIB play distinct roles during cytokinesis. NMIIA is required for the proper formation of the contractile ring and initial ingression of the cleavage furrow, and NMIIB is required for the completion of cytokinesis, as well as maintaining the integrity of the cell cortex throughout mitosis. 3) Overview of future research program. We propose to continue our research on how myosin II filaments create larger contractile arrays by addressing three main themes. 1) We will continue to use migrating cells as a model system to investigate the molecular mechanisms controlling the assembly/disassembly of NMII filament-stacks. 2) We will also explore the different roles of NMIIA and NMIIB during cytokinesis with a particular focus on their cooperation in creating the contractile arrays in the cleavage furrow and cell cortex. 3) Finally, we will further test the universality of our expansion model by investigating how muscle myosin II isoforms create filament- stacks within cardiac muscle cells. Our ultimate goal is to develop a universal model of myosin II filament-stack formation that can be applied to the study of diverse contractile systems.

用超分辨显微镜破译肌球蛋白II亚型的功能 1)背景和我们认识上的主要差距。细胞修改它们的形状和环境以驱动 对真核生物生命至关重要的过程，包括细胞分裂、细胞迁移和肌肉收缩。力量 由分子马达肌球蛋白II产生，驱动这些过程。因此，肌球蛋白II是如何组装细丝的 能够在细胞内产生力量是我们理解发育过程和 疾病的发展依赖于外力，如癌症。以前的研究已经提供了优雅的细节 肌球蛋白II的单丝是如何组装的。空间位阻限制了肌球蛋白II分子的数量 可以添加到单个灯丝中。为了增加细胞内的力量生成规模，肌球蛋白II 细丝被组织成阵列，称为“堆叠”。虽然有充分的证据表明，肌肉和 肌球蛋白II的非肌肉亚型存在于Stack中，我们不知道Stack是如何组装的。2)描述 关于私家侦探的最新进展。在他博士后工作结束时，PI显示出超分辨率 显微镜可以用来解析非肌肉肌球蛋白IIA(NMIIA)细丝的结构，并且 一堆细丝以某种方式从迁移中的癌细胞边缘的单个细丝生长(Burnette等人， JCB 2014)。Burnette实验室的第一篇独立论文随后定义了 NMIIA灯丝在物理上以一种我们称为“膨胀”的机制生长成堆叠，这是由 NMIIA的运动活性、周围肌动蛋白细丝的密度以及Rho GTP酶信号(Fenix等人， 中国央行2016年)。在间期和收缩环中，扩张发生在迁移细胞的边缘 在细胞分裂过程中。伯内特实验室的第二篇论文显示了肌球蛋白II与肌球蛋白II之间的力量平衡 收缩性和粘附性控制着乳沟的形状(Taneja等人，科学报告 2016)，类似于爬行细胞的前沿如何获得其形状(Burnette等人)。JCB 2014)。我们现在 有数据表明，NMIIA和NMIIB在细胞质分裂过程中发挥着不同的作用。NMIIA是正确的 收缩环的形成和解理沟的初始内移，需要NMIIB 完成胞质分裂，以及在整个有丝分裂过程中保持细胞皮质的完整性。3)概述 未来的研究计划。我们建议继续研究肌球蛋白II细丝如何产生更大的 通过解决三个主要主题来实现收缩阵列。1)我们将继续使用迁移细胞作为模型 系统，以研究控制NMII丝堆叠组装/拆卸的分子机制。 2)我们还将探讨NMIIA和NMIIB在细胞质分裂过程中的不同作用，特别是它们的 在卵裂沟和细胞皮质中创建收缩阵列的合作。3)最后，我们将进一步 通过研究肌肉肌球蛋白II亚型如何产生细丝来测试我们扩张模型的普适性- 堆积在心肌细胞内。我们的最终目标是开发一个通用的肌球蛋白II纤维堆叠模型 可用于研究不同收缩系统的构型。