Mechanisms of Nuclear Migration

核迁移机制

• 批准号: 10077853
• 负责人: DANIEL A STARR
• 金额: $ 37.69万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10077853
• 关键词: ATP phosphohydrolase; Actins; Address; Aneurysm; Architecture; Auxins; Biological Assay; Biophysics; Blindness; Caenorhabditis elegans; Cell Nucleus; Cell physiology; Cells; Complement; Complex; Cytoplasm; Cytoskeleton; DNA Repair; Data; Defect; Development; Developmental Process; Disease; Dynein ATPase; Eukaryota; Event; Evolution; Future; Genetic Screening; Giant Cells; Homologous Gene; In Vitro; Inflammation; Kinesin; Knowledge; Malignant Neoplasms; Mediating; Meiosis; Microtubules; Minus End of the Microtubule; Mitochondria; Modeling; Molecular; Motor; Motor Activity; Movement; Muscular Dystrophies; Mutation; Neoplasm Metastasis; Nuclear; Nuclear Envelope; Nuclear Outer Membrane; Nuclear Pore Complex; Organelles; Play; Positioning Attribute; Progeria; Protein Disulfide Isomerase; Protein Isoforms; Proteins; Public Health; Reagent; Research; Spermatogenesis; Sterility; Surface; System; Tissues; Total Internal Reflection Fluorescent; cancer cell; constriction; disulfide bond; env Gene Products; genetic approach; hearing impairment; human disease; innovation; migration; mutant; nervous system disorder; tissue/cell culture; transmission process; wound healing

Project Summary Nuclear migration and anchorage are central to many cellular events. We uncovered a conserved network of nuclear envelope proteins and force generators that mediate nuclear positioning. LINC (linker of nucleoskele- ton and cytoskeleton) complexes, which we discovered, maintain nuclear envelope architecture, mark the surface of nuclei distinctly from the contiguous ER, and were instrumental in the early evolution of eukaryotes. We address four gaps in our knowledge of the mechanisms regulating nuclear positioning. (1) How is the developmental switch between nuclear migration and anchorage mediated? We hypothesize that different LINC complexes are required for a nucleus to switch from migrating to being anchored. We propose that an intermolecular disulfide bond, which could be regulated by protein disulfide isomerases and/or the AAA+ ATPase torsin, is central to the switch. We further hypothesize that LINC directly interacts with the outer nuclear membrane to optimize the transfer of forces across the nuclear envelope. (2) How are nuclei anchored in large syncytial cells? It is important for nuclei to be evenly spaced so that multi-nucleated syncytia are able to act as a single unit. We recently found that ANC-1 anchors syncytial nuclei and mitochondria through unknown, LINC-independent mechanisms, and hypothesize that ANC-1 organizes the cytoplasm through microtubules. (3) How do nuclei favor one microtubule motor over another at different stages of development? The KASH protein UNC-83 mediates nuclear movements toward plus or minus ends of microtubules at differ- ent stages of development. We hypothesize that the choice is regulated by alternative isoforms of UNC-83 that differentially activate kinesin-1 motor activity. (4) How do nuclei deform to migrate through narrow spaces? Our data support a model where LINC complexes function parallel to branched actin networks to deform nuclei as they squeeze through narrow constrictions. Our experimental system is innovative because we can view live nuclei throughout development, including a tissue where 139 nuclei are in a single hypodermal syncytium and a second tissue where nuclei migrate through narrow constrictions as a normal part of development. Further- more, we have developed reagents essential to our future plans, including an array of point mutants in LINC complexes that separate function, cell-specific markers, a tissue-specific auxin-induced degron system, and over ten mutant lines from a forward genetic screen for defects in nuclear migration through constrictions. To complement our C. elegans genetic approaches, we also collaborate to confirm our findings in mammalian tissue culture cells and an in vitro microtubule motor assay with TIRF microscopy. Our studies are expected to determine how LINC complexes are regulated at molecular and biophysical levels, how the outer nuclear membrane is involved in force transmission, how giant KASH proteins organize the global cytoskeleton and position organelles, how UNC-83 mediates the choice between dynein and kinesin-directed nuclear move- ments throughout development, and how actin helps nuclei squeeze through constricted spaces.

项目概要 核迁移和锚定是许多细胞事件的核心。我们发现了一个保守的网络 核膜蛋白和介导核定位的力发生器。 LINC（核骨架连接子） 我们发现，吨和细胞骨架）复合物维持核膜结构，标记 细胞核表面与邻近的内质网明显不同，并且在真核生物的早期进化中发挥了重要作用。 我们解决了我们在调节核定位机制方面的四个知识空白。 (1) 情况如何 核迁移和锚定之间的发育转换是介导的吗？我们假设不同 LINC 复合物是细胞核从迁移转变为锚定所必需的。我们建议 分子间二硫键，可由蛋白质二硫键异构酶和/或 AAA+ 调节 ATPase Torsin 是开关的核心。我们进一步假设 LINC 直接与外部相互作用 核膜以优化穿过核膜的力的传递。 (2) 原子核是如何锚定的 在大合胞体细胞中？细胞核均匀分布很重要，这样多核合胞体才能形成 作为一个整体。我们最近发现 ANC-1 通过锚定合胞核和线粒体 未知的、不依赖于 LINC 的机制，并假设 ANC-1 通过以下方式组织细胞质： 微管。 (3) 在不同的发育阶段，细胞核如何有利于一种微管运动而不是另一种微管运动？ KASH 蛋白 UNC-83 介导核运动向微管正端或负端的不同方向运动。 耳鼻喉科的发展阶段。我们假设该选择受到 UNC-83 的替代亚型的调节， 差异性激活 kinesin-1 运动活动。 (4) 原子核如何变形以在狭窄的空间中迁移？我们的 数据支持一个模型，其中 LINC 复合物与分支肌动蛋白网络平行发挥作用，使细胞核变形为 他们挤过狭窄的狭窄地带。我们的实验系统是创新的，因为我们可以实时观看 整个发育过程中的细胞核，包括一个皮下合胞体中有 139 个细胞核的组织， 第二种组织，作为发育的正常部分，细胞核通过狭窄的狭窄处迁移。更远- 此外，我们还开发了对我们未来计划至关重要的试剂，包括 LINC 中的一系列点突变体 分离功能、细胞特异性标记、组织特异性生长素诱导的降解决定子系统的复合物，以及 来自正向遗传筛选的十多个突变系，用于通过收缩进行核迁移缺陷。到 为了补充我们的线虫遗传方法，我们还合作确认我们在哺乳动物中的发现 组织培养细胞和使用 TIRF 显微镜进行体外微管运动测定。我们的研究预计 确定 LINC 复合物如何在分子和生物物理水平上受到调节，外核如何 膜参与力传递，巨型 KASH 蛋白如何组织整体细胞骨架以及 位置细胞器，UNC-83如何介导动力蛋白和驱动蛋白定向核运动之间的选择- 整个发育过程中的变化，以及肌动蛋白如何帮助细胞核挤过狭窄的空间。