Mechanisms of Nuclear Migration

核迁移机制

• 批准号: 10543175
• 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/10543175
• 关键词: ATP phosphohydrolase; Actins; Address; Aneurysm; Architecture; Auxins; Biological Assay; Biophysics; Blindness; Caenorhabditis elegans; Cell Nucleus; Cell physiology; Cells; Collaborations; 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; 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.

项目摘要 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 核包络蛋白和介导核定位的力发电机。 LINC（核克莱（Nucleoskele）的接头 我们发现的吨和细胞骨架）配合物，维护核包膜结构，标记 核表面与连续ER明显，并在真核生物的早期演变中发挥了作用。 在对调节核定位的机制的了解中，我们解决了四个差距。 （1）如何 核迁移与锚定介导的发展切换？我们假设这是不同的 核构成核心需要从迁移到锚定。我们建议 分子间二硫键，可以通过蛋白质二硫化物异构酶和/或AAA+调节 ATPase Torsin，是开关的中心。我们进一步假设LINC直接与外部相互作用 核膜以优化力在整个核包膜中的转移。 （2）核如何锚定 在大型合胞细胞中？对于核的间距均匀至关重 充当一个单元。我们最近发现ANC-1通过合成核和线粒体锚定 未知的，独立于LINC的机制，并假设ANC-1通过 微管。 （3）在不同发育阶段，核如何偏向一个微管电动机而不是另一个微管电动机？ Kash蛋白UNC-83介导了核运动朝着微管的加上或负末端的核运动 - 开发阶段。我们假设该选择是由UNC-83的替代同工型调节的 差异激活驱动蛋白-1运动活性。 （4）核如何通过狭窄的空间迁移？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起作用，以将核变形为 他们通过狭窄的收缩挤压。我们的实验系统具有创新性，因为我们可以看现场 整个发育过程中的核，包括一个组织，其中139个核位于单个皮下综合体中 第二组织，其中核通过狭窄的收缩作为发育的正常部分迁移。更远- 更重要的是，我们开发了对未来计划至关重要的试剂，包括Linc的一系列点突变体 分离功能，细胞特异性标记的复合物，组织特异性生长素诱导的degron系统和 从正向遗传筛查中的十多种突变型线通过收缩来解决核迁移的缺陷。到 补充我们的秀丽隐杆线虫遗传方法，我们还合作以确认我们在哺乳动物中的发现 组织培养细胞和带有TIRF显微镜的体外微管运动测定法。我们的研究预计将 确定如何在分子和生物物理水平下调节林络合物，外核如何调节 膜参与力传播，巨型Kash蛋白如何组织全局细胞骨架和 定位细胞器，UNC-83如何介导动力蛋白和动力蛋白定向的核运动之间的选择 - 整个发育过程中，以及肌动蛋白如何帮助核通过狭窄的空间挤压。