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 调节核定位的核膜蛋白和力发生器。LINC(核骨架连接物- Ton和细胞骨架)复合体，我们发现，保持了核膜结构，标志着 细胞核的表面明显不同于毗邻的ER，在真核生物的早期进化中起着重要作用。 我们解决了我们对规范核定位的机制的认识中的四个空白。(1)情况如何？ 核迁移和锚定之间的发育转换？我们假设不同的是 LINC复合体是原子核从迁移切换到锚定所必需的。我们建议一个 分子间二硫键，可由蛋白质二硫键异构酶和/或AAA+调节 ATPase Torsin是开关的中心。我们进一步假设LINC直接与外部 核膜，以优化跨核膜的力量传递。(2)原子核是如何锚定的 在大型合体细胞中？重要的是，细胞核要均匀分布，这样多核合胞体才能 作为一个整体行动。我们最近发现ANC-1通过锚定合体细胞核和线粒体 未知的，LINC非依赖的机制，并假设ANC-1通过 微管。(3)在发育的不同阶段，细胞核如何比另一个更有利于微管运动？ Kash蛋白UNC-83介导核向微管正端或负端的移动。 处于发展的各个阶段。我们推测，这种选择是由UNC-83的替代亚型调控的 差异激活激动素-1的运动活性。(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，在该模型中，LINC复合体与分支的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，因为我们可以观看现场 整个发育过程中的核，包括一个组织，其中139个核在一个皮下合胞体中，以及 第二种组织，细胞核通过狭窄的收缩进行迁移，这是发育的正常部分。进一步- 此外，我们已经开发出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 将功能、细胞特异性标记、组织特异性生长素诱导的降解系统分开的复合体，以及 来自正向遗传筛选的十多个突变系，以寻找通过收缩的核迁移缺陷。至 除了补充我们的线虫遗传方法，我们还合作在哺乳动物身上证实了我们的发现 组织培养细胞和TIRF显微镜下体外微管运动测定。我们的研究有望 确定LINC复合体如何在分子和生物物理水平上被调节，外核如何 膜参与了力的传递，巨型Kash蛋白如何组织全球细胞骨架和 定位细胞器，UNC-83如何在动力蛋白和动力蛋白指导的核移动之间进行选择- 在整个发育过程中，肌动蛋白如何帮助细胞核挤压狭窄的空间。