Mechanisms of Nuclear Migration

核迁移机制

• 批准号: 10337237
• 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/10337237
• 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 核被膜蛋白和力发生器介导的核定位。LINC（核苷酸- 吨和细胞骨架）复合物，我们发现，维持核膜结构，标志着 细胞核的表面与相邻的ER明显不同，并且在真核生物的早期进化中起着重要作用。 我们处理我们的知识的四个差距的机制，规范核定位。(1)怎么样 核迁移和锚定介导的发育转换？我们假设不同的 LINC复合体是细胞核从迁移到锚定所必需的。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 ATP扭转蛋白是这种转变的核心。我们进一步假设，LINC直接与外部相互作用， 核膜，以优化跨核膜的力的传递。(2)原子核是如何固定的 在大的合胞体细胞中细胞核的均匀分布是很重要的，这样多核合胞体就能够 作为一个整体行动我们最近发现，ANC-1锚定合胞核和线粒体， 未知的，LINC独立的机制，并假设ANC-1组织细胞质通过 微管(3)在不同的发育阶段，细胞核是如何偏爱一种微管马达的？ KASH-83蛋白介导细胞核向微管正负端的运动， 进入发展阶段。我们假设，这种选择是由β-83的替代亚型调节的， 差异激活驱动蛋白-1运动活性。(4)原子核是如何变形以通过狭窄的空间迁移的？我们 数据支持LINC复合物与分支肌动蛋白网络平行发挥作用的模型， 它们从狭窄的收缩处挤出来我们的实验系统是创新的，因为我们可以实时查看 在整个发育过程中，包括一个组织，其中139个细胞核在一个皮下合胞体中， 第二个组织，细胞核通过狭窄的收缩迁移，作为正常发育的一部分。此外─ 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中的一系列点突变体 分离功能、细胞特异性标记物、组织特异性生长素诱导的降解决定子系统的复合物，以及 通过收缩核迁移缺陷的正向遗传筛选获得的十多个突变株系。到 补充我们的C。我们还合作证实了我们在哺乳动物中的发现， 组织培养细胞和用TIRF显微镜进行的体外微管运动测定。我们的研究有望 确定LINC复合物在分子和生物物理水平上是如何调节的，外核是如何调节的， 膜参与力的传递，巨大的KASH蛋白如何组织全球细胞骨架， 位置细胞器，如何在动力蛋白和驱动蛋白定向的核移动之间介导的选择， 以及肌动蛋白如何帮助细胞核挤压狭窄的空间。