Mechanisms of Nuclear Migration

核迁移机制

• 批准号: 10797575
• 负责人: DANIEL A STARR
• 金额: $ 22.13万
• 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/10797575
• 关键词: ATP phosphohydrolase; Actins; Address; Adult; Aneurysm; Animals; Architecture; Auxins; Binding; Biological Assay; Biophysics; Blindness; Caenorhabditis elegans; Cell Nucleus; Cell physiology; Cells; Collaborations; Complement; Complex; Cytoplasm; Cytoskeletal Modeling; Cytoskeleton; DNA Repair; Data; Defect; Development; Developmental Process; Disease; Drosophila genus; Dynein ATPase; Endothelial Cells; Eukaryota; Event; Evolution; Experimental Models; Extracellular Matrix; Extravasation; Future; Genetic; Genetic Screening; Giant Cells; Grant; Homologous Gene; Image; In Vitro; Inflammation; Kinesin; Knowledge; Lamins; Leukocytes; Malignant Neoplasms; Mammals; Mediating; Meiosis; Membrane; 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; Process; Progeria; Protein Disulfide Isomerase; Protein Isoforms; Proteins; Public Health; Reagent; Research; Role; Spermatogenesis; Sterility; Surface; System; Tissues; Total Internal Reflection Fluorescent; Work; cancer cell; cell motility; constriction; disulfide bond; env Gene Products; genetic approach; genetic manipulation; hearing impairment; human disease; innovation; migration; molecular dynamics; mutant; nervous system disorder; single molecule; tissue/cell culture; transmission process; wound healing

Project Summary (From R35 MIRA original proposal) 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 nucleoskeleton 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 ad- dress four gaps in our knowledge of the mechanisms regulating nuclear positioning. (1) How is the developmen- tal 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 disul- fide 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 medi- ates nuclear movements toward plus or minus ends of microtubules at different 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. Furthermore, we have developed rea- gents 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 micro- tubule 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 movements throughout development, and how actin helps nuclei squeeze through constricted spaces.

项目摘要（来自R35 MIRA原始提案） 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。核骨架连接体 和细胞骨架）复合物，我们发现，维持核膜结构，标记表面 的细胞核明显从相邻的ER，并在真核生物的早期进化工具。我们- 填补了我们对核定位机制的认识中的四个空白。(1)发展如何- 核迁移和锚定介导之间的对话转换？我们假设不同的LINC复合物 是细胞核从迁移到锚定所必需的。我们认为分子间的双硫- fide键是重要的，它可以被蛋白质二硫键异构酶和/或AAA+ ATP酶torsin调节， 到开关。我们进一步假设LINC直接与外核膜相互作用，以优化 力在核膜上的传递(2)细胞核是如何固定在大的合胞体细胞中的？是 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单一的单位。我们 最近发现ANC-1通过未知的、非LINC依赖的方式锚定合胞核和线粒体， 机制，并假设ANC-1通过微管组织细胞质。(3)原子核如何 在不同的发育阶段更倾向于一种微管马达KASH蛋白的表达 在不同的发育阶段，细胞核向微管的正端或负端运动。我们 假设这种选择是由不同激活驱动蛋白-1的β-83的替代亚型调节的 运动活动(4)原子核是如何变形以通过狭窄的空间迁移的？我们的数据支持一个模型， LINC复合物的功能平行于分支肌动蛋白网络，使细胞核变形，因为它们挤压通过狭窄的 收缩。我们的实验系统是创新的，因为我们可以在整个发育过程中观察活细胞核， 包括其中139个细胞核在单个皮下合胞体中的组织和其中细胞核在单个皮下合胞体中的第二组织 作为正常发育的一部分通过狭窄的收缩迁移。此外，我们还开发了一种... 这对我们未来的计划至关重要，包括在LINC复合物中分离功能的一系列点突变体， 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，以及来自一个正向突变体的十多个突变株系， 通过收缩进行核迁移缺陷的遗传筛查。为了补充我们的C。线虫遗传 方法，我们还合作证实了我们在哺乳动物组织培养细胞和体外微 用TIRF显微镜进行小管运动测定。我们的研究有望确定LINC复合物是如何 在分子和生物物理水平上进行调节，外核膜如何参与力传递， 巨大的KASH蛋白如何组织全球细胞骨架和定位细胞器， 在整个发育过程中动力蛋白和驱动蛋白指导的核运动之间的选择，以及肌动蛋白如何帮助 原子核在狭窄的空间中挤压。

项目成果

tmem-258 is dispensable for both nuclear anchorage and migration in C. elegans.

tmem-258 对于线虫的核锚定和迁移都是可有可无的。

• DOI: 10.17912/micropub.biology.000208
• 发表时间: 2020
• 期刊: microPublication biology
• 作者: Gregory,Ellen;Starr,Daniel
• 通讯作者: Starr,Daniel

Caenorhabditis elegans models for striated muscle disorders caused by missense variants of human LMNA.

• DOI: 10.1371/journal.pgen.1010895
• 发表时间: 2023-08
• 期刊: PLoS genetics
• 影响因子: 4.5

FLN-2 functions in parallel to LINC complexes and Cdc42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

在秀丽隐杆线虫 P 细胞核迁移过程中，FLN-2 与 LINC 复合物和 Cdc42/肌动蛋白通路平行发挥作用。

• DOI: 10.1101/2023.08.04.552041
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Ma,Linda;Kuhn,Jonathan;Chang,Yu-Tai;Elnatan,Daniel;Luxton,GWGant;Starr,DanielA
• 通讯作者: Starr,DanielA

A Nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells.

• DOI: 10.3389/fcell.2022.974168
• 发表时间: 2022
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5