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Mechanisms of Nuclear Migration

核迁移机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要（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，细胞骨架）复合物维持核膜结构，标记表面细胞核与邻近的内质网截然不同，并且在真核生物的早期进化中发挥了重要作用。我们做广告—— 弥补我们在核定位调节机制知识方面的四个空白。（一）发展情况如何核迁移和锚定之间的转换是介导的吗？我们假设不同的 LINC 复合物细胞核从迁移转变为锚定所需要的。我们建议分子间二硫 fide 键是核心，可通过蛋白质二硫键异构酶和/或 AAA+ ATPase torsin 进行调节到开关。我们进一步假设 LINC 直接与外核膜相互作用以优化力穿过核膜的转移。 (2) 细胞核如何锚定在大合胞体细胞中？这是对于细胞核均匀分布很重要，这样多核合胞体才能作为一个单元发挥作用。我们最近发现 ANC-1 通过未知的、不依赖于 LINC 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，更喜欢一种微管马达而不是另一种微管马达？ KASH 蛋白 UNC-83 介质在不同的发育阶段，核向微管的正端或负端运动。我们假设该选择受到 UNC-83 的替代亚型的调节，这些亚型差异性地激活驱动蛋白-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，在细胞核挤过狭窄区域时使细胞核变形限制。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核，包括一个组织，其中 139 个细胞核位于单个皮下合胞体中，以及第二个组织，其中细胞核作为发育的正常部分，通过狭窄的狭窄区域迁移。此外，我们还开发了重新对我们未来计划至关重要的男性，包括 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前向的十多个突变系通过收缩进行核迁移缺陷的遗传筛查。为了补充我们的秀丽隐杆线虫遗传方法，我们还合作证实了我们在哺乳动物组织培养细胞和体外显微实验中的发现使用 TIRF 显微镜进行肾小管运动测定。我们的研究预计将确定 LINC 复合物是如何发挥作用的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，巨型 KASH 蛋白如何组织整体细胞骨架和定位细胞器，UNC-83 如何介导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，以及肌动蛋白如何提供帮助原子核挤过狭窄的空间。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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
作者：
通讯作者：

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
影响因子：
0
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.