Mechanisms of Nuclear Migration

项目摘要(来自R35 Mira原始建议书) 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 和细胞骨架)复合体，我们发现，保持核膜结构，标记表面 与相邻的内质网明显不同，在真核生物的早期进化中起到了重要作用。我们的广告- 填补我们对核定位调控机制的知识中的四个空白。(1)发展情况如何- TAL在核迁移和锚定之间的转换？我们假设不同的LINC复合体 是原子核从迁移转换到锚定所必需的。我们提出了分子间二价体-- 硫键是中心，它可以由蛋白质二硫键异构酶和/或AAA+ATPase Torsin调节 到交换机。我们进一步假设LINC直接与外核膜相互作用以优化 跨核包层的力量转移。(2)核是如何定位在大的合体细胞中的？它是 重要的是核要均匀分布，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LINC非依赖性锚定合体细胞核和线粒体 机制，并假设ANC-1通过微管组织细胞质。(3)原子核是如何 在不同的发育阶段偏爱一种微管马达而不是另一种？Kash蛋白UNC-83-Medi- 在不同发育阶段，阿特斯核向微管正端或负端移动。我们 假设这种选择是由UNC-83的不同亚型调控的，这些亚型可以不同地激活Kinesin-1 运动活动。(4)原子核如何变形以在狭窄的空间中迁移？我们的数据支持这样一个模型 LINC复合体的功能与分支肌动蛋白网络平行，当它们挤过狭窄的细胞核时会使细胞核变形 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看到活的细胞核， 包括其中139个核在单个皮下合体中的组织以及其中核的第二组织 在狭窄的狭窄地带迁徙是正常发展的一部分。此外，我们还开发了ReA- 对我们的未来计划至关重要的绅士，包括在LINC复合体中分离功能的一系列点突变， 细胞特异性标记物，组织特异性生长素诱导的降解系统，以及来自 通过收缩进行核迁移缺陷的基因筛查。来补充我们线虫的基因 方法，我们还合作确认了我们在哺乳动物组织培养细胞和体外微型细胞中的发现。 TIRF显微镜下肾小管运动测定。我们的研究有望确定LINC复合体是如何 在分子和生物物理水平上进行调控，外核膜如何参与力量传递， 巨大的Kash蛋白如何组织全球细胞骨架和定位细胞器，UNC-83如何介导 在整个发育过程中在动力蛋白和动力蛋白指导的核运动之间的选择，以及肌动蛋白如何帮助 原子核挤过狭窄的空间。

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.