Mechanisms of how nuclear envelope bridges link nuclei to the cytoskeleton.

描述（由申请人提供）：各种细胞过程，包括受精、细胞分裂、细胞迁移和细胞极性，都取决于核迁移事件。内核膜SUN蛋白和外核膜KASH蛋白将细胞核偶联至细胞骨架。在了解喀什-SUN桥梁如何形成和运作方面仍然存在差距。具体而言，蛋白质如何被运输到内核质膜、微管和马达如何协调以移动细胞核以及KASH和SUN蛋白如何相互作用以将细胞质力连接到细胞核的分子机制仍然未知。我们的假设是，细胞质中微管马达产生的力通过保守的KASH和SUN蛋白的桥连接到细胞核。了解力如何在核膜上传递将使我们能够阐明细胞核如何在细胞中定位，染色体如何在细胞核内移动以及这些过程的扰动如何破坏细胞和发育过程的机制。我们的模型将通过三个具体的目标进行检验：（目标1）阐明内核膜生物发生的机制。目前的范例是膜蛋白在ER膜内扩散到核膜。我们的初步数据支持一种替代的主动运输模型的内核膜贩运，使用相结合的可溶性核进口机械，膜结合的进口，和高尔基体贩运中间。我们假设，多个核膜内定位信号的功能，首先积极运输ESTA-84从外周ER到核膜，然后介导移动通过核孔。(Aim 2）确定驱动蛋白、动力蛋白和微管如何起作用以移动细胞核。拔河，相互依存的监管，双向运动提出的模型来解释如何电机的相反极性的功能一起移动货物。我们的假设是，驱动蛋白-1提供的力量，移动核和动力蛋白介导的向后运动和滚动绕过路障。我们将区分两种模式NOCA-1如何调节极化微管阵列，通过调节无论是加端尖端动力学或微管成核。(Aim 3）确定细胞质中产生的力如何耦合到细胞核。有两种模型可以解释KASH-SUN桥在核迁移中的作用;它们可以简单地作为外核对接点，也可以作为穿过核膜的力的传感器。我们假设，在细胞质中产生的力直接连接到核板的KASH-SUN桥。我们的方法是创新的，因为它利用了C。elegans模型具有独特的遗传和分子优势，能够拍摄和量化核迁移。所提出的研究是重要的，因为它有望（A）阐明蛋白质运输到内核膜的机制，（B）阐明将适用于其他大型货物的双向核迁移沿着极化微管的机制，以及（C）确定移动核的力如何穿过核膜转移。 公共卫生相关性：拟议的研究与公共卫生有关，因为KASH和SUN蛋白的突变已被证明会导致或与肌肉萎缩症，共济失调，无脑畸形，早衰症和多种癌症有关。此外，核迁移的缺陷导致这些疾病的许多神经肌肉缺陷。因此，拟议的研究与NIH的使命有关，该使命促进基础细胞和发育生物学的基本发现，对人类健康产生积极影响的潜力很大。

DESCRIPTION (provided by applicant: A wide variety of cellular processes, including fertilization, cell division, cell migration, and cell polarity, depend on nuclear migration events. Inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins couple nuclei to the cytoskeleton. Gaps remain in understanding how KASH-SUN bridges are formed and function. Specifically, the molecular mechanisms of how proteins are trafficked to the inner nuclear mem- brane, how microtubules and motors are coordinated to move nuclei, and how KASH and SUN proteins interact to connect cytoplasmic forces to nuclei remain unknown. Our hypothesis is that forces generated by microtubule motors in the cytoplasm are connected to the nucleus by a bridge of conserved KASH and SUN proteins. Understanding how forces are transferred across the nuclear envelope will allow us to elucidate mechanisms of how nuclei are positioned in a cell, how chromosomes are moved inside the nucleus, and how perturbations of these processes disrupt cell and developmental processes. Our model will be tested by three specific aims: (Aim 1) Elucidate mechanisms of inner nuclear membrane biogenesis. The current paradigm is that membrane proteins diffuse within the ER membrane to the nuclear envelope. Our preliminary data support an alternative active transport model for inner nuclear membrane trafficking, using a combination of the soluble nuclear import machinery, membrane-bound importins, and a Golgi trafficking intermediate. We hypothesize that multiple inner-nuclear-membrane-localization signals function to first actively transport UNC-84 from the peripheral ER to the nuclear envelope and to then mediate movement across the nuclear pore. (Aim 2) Deter- mine how kinesin, dynein, and microtubules function to move nuclei. Tug-of-war, interdependent regulation, and bi-directional movement are proposed models to explain how motors of opposite polarity function together to move a cargo. Our hypothesis is that kinesin-1 provides the force to move nuclei and that dynein mediates backwards movements and rolling to bypass roadblocks. We will distinguish between two models for how NOCA-1 regulates polarized microtubule arrays-by regulating either plus-end tip dynamics or nucleation of microtubules. (Aim 3) Determine how forces generated in the cytoplasm are coupled to the nucleus. Two models could explain the role of the KASH-SUN bridge in nuclear migration; they could serve simply as outer nuclear docking sites or, also as transducers of force across the nuclear envelope. We hypothesize that forces generated in the cytoplasm are directly linked to the nuclear lamina by KASH-SUN bridges. Our approach is innovative because it takes advantage of a C. elegans model with unique genetic and molecular strengths with the ability to film and quantify nuclear migration. The proposed research is significant because it is expected to (A) elucidate mechanisms of protein transport to the inner nuclear membrane, (B) elucidate mechanisms of bi- directional nuclear migration along polarized microtubules that will be applicable to other large cargos, and (C) determine how the forces that move nuclei are transferred across the nuclear envelope. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because mutations in KASH and SUN proteins have been shown to cause or to be linked to muscular dystrophies, ataxias, lissencephaly, progeria, and multiple cancers. Moreover, defects in nuclear migration contribute to many of the neuromuscular defects of these diseases. Thus, the proposed research is relevant to the part of the NIH's mission that fosters fundamental discoveries in basic cell and developmental biology with great potential for a positive impact on human health.