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

描述（由申请人提供）：多种细胞过程，包括受精、细胞分裂、细胞迁移和细胞极性，都依赖于核迁移事件。内核膜SUN蛋白和外核膜KASH蛋白将细胞核偶联到细胞骨架上。在了解喀什-孙桥的形成和功能方面仍然存在空白。具体来说，蛋白质如何运输到内核膜的分子机制，微管和马达如何协调移动细胞核，以及KASH和SUN蛋白如何相互作用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的微管马达产生的力通过保守的KASH和SUN蛋白的桥梁连接到细胞核。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的机制，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。我们的模型将通过三个具体目标进行测试：（目的1）阐明核膜生物发生机制。目前的范式是膜蛋白在内质网膜内扩散到核膜。我们的初步数据支持核膜内转运的另一种主动转运模型，该模型使用可溶性核进口机制、膜结合进口蛋白和高尔基转运中间体的组合。我们假设多个内核膜定位信号首先主动将UNC-84从外周内质网转运到核包膜，然后介导通过核孔的运动。（目标2）威慑-了解如何运动蛋白，动力蛋白和微管的功能，以移动细胞核。拔河、相互调节和双向运动被提出模型来解释相反极性的马达如何一起工作来移动货物。我们的假设是动力蛋白-1提供了移动细胞核的力量，而动力蛋白介导了向后运动和绕过路障的滚动。我们将区分NOCA-1如何通过调节微管的正端动力学或成核来调节极化微管阵列的两种模型。（目标3）确定细胞质中产生的力如何与细胞核耦合。两个模型可以解释喀什-孙桥在核迁移中的作用；它们可以简单地作为外核对接点，也可以作为跨核膜的力传感器。我们假设细胞质中产生的力通过kashh - sun桥直接与核层相连。我们的方法是创新的，因为它利用秀丽隐杆线虫模型具有独特的遗传和分子优势，能够拍摄和量化核迁移。这项拟议的研究意义重大，因为它有望(A)阐明蛋白质向核膜内运输的机制，(B)阐明沿极化微管的双向核迁移机制，这将适用于其他大型货物，以及(C)确定移动细胞核的力如何在核膜上转移。

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.