非中心体微管自身调控机制以及其调控胞内细胞器定位的分子机制的研究

In animal cells, two populations of microtubules, centrosomal and non-centrosomal, are detected at the interphase of cell cycle. Centrosomal microtubules are radially nucleated from the centrosome via the??-tubulin ring complex, with their minus ends anchored to this subcellular structure. Their plus ends, on the other hand, are subjected to dynamic instability, and the stability of the plus ends is regulated by +TIPs or plus-end tracking proteins. Non-centrosomal microtubules are produced by various mechanisms, and their minus ends are located in the cytoplasm. How these minus ends are stabilized is, however, little understood. A centrosomal protein, ninein, has been shown to relocate into the non-centrosomal sites, and anchors MT minus ends there. Recent studied identified novel proteins that bind to the minus ends, Nezha and Drosophila Patronin, which are structurally related to one another. It was shown that Nezha tethers non-centrosomal microtubules to the cell junctions, although a non-junctional cytoplasmic population of Nezha/CAMSAP was also detectable; and Patronin can stabilize the minus ends by protecting them against Kinesin 13-mediated depolymerization. .In the present study, we analyzed the functions of Nezha, whi, using Caco2 epithelial cells. We found that Nezha is important for nucleating non-centrosomal microtubules. Depletion of Nezha affected microtubule assembly, causing an increase of centrosomal microtubules. In Nezha depleted cells, the Golgi apparatus was fragmented, and other organelles, such as mitochondria and endosomes, also displayed altered distributions. Further analysis revealed that Nezha depletion caused the suppression of active migration of ERGIC components between Golgi and ERES. From these observations, we conclude that the Nezha fimily/CAMSAPs-tethered microtubules play a crucial role for organelle assembly, whose actions are distinct from those of centrosomal microtubules. In future studies, we are going to deepen the current findings and to study the molecular mechanisms of its regulation.

微管作为细胞骨骼的一个重要成员，对维持细胞以至机体的正常的生理功能有不可缺少的作用。微管分为中心体微管与非中心体微管。由于非中心体微管具有很高的动态特征，难以观察，目前对非中心体微管的认识和研究非常有限。在近来的研究中，我们首次发现了非中心体微管负端蛋白并命名为 Nezha。前期研究结果表明，Nezha显示出了对非中心体微管的负端安定机制的维持起着至关重要的作用，利用此蛋白不仅可以标识非中心体微管，与中心体微管进行区分，还为我们继续深入研究非中心体微管提供了一个难得的切入点。同时，我们研究发现Nezha家族蛋白不仅对于非中心体微管的安定有重要的作用，在其敲降细胞中还发现高尔基体、线粒体和内质网等细胞小器官的定位异常，因此，我们拟通过其作为模型来进一步研究非中心体微管的功能。. 本项研究目标在于揭示非中心体微管在细胞内的功能的分子机制，为研究其在机体中的作用打下基础。

本项目主要研究Nezha及其家族CAMSAPs的功能。通过研究Nezha及其家族分子潜在相互作用分子，明确Nezha对非中心体微管的调控，进一步对微管锚定、胞内运输、细胞周期以及细胞连接等调控的分子机制。.我们通过构建一系列缺失突变体发现 Nezha的CC (1, 2) 结构域对非中心体微管负端的锚定和分布是必需的。质谱分析及免疫共沉淀证实Nezha通过CC (1, 2) 结构域与ACF7第19个spectrin结构域互作。免疫荧光染色实验确认了ACF7依赖于Nezha定位于非中心体微管负端，ACF7依靠其N端两个CH结构域将非中心体微管负端锚定在微丝上。在细胞前沿，Myosin II介导横向应力纤维的形成并帮助其向胞内逆向流动。在此过程中，非中心体微管负端Nezha可通过在微丝上的锚定跟随横向纤维一同向胞内发生逆向流动且依赖于ACF7及Myosin II的活性。同时，微丝通过锚定Nezha并带动其逆向流动对于调整Nezha的定位、维持细胞前沿平行微管的长度及稳定微管的生长方向是必须的。非中心体微管负端如果锚定失败，不论是ACF7敲降或Nezha缺失CC (1, 2) 结构域，都会导致从Nezha发出的EB2方向变紊乱，进而导致微管到达粘着斑的频率下降，最终影响了MAP4K4在粘着斑的定位及细胞迁移受阻。本研究首次揭示了非中心体微管和微丝之间通过Nezha-ACF7复合体建立的新的结构联系，使非中心体微管负端跟随横向应力纤维逆向流动，维持微管的生长方向以正确达到粘着斑处，从而促进细胞迁移。.我们发现在细胞进入分裂期时，Nezha会失去其在微管负端的定位，并伴随着微管骨架的重排。我们筛选找到了与有丝分裂密切相关的蛋白importin-β，经免疫共沉淀实验证明importin-β通过其importin-α结合域与Nezha相互作用，调节来调控细胞分裂前期微管骨架的重排。.我们发现在上皮细胞中Nezha通过与CG-NAP结合互作，而对高尔基体聚合和分布起着重要的作用。敲降Nezha或者CG-NAP均会造成高尔基体产生碎片化。进一步的研究发现依赖于微管解聚因子stathmin的微管动态分布沿着细胞轴方向在核周区域活性最高，抑制这种梯度将会扰乱高尔基体在核周区域的分布。我们认为高尔基体的装配是由多种因素诱导的，其中包括依赖Nezha的高尔基体囊泡簇和微管的动态分布梯度。

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