Centrosome structure and mechanism of MT nucleation by gamma-tubulin complexes

描述（由申请人提供）：跨越从原子到整个细胞器的大小尺度，我们的长期目标是合成所有相关结构和功能之间的相互作用的原子分辨率图片。微管蛋白复合物、调节蛋白和中心体基质。在这里，我们专注于确定微管（MT）成核的分子机制，并了解成核机械的组装和调节。我们的实验室是唯一准备使用层次结构的方法（x射线晶体学，cryoEM单粒子重建，cryoEM断层扫描），以确定结构？-微管蛋白复合物在体外和原位，并通过功能研究和创新的动力学建模了解其作用机制。以前我们发现酵母？-微管蛋白小复合物（？TuSC）可以组装成环，并获得了环的6.5A cryoEM结构，解释了MT 13重对称的起源，并发现了一种意想不到的调节和组装模式。建议的实验扩展这些结果，提供了一个详细的了解MT成核的物理起源，和细胞机制，支配it. Specifically我们解决以下问题：1）确定酵母和果蝇环复合物的结构和它们与MT的相互作用：我们将提高我们的酵母的分辨率？TuSC环，并将产生一个完整的伪原子结构。我们将扩大我们初步的分离果蝇的cryoEM结构（3.5nm）的分辨率（目标~ 1 nm）？TuRCs（2.2MDa？-微管蛋白环复合物），并确定不同的GCP如何围绕环组装。结构？TuSC戒指？将测定与MT或1层非聚合酵母微管蛋白结合的TuRC，并与来自cryoEM断层扫描的原位加帽MT减去末端的结构进行比较。2)Spc 110/72促进组装的机制？TuSC环和磷酸化调节：我们知道Spc 110稳定酵母的形成？TuSC环。我们将使用一种新开发的FRET检测，以有效地测量环组装在体外，并确定什么样的Spc 110和Spc 72的结构域所需的组装和Spc 110/72磷酸化的作用。3)酵母激活成核？TuSC环：虽然必要，但组装成环不足以实现有效的MT成核，两者都需要？TuSC闭合以匹配MT对称性和变构激活。将确定PTM或其他结合伴侣在此过程中的作用。4)中心体的结构组织-非？-微管蛋白组分：我们的基底体cryoEM断层扫描显示了装饰三联体的新的非微管蛋白结构。我们提出了SIM和STORM显微镜和cryoTomography的组合，以确定这些新结构的分子身份。我们的SIM/STORM成像揭示了中心体的中心粒周围物质中意想不到的结构域。我们继续这些努力评估的相互作用和组装，包括结构分析的PLP。

DESCRIPTION (provided by applicant): Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between ¿¿- and ?-tubulin complexes, regulatory proteins, and the centrosomal matrix. Here we focus on determining the molecular mechanism of microtubule (MT) nucleation, and understanding the assembly and regulation of the nucleating machinery. Our laboratory is uniquely poised to use a hierarchy of structural approaches (x-ray crystallography, cryoEM single particle reconstruction, cryoEM Tomography) to determine the structures of ?-tubulin complexes in vitro and in situ, and to understand their mechanism of action through functional studies and innovative kinetic modeling. Previously we discovered that yeast ?-tubulin small complex (?TuSC ) can assemble into rings and obtained a 6.5A cryoEM structure of the rings, explaining the origins of MT 13-fold symmetry and discovering an unexpected mode of regulation and assembly. The proposed experiments expand upon these results, providing a detailed understanding of the physical origins of MT nucleation, and the cellular machinery that dictates it. Specifically we address the following questions: 1) Determine the structure of yeast and Drosophila ring complexes and their interactions with MTs: We will improve the resolution of our yeast ?TuSC rings and will generate a complete pseudo-atomic structure. We will extend the resolution (goal ~ 1nm) of our preliminary of cryoEM structure (3.5nm) of isolated Drosophila ?TuRCs (2.2MDa ?-tubulin ring complex) and identify how the different GCPs assemble around the ring. Structures of ?TuSC rings ?TuRCs bound to MTs or 1 layer of non-polymerizing yeast ¿¿-tubulin will be determined and compared to structures of in situ capped MT minus ends from cryoEM tomography. 2) Mechanism of Spc110/72 facilitated assembly of ?TuSC rings and regulation by phosphorylation: We know that Spc110 stabilizes formation of yeast ?TuSC rings. We will use a newly developed FRET assay to efficiently measure ring assembly in vitro and determine what domains of Spc110 and Spc72 are required for assembly and the role of Spc110/72 phosphorylation. 3) Activation of nucleation by yeast ?TuSC rings: While necessary, assembly into rings is insufficient for potent MT nucleation, with a need for both ?TuSC closure to match MT symmetry and an allosteric activation. The role of PTMs or other binding partners in this process will be determined. 4) Structural organization of centrosomes - role of non ?-tubulin components: Our cryoEM tomography of basal bodies revealed new non-tubulin structures decorating the triplets. We propose a combination of SIM and STORM microscopy and cryoTomography to determine the molecular identity of these novel structures. Our SIM/STORM imaging has revealed unexpected structural domains within the centrosomal pericentriolar material. We continue these efforts assessing interactions and assembly including a structural analysis of Plp.