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αβ-和γ-微管蛋白复合物，调节蛋白和中心体基质之间所有相关结构和功能相互作用的原子分辨率图像。本文主要研究微管（MT）成核的分子机制，了解成核机制的组装和调控。我们的实验室是唯一准备使用层次结构方法（x射线晶体学，低温电子显微镜单粒子重建，低温电子显微镜断层扫描）来确定体外和原位γ-微管蛋白复合物的结构，并通过功能研究和创新的动力学建模来了解其作用机制。此前我们发现酵母γ-微管蛋白小复合物（γTuSC）可以组装成环状，并获得了6.5A的低温电镜结构，解释了MT 13倍对称的起源，并发现了一种意想不到的调节和组装模式。提出的实验扩展了这些结果，提供了MT成核的物理起源的详细理解，以及决定它的细胞机制。具体来说，我们解决了以下问题：1)确定酵母和果蝇环复合物的结构及其与MTs的相互作用：我们将提高酵母γTuSC环的分辨率，并生成完整的伪原子结构。我们将扩大我们分离的果蝇γ- turcs （2.2MDa γ-微管蛋白环复合物）的初步低温电镜结构（3.5nm）的分辨率（目标~ 1nm），并确定不同的gcp如何在环周围组装。与MT或1层非聚合酵母αβ-微管蛋白结合的γ - tusc环γ - turc的结构将被确定，并与冷冻电镜断层扫描中原位覆盖的MT负端结构进行比较。2) Spc110/72促进γTuSC环组装和磷酸化调控的机制：我们知道Spc110稳定酵母γTuSC环的形成。我们将使用新开发的FRET实验来有效地测量体外环组装，并确定Spc110和Spc72的哪些结构域是组装所必需的，以及Spc110/72磷酸化的作用。3)酵母γTuSC环激活成核：虽然有必要，但组装成环不足以实现有效的MT成核，需要γTuSC闭合以匹配MT对称性和变构激活。ptm或其他绑定伙伴在此过程中的作用将被确定。4)中心体的结构组织-非γ-微管蛋白成分的作用：我们对基底体的低温断层扫描发现了新的非微管蛋白结构修饰三联体。我们建议结合SIM和STORM显微镜和冷冻断层扫描来确定这些新结构的分子身份。我们的SIM/STORM成像显示了中心体中心周围物质中意想不到的结构域。我们继续这些努力评估相互作用和组装，包括Plp的结构分析。

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 αβ- and γ-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 γ-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 γ-tubulin small complex (γTuSC ) 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 γTuSC 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 γTuRCs (2.2MDa γ-tubulin ring complex) and identify how the different GCPs assemble around the ring. Structures of γTuSC rings γTuRCs bound to MTs or 1 layer of non-polymerizing yeast αβ-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 γTuSC rings and regulation by phosphorylation: We know that Spc110 stabilizes formation of yeast γTuSC 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 γTuSC rings: While necessary, assembly into rings is insufficient for potent MT nucleation, with a need for both γTuSC 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 γ-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.