微管细胞骨架在细胞分裂及胞内运输等许多基本细胞生命活动过程发挥关键功能。微管形成的最初步骤微管成核极其迅速、涉及多蛋白质协同调控、在细胞内不同部分发生，因此深入进行其分子机制研究具有许多挑战。目前，微管成核的调控机制仍然不甚清楚，是细胞生物学研究的热点问题之一。近期，我们在研究进化上保守的非中心体微管成核关键调控蛋白mto1p/CDK5RAP2时，鉴定到其新的结合蛋白rsp1p，并发现rsp1p协同驱动蛋白klp2p通过mto1p/CDK5RAP2调控非中心体微管成核。为此，本项目将以mto1p/CDK5RAP2为切入点，通过综合利用包括高分辨率激光共聚焦活细胞显微镜观察等多种技术手段，在裂殖酵母和人类细胞系中研究进化上保守的蛋白rsp1p与mto1p和klp2p的互作机制及其非中心体微管成核的具体调控功能。本项目的研究成果将使人们深刻理解非中心体微管成核过程的分子调控机理。

The microtubule cytoskeleton plays important roles in many fundamental cellular events including cell division and vesicle/organelle trafficking. The initial step of microtubule assembly is microtubule nucleation, taking place rapidly at multiple cellular compartments and being mediated by a large number of proteins. These post a challenge on investigating the molecular mechanisms underlying microtubule nucleation. While taking this challenge, we identified an evolutionarily conserved J-domain protein rsp1p as a binding protein of mto1p, the known protein responsible for initiating microtubule nucleation, and further identified the kinesin klp2p as a binding protein of rsp1p. Preliminary functional characterization suggested that klp2p, rsp1p, and mto1p work in concert to orchestrate microtubule nucleation. In this proposal, we will employ a combination of molecular, cell biological, and biochemical approaches and live-cell microscopy to investigate the interplay among klp2p, rsp1p, and mto1p, as well as their human homologs, in microtubule nucleation. Since the human homolog of mto1p, CDK5RAP2, is associated with microcephaly, this work will not only provide molecular insights into microtubule nucleation, but also pave the way for understanding the pathophysiology of microcephaly.