A Microtubule Interactome: Complexes with Roles in Cell Cycle and Mitosis

The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.微管（microtubule, MT）细胞骨架参与诸多细胞生命活动过程，包括胞内物质运输、细胞极性维持以及有丝分裂调控。上述功能由微管相关蛋白（MT-associated proteins, MAPs）协同调控，这类蛋白可结合微管并改变其自身特性，且彼此间相互配合。我们采用微管共沉淀测定法，结合一维（1D）与二维（2D）聚丙烯酰胺凝胶电泳（PAGE）及质谱分析，从早期果蝇胚胎中鉴定出超过250种微管相关蛋白。针对本研究中通过该方法分离得到的新型微管相关蛋白，我们采用两种互补策略分析其细胞功能：其一，开展RNA干扰（RNA interference, RNAi）筛选，鉴定出21个此前未被表征的、参与微管组织的基因；其二，基于二元蛋白质相互作用数据进行生物信息学分析，构建微管相关蛋白的潜在相互作用网络。通过整合两种研究策略，我们鉴定并验证了可能在细胞周期调控与有丝分裂中发挥重要作用的微管相关蛋白复合物。本研究证实，采用此类多学科方法可获取具有生物学意义的研究数据，并鉴定出一批新的微管相关蛋白，其中多数似乎在细胞分裂过程中发挥关键作用。