Regulation of microtubule dynamics by a protein interaction network at microtubule ends consisting of EB1, CLIP-170, XKCM1 and XMAP215 - an in vitro reconstitution

微管动力学的调节对于真核细胞的内部组织至关重要。几种微管相关蛋白调节微管的动力学，并且已知对于细胞和发育生物学中的基本过程至关重要，例如适当的细胞分裂或细胞极性的建立。越来越多的证据表明，这些调节剂在动态微管末端形成蛋白质相互作用网络。直到今天，人们还不清楚这种调节微管功能的网络是如何作为一个系统发挥作用的。最近，末端结合蛋白1（EB1）已被确定为该网络的枢纽，使用体外重建的方法。这为研究EB 1如何控制该网络中微管动力学的其他调节因子提供了可能性。我们将在体外重建这个调控网络的核心。我们将集中在阐明的细胞周期依赖控制这个网络的活动的分子机制。生物化学方法和定量时间分辨荧光显微镜的组合下降到单分子水平将被用来分析在一个最小的和良好的控制系统的微管动力学的生理调节的规则。这项工作将导致在系统水平上对动态蛋白质相互作用网络的功能的分子理解。

The regulation of microtubule dynamics is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins modulate the dynamics of microtubules and are known to be crucial for fundamental processes in cell and developmental biology, such as proper cell division or the establishment of cell polarity. There is increasing evidence that these regulators form a protein interaction network at dynamic microtubule ends. Until today it is not understood how this network regulating microtubule function acts as a system. Recently, end binding protein 1 (EB1) has been identified as the hub of this network using an in vitro reconstitution approach. This opens now the possibility to study how EB1 controls the other regulators of microtubule dynamics in this network. We will reconstitute here the core of this regulatory network in vitro. We will focus on the elucidation of the molecular mechanism of cell cycle dependent control of the activity of this network. A combination of biochemical methods and quantitative time-resolved fluorescence microscopy down to the single molecule level will be used to analyze in a minimal and well-controlled system the rules underlying physiological regulation of microtubule dynamics. This work will lead to a molecular understanding of the functioning of a dynamic protein interaction network at a systems level.