Molecular mechanism of the selective recognition of growing microtubule ends by vertebrate +TIPs

微管细胞骨架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提供与染色体、膜细胞器或细胞皮层的连接。这些蛋白质的一个子类，即所谓的微管正端结合蛋白（TIP），具有选择性结合微管生长正端的显着特性。这些蛋白质具有多种重要功能。它们对于正确的细胞分裂至关重要，参与信号传导或负责细胞极性的建立。这些蛋白质的缺陷形式可能导致癌症。然而，这些蛋白质识别动态微管末端的分子机制以及它们如何影响微管动力学尚不清楚。这是由于微管正端追踪系统的复杂性造成的，该系统由体内几种相互作用的蛋白质组成。另一个问题是正端追踪无法在体外重建。在这里，我们将用几种脊椎动物 TIP 重建体外动态加端跟踪。使用生化技术和延时荧光显微镜直至单分子水平，我们将在最小且控制良好的系统中定量分析微管正端跟踪现象背后的分子相互作用和动力学。我们的目标是阐明相互作用的 TIP 之间的组合效应如何在分子水平上定义多组分正端跟踪系统的功能。这项工作将导致对显示复杂动态行为的动态蛋白质相互作用网络的功能的分子理解。

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins modulate the dynamics of microtubules and provide a link to chromosomes, membrane organelles or the cell cortex. A subclass of these proteins, the so-called microtubule plus-end binding proteins (+TIPs), have the remarkable property of selectively binding to the growing plus ends of microtubules. These proteins have several essential functions. They are crucial for proper cell division, involved in signalling or responsible for the establishment of cell polarity. Defective forms of these proteins can lead to cancer. The molecular mechanism, however, by which these proteins recognize the dynamic microtubule end and how they affect microtubule dynamics is not understood. This is a result of the complexity of the microtubule plus-end tracking system that consists of several interacting proteins in vivo. Another problem has been that plus-end tracking could not be reconstituted in vitro. Here we will reconstitute with several vertebrate +TIPs dynamic plus-end tracking in vitro. Using biochemical techniques and time-lapse fluorescence microscopy down to the single molecule level, we will analyze quantitatively in a minimal and well-controlled system the molecular interactions and the dynamics underlying the phenomenon of microtubule plus-end tracking. We aim at elucidating how combinatorial effects between mutually interacting +TIPs define the functioning of the multi-component plus-end tracking system at a molecular level. This work will lead to a molecular understanding of the functioning of a dynamic protein interaction network showing complex dynamic behavior.