How does MICROTUBULE ORGANIZATION 1 (MOR1) regulate microtubule dynamics in Arabidopsis thaliana?

所有真核细胞都依赖于被称为微管(MTS)的蛋白质细丝的动态阵列来进行细胞内运输、细胞形状和分裂。MTS的快速重排需要XMAP215/Dis2家族的微管相关蛋白的活性，这是一类高度保守的蛋白质。这些蛋白质同时促进MT的生长和收缩，并随着MT末端的连续移动，从而能够催化多种反应。和其他XMAP215/Dis2成员一样，植物同源基因MOR1预计会形成一个具有多个N端TOG结构域的长线性分子。正向遗传学筛查中发现的导致MT动力学随温度变化的MOR1突变等位基因，始终只替换第一个TOG结构域中的保守氨基酸，表明这个N-末端结构域对聚合酶活性至关重要。因此，我们打算专门测试这一假设，即第一个TOG结构域在将游离微管蛋白二聚体募集到MTS的生长正端起着至关重要的作用。我们将利用高等植物模型系统拟南芥来阐明使XMAP215/Dis2家族蛋白作为进程聚合酶发挥作用的分子机制。通过结合体外聚合分析、单分子和活细胞成像，我们将比较携带特定点突变和结构域缺失的基因工程MOR1构建。这些发现将激发对真核细胞功能至关重要的过程的新想法和理解。

All eukaryotic cells depend on dynamic arrays of protein filaments called microtubules (MTs) for intracellular transport, cell shape and division. Rapid rearrangement of MTs requires the activity of microtubuleassociated proteins of the XMAP215/Dis2 family, a highly conserved class of proteins. These proteins promote both MT growth and shrinkage and move processively with the MT ends, thus enabling catalysis of multiple reactions. Like other XMAP215/Dis2 members, the plant homologue MOR1 is predicted to form a long linear molecule with multiple N-terminal TOG domains. Mutant alleles of MOR1 identified in forward genetics screens that cause temperature-dependent loss of MT dynamics, consistently substitute conserved amino acids in only the first TOG domain, suggesting that this N-terminal domain is crucial for polymerase activity. We therefore intend to specifically test the hypothesis that the first TOG domain plays a crucial role in recruitment of free tubulin dimers to the growing plus end of MTs. We will utilize the higher plant model system Arabidopsis thaliana to elucidate the molecular mechanisms that enable XMAP215/Dis2 family proteins to work as processive polymerases. By combining in vitro polymerization assays, single molecule and live cell imaging, we will compare genetically engineered MOR1 constructs carrying specific point mutations and domain deletions. These findings will stimulate new ideas and understanding of a process that is central to eukaryotic cell function.