Microtubule Organization by Kinesin-12 in the Phragmoplast

知识的优点。本项目致力于了解植物细胞分裂机制。植物细胞通过在两个子细胞核之间放置一个称为细胞板的物理屏障进行分裂。为了实现这一目标，高级绿藻和陆生植物生物已经发展出一种共同的细胞动力学装置，称为片质体，这是一个关键的进化里程碑。膜质体是由微管的细胞骨架元素的框架建立的，微管与未来的细胞板垂直排列。微管是动态极性细丝，其正端比负端表现出更快的生长能力。膜质体微管被组织成双极阵列，其正极位于分裂位点或附近。高尔基体产生的囊泡被运送到微管正端，导致细胞板的组装。微管是如何组织成这种高度有序的形式是这个项目在未来三年要解决的核心问题。许多研究表明，微管相关蛋白和基于微管的运动激酶是膜质体中微管组织的关键参与者。我们一直以芥蓝植物拟南芥为研究对象，发现三个激酶蛋白超家族的激酶蛋白-12成员只出现在发育中的片质体的微管+端，并在微管组织中发挥关键作用。计划中的研究旨在验证这样一种假设，即Kinesin-12马达及其结合蛋白形成马达复合物，以调节隔膜微管的双极性组织。为了验证这一假设，第一个目标是应用遗传学方法来解剖三个Kinesin-12马达如何在膜质体中协同工作。第二个目标是通过使用生化、细胞生物学和遗传学工具来了解两种驱动蛋白-12结合蛋白的功能。最后一个目标是了解Kinesin-12的功能如何与微管相关蛋白MAP65-3的功能联系在一起。即将取得的发现将提供在膜质体微管+端蛋白质-蛋白质相互作用的图像。更广泛的影响。在这个项目中，模式生物拟南芥的使用将带来对在陆地植物进化过程中负责层质体发明的分子机制的见解。实验结果将在期刊刊物上公布。它们还将被翻译成讲座材料，供250多名本科生在本项目首席研究员每年讲授的细胞生物学课程中使用。同时，参与的研究生接受生物化学、细胞生物学和遗传学领域的跨学科培训。此外，该项目通过为高中生和本科生提供在实验室的动手研究经验来吸引他们。通过直接为发现做出贡献，这些学生分享了可能激励他们从事科学事业的兴奋。

Intellectual merits. This project is devoted to understanding cell division mechanisms in plants. Plant cells divide by laying down a physical barrier called the cell plate between two daughter nuclei. In order to achieve this goal, organisms of advanced green algae and land plants have developed a common cytokinetic apparatus named the phragmoplast which is a key evolutionary landmark. The phragmoplast is established by a framework of the cytoskeletal element of microtubules, which are aligned perpendicularly to the future cell plate. Microtubules are dynamic polar filaments with their plus ends exhibiting more rapid growth capability than their minus ends. Phragmoplast microtubules are organized into a bipolar array with their plus ends located at or near the division site. Vesicles generated by the Golgi apparatus are transported toward microtubule plus ends, resulting in the assembly of the cell plate. How microtubules are organized into this highly ordered form is the central question being addressed in this project for the next three years. A number of studies suggest that microtubule-associated proteins and the microtubule-based motor kinesins are critical players for microtubule organization in the phragmoplast. We have been using the cress plant Arabidopsis thaliana as a model for our studies, and have discovered that three Kinesin-12 members of the kinesin superfamily exclusively appear at microtubule plus ends in the developing phragmoplast, and play a critical role in microtubule organization. The planned studies are aimed at testing the hypothesis that the Kinesin-12 motors and their binding proteins form a motor complex to regulate the bipolar organization of phragmoplast microtubules. In order to test this hypothesis, the first objective is to apply genetic approaches to dissect how the three Kinesin-12 motors work together in the phragmoplast. The second objective is to understand functions of two Kinesin-12-binding proteins by using biochemical, cell biological, and genetic tools. The last objective focuses on understanding how the function of Kinesin-12 is linked to that of the microtubule-associated protein MAP65-3. Discoveries to be made are going to provide a protein-protein interactive picture at microtubule plus ends in the phragmoplast.Broader impacts. In this project, the use of the model organism Arabidopsis thaliana will bring insights into molecular mechanisms that are responsible for the invention of the phragmoplast during the evolution toward land plants. Experimental results will be made public by journal publications. They will also be translated into lecture materials to reach 250+ undergraduate students in a Cell Biology class taught each year by the Principal Investigator of this project. In the meantime, the participating graduate student receives interdisciplinary training in areas of biochemistry, cell biology and genetics. In addition, the project engages high school students and undergraduate students by providing them with hands-on research experience in the laboratory. By directly contributing to discoveries, these students share the excitement that could inspire them to pursue a career in science.