Assembling the Phragmoplast Microtubule Array

植物细胞分裂的分子机制细胞通过复制其基因组内容，然后将基因组分裂成两个相同的子细胞来繁殖。分裂的最后一步是胞质分裂，两个子细胞在物理上彼此分离;它的成功对于所有生物的正常生长和发育以及繁殖都是绝对必要的。来自不同界域的真核生物已经发展了胞质分裂机制，这些机制具有某些共同特征，但在其他方面有所不同。 在高级绿色藻类和植物中，胞质分裂是由一种称为成膜体的复杂装置引起的，该成膜体包含结构微管的核心框架。 成膜体微管作为运输用于组装细胞板的囊泡的轨道，细胞板是物理上分离两个子细胞的结构。 微管经历快速重塑，而发育中的细胞板向外扩展到细胞皮质。 成膜体被认为是地球上陆地植物出现的进化里程碑，但人们对植物细胞如何组装这种结构知之甚少。 为了了解植物胞质分裂，本计画将剖析成膜体中微管重组的调控机制。 这项工作不仅将推进植物细胞分裂的知识，而且还揭示了真核细胞如何利用蛋白质为基础的机器来完成复杂的任务。这里获得的知识将适用于所有植物，并帮助了解海洋绿色藻类如何在进化过程中转变为陆地植物。 来自加州大学戴维斯分校的本科生和高中生将加入这一发现，并接受现代细胞生物学的实践培训。 PI Co-PI强调培养本科生，他们的好奇心和研究天赋被相对较低的GPA所掩盖。& 拟议项目的影响将扩大代表性不足群体的参与，并旨在为他们提供毕业后的高级培训，或使他们在学术界和生物技术行业寻找工作时具备技术竞争力。 PI的研究小组使用芥菜植物拟南芥作为模型系统来剖析胞质分裂，因为它具有先进的遗传学/基因组学和发达的细胞生物学工具。作为初步工作，PI已经准备了必要的试剂、有洞察力的突变体和信息丰富的荧光标记线，以使系统为拟议的实验做好准备。 先前的成就已经导致了一个模型，假设成膜体以模块化形式组装，其中交错微管的核心被非交错微管包围。 目前的项目将测试这个模块化模型，以了解这个装置是如何组装来执行胞质分裂的，以及在细胞分裂完成后如何被拆开。 具体来说，该项目将检查在成膜体中作用于微管加末端的蛋白质，并整合它们的功能以生成动态阵列。 本工作有两个目标：1）建立成膜体微管结构的定量模型。本论文将致力于利用最先进的活细胞成像技术来分析成膜体微管阵列的动力学。 研究将使用光开关微管标记来测试微管移位是否发生在成膜体中。 可用的突变体将用于测试微管捆绑蛋白MAP65 - 3、马达驱动蛋白-12和激酶MPK 4在阵列的组装和拆卸中的特定功能。 从这些研究的结果将被整合到一个定量模型概括微管动力学和胞质分裂过程中的重组。 2)揭示MAP65 - 4在成膜体中的新功能。该项目将通过免疫荧光和活细胞成像在转基因系中检查MAP65 - 4的定位。 随后进行遗传实验以测试成膜体中MAP 65 - 3和MAP 65 - 4的任何冗余功能。 这些调查的结果将推进我们的知识如何建立反平行微管阵列。总的来说，这个项目将为本科生和研究生提供培训机会

Towards a molecular mechanism of how plant cells divideCells reproduce by duplicating their genomic content and then dividing the genome into two identical daughter cells. The final step of division is cytokinesis, where the two daughters are physically separated from each other; its success is absolutely essential for normal growth and development, as well as reproduction in all organisms. Eukaryotic organisms from different kingdoms have developed cytokinesis mechanisms that share certain features but differ in others. In advanced green algae and plants, cytokinesis is brought about by a sophisticated apparatus called the phragmoplast, which contains a core framework of structural microtubules. Phragmoplast microtubules serve as tracks that transport vesicles used to assemble the cell plate, the structure that physically separates the two daughter cells. Microtubules undergo rapid remodeling while the developing cell plate expands outward toward the cell cortex. The phragmoplast is considered to be an evolutionary landmark that enabled the emergence of land plants on earth, but little is known about how plant cells assemble this structure. In order to understand plant cytokinesis, this project will dissect mechanisms that regulate microtubule reorganization in the phragmoplast. This work will not only advance knowledge of plant cell division but also shed light on how eukaryotic cells harness protein-based machineries to accomplish sophisticated tasks. Knowledge obtained here will be applicable to all plants, and aid understanding of how marine green algae transitioned into land plants during evolution. Undergraduate and high school students from the UC Davis campus area will join the discovery, and receive hands-on training in modern cell biology. The PI & Co-PI emphasize training undergraduate students whose curiosity and talent in research are shadowed by their relatively low GPAs. The impacts of the proposed project will broaden participation of underrepresented groups, and aim for their advanced training upon graduation or prepare them to be technically competitive when seeking jobs in academia and the biotechnology industry. The PI's group uses the mustard plant Arabidopsis thaliana as a model system to dissect cytokinesis because of its advanced genetics/genomics and well developed cell biology tools. As preliminary work, the PI has prepared necessary reagents, insightful mutants, and informative fluorescent marker lines to make the system ready for the proposed experiments. Prior accomplishments have resulted in a model hypothesizing that the phragmoplast is assembled in modular form, where a core of interdigitating microtubules are surrounded by non-interdigitating ones. The current project will test this modular model to learn how this apparatus is assembled to execute cytokinesis and taken apart upon the completion of cell division. Specifically, the project will examine proteins that act at microtubule plus ends in the phragmoplast and integrate their functions in order to generate the dynamic array. The proposed work has two objectives: 1) Towards establishing a quantitative model of microtubule organization in the phragmoplast. Efforts will be devoted to analyzing the kinetics of the phragmoplast microtubule array by the state-of-the-art live-cell imaging technologies. Research will use a photoswitchable microtubule marker to test whether microtubule translocation takes place in the phragmoplast. Available mutants will be employed to test specific functions of the microtubule-bundling protein MAP65-3, the motor Kinesin-12, and the kinase MPK4 in the assembly and disassembly of the array. Results from these studies will be integrated into a quantitative model recapitulating microtubule dynamics and reorganization during cytokinesis. 2) To uncover the novel function of MAP65-4 in the phragmoplast. The project will examine MAP65-4 localization by immunofluorescence and live-cell imaging in transgenic lines. Genetic experiments will be followed to test for any redundant functions of MAP65-3 and MAP65-4 in the phragmoplast. The outcome of these investigations will advance our knowledge on how the anti-parallel microtubule array is established. Overall, this project will provide training opportunities for undergraduate and graduate students