Assembling the Phragmoplast Microtubule Array

研究植物细胞如何分裂的分子机制细胞通过复制它们的基因组内容，然后将基因组分裂成两个相同的子细胞进行繁殖。分裂的最后一步是胞质分裂，即两个女儿在身体上相互分离；它的成功对所有有机体的正常生长和发育以及繁殖都是绝对必要的。来自不同王国的真核生物已经发展出了细胞质分裂机制，这些机制具有某些共同的特征，但在其他方面又有所不同。在高级绿藻和植物中，细胞质分裂是由一种名为膜质的复杂装置实现的，它包含一个结构微管的核心框架。成纤维细胞微管作为运输用于组装细胞板的小泡的轨道，该结构在物理上分隔两个子细胞。当发育中的细胞板向外向细胞皮质扩张时，微管经历快速重塑。成膜体被认为是使陆地植物在地球上出现的进化里程碑，但人们对植物细胞如何组装这种结构知之甚少。为了了解植物的细胞质分裂，本项目将剖析调控膜质体内微管重组的机制。这项工作不仅将促进植物细胞分裂的知识，还将阐明真核细胞如何利用基于蛋白质的机械来完成复杂的任务。在这里获得的知识将适用于所有植物，并有助于理解海洋绿藻在进化过程中如何转变为陆地植物。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&amp；Co-Pi强调培养本科生，他们的好奇心和研究天赋因GPA相对较低而黯然失色。拟议项目的影响将扩大代表性不足群体的参与，并旨在使他们在毕业后接受高级培训，或使他们在学术界和生物技术行业寻找工作时具有技术竞争力。由于其先进的遗传学/基因组学和发达的细胞生物学工具，Pi的团队使用芥菜植物拟南芥作为模式系统来剖析细胞质分裂。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系，为拟议的实验做好了准备。以前的研究成果已经导致了一种模型假设，即成膜体是以模块的形式组装的，其中交错的微管的核心被非交错的微管包围。目前的项目将测试这一模块化模型，以了解该装置如何组装以执行胞质分裂，并在细胞分裂完成时拆开。具体地说，该项目将研究作用于膜质体内微管+末端的蛋白质，并整合它们的功能，以生成动态阵列。这项拟议的工作有两个目标：1)建立成纤维细胞微管组织的定量模型。将致力于利用最先进的活细胞成像技术来分析膜质微管阵列的动力学。研究将使用一种可光切换的微管标记来测试微管是否在膜质体内发生移位。可用的突变体将被用来测试微管捆绑蛋白MAP65-3、运动激动素-12和激酶MPK4在阵列组装和拆卸中的特定功能。这些研究的结果将被整合到一个定量模型中，重述细胞质分裂过程中微管的动力学和重组。2)揭示MAP65-4在成纤维细胞中的新功能。该项目将通过免疫荧光和活细胞成像在转基因品系中检查MAP65-4的定位。随后将进行遗传学实验，以测试MAP65-3和MAP65-4在膜质体内的任何冗余功能。这些研究的结果将促进我们对反平行微管阵列如何建立的了解。总的来说，该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