Assembling the Phragmoplast Microtubule Array

植物细胞如何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进行繁殖。分裂的最后一步是细胞质分裂，两个子细胞在物理上彼此分离；它的成功对所有生物的正常生长和发育以及繁殖都是绝对必要的。来自不同王国的真核生物已经发展出细胞分裂机制，这些机制具有共同的某些特征，但在其他方面有所不同。在高级绿藻和植物中，细胞分裂是由一种叫做片质体的复杂装置带来的，它包含一个结构微管的核心框架。膜质体微管作为轨道运输用于组装细胞板的囊泡，细胞板是物理上分离两个子细胞的结构。当发育中的细胞板向外扩展到细胞皮层时，微管经历快速重塑。膜质体被认为是地球上陆地植物出现的一个进化里程碑，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分裂，本项目将剖析调控膜质体微管重组的机制。这项工作不仅将推进植物细胞分裂的知识，而且还将阐明真核细胞如何利用基于蛋白质的机制来完成复杂的任务。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过渡到陆地植物。来自加州大学戴维斯分校校区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强调培养那些在研究方面的好奇心和天赋被相对较低的gpa所掩盖的本科生。拟议项目的影响将扩大代表性不足的群体的参与，并旨在使他们在毕业时接受高级培训，或使他们在寻找学术界和生物技术行业的工作时具备技术竞争力。PI的小组使用芥菜植物拟南芥作为模型系统来解剖细胞动力学，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，使系统为拟议的实验做好准备。先前的研究成果已经产生了一个模型，假设片质体是以模块形式组装的，其中交叉的微管核心被非交叉的微管包围。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细胞分裂，并在细胞分裂完成时拆开的。具体来说，该项目将检查在膜质体微管+端起作用的蛋白质，并整合它们的功能，以产生动态阵列。提出的工作有两个目标：1)建立膜内微管组织的定量模型。努力将致力于通过最先进的活细胞成像技术分析膜质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易位是否发生在膜质体中。可用的突变体将被用来测试微管捆绑蛋白MAP65-3、马达激酶激酶激酶激酶激酶激酶激酶激酶激酶激酶激酶激酶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，重现细胞分裂过程中的微管动力学和重组。2)揭示MAP65-4在phragmoplast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测MAP65-4的定位。后续的遗传实验将检测MAP65-3和MAP65-4在片质体中是否存在冗余功能。这些研究的结果将促进我们对如何建立反平行微管阵列的认识。总体而言，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