Cytoskeletal Regulation of Cell Wall Biosynthesis and Cell Morphogenesis

智力上的功绩。植物以不同的形状构建细胞，这对特殊的细胞功能和植物本身的结构都是重要的。为了创造这些形状，植物细胞必须组织包裹它们的坚硬墙壁的分子结构和机械性能。一种被称为皮质微管细胞骨架的高度有序的蛋白质聚合物网络被用来组织细胞壁和建立特定的细胞形状。皮质微管细胞骨架已被证明：(1)指导纤维素合成酶复合体沉积纤维素(细胞壁的主要结构成分)的轨迹；(2)将这些复合体的传递定位到它们在细胞膜上的作用部位；(3)在胁迫条件下，在细胞的皮质中系留含有纤维素合成酶的细胞器。微管细胞骨架执行这些活动的分子机制尚不清楚，所有这些过程在细胞生长和形态发生中的确切生物学功能也不是很清楚。为了研究这些问题，Ehrhardt实验室设计了一种突变株的遗传筛查，这些突变株在细胞皮质和细胞膜的纤维素合成酶及其组织的运输方面存在缺陷。这一屏幕显示了一类在CSC引导方面有缺陷的突变体，他们失去了皮质拴系，即丢失的突变体。这些突变体是令人兴奋的工具，可以发现参与细胞骨架功能的新分子，并对组织细胞壁的相互作用网络有新的见解。该项目有三个主要的实验目标：(1)通过在亚细胞蛋白质和细胞器动力学、细胞和组织生长以及细胞壁结构水平上表征这些突变体来研究功能丧失；(2)识别这些基因并确定蛋白质产物在细胞中的位置和它们的行为；以及(3)识别其他与丢失的蛋白质相互作用的蛋白质，以建立从微管细胞骨架到细胞壁生物合成机制的分子相互作用和/或调控网络。这些研究将为植物细胞生物学中的几个核心问题提供新的见解：皮质细胞骨架的功能，细胞形态发生的机制，以及纤维素和细胞壁的生物合成和组织机制-生物燃料和生物材料研究的主要目标。更广泛的影响。把研究和教育结合起来。该项目的研究活动将有助于培养一名年轻的博士后科学家，并将使几名高中生第一手接触到基础研究。这些学生将参加卡内基暑期实习计划，通过卡内基教职员工和研究人员每周一次的研究讲座，并让他们有机会在公共论坛上展示他们的发现，来增强学生的体验。这项研究的材料将用于斯坦福大学的高级成像课程。本课程为学生提供制造激光陷阱和TIRF显微镜等先进仪器的实践经验。对科学方面代表性不足的群体进行培训。暑期研究助理将从当地有相当多少数族裔代表的高中招聘。其他外展活动包括与只培训本科生的部门和机构的研究人员建立合作。研究的交流。研究结果将被纳入Ehrhardt实验室的网站Deepgreen.stanford.edu。这个网站被世界各地的几所大学用来进行课堂教学。该实验室的图像和结果此前已被纳入主要教科书。基础设施建设。Ehrhardt实验室已经为植物细胞和相关的分子资源建立了一个荧光蛋白质标记的数据库。这项研究产生的材料将被添加到这个数据库中，并由实验室的网站和拟南芥库存中心分发。其他社会福利。纤维素为陆地植物提供机械支持的基础，也是光合作用输出的主要汇。控制纤维素生物合成和细胞壁组织的分子机制与作物改良、生物材料工程和生物燃料开发有关。

Intellectual Merit. Plants build cells in a diverse range of shapes that are important both for specialized cell function and for creating the structure of the plant itself. To create these shapes, plant cells must organize the molecular structure and mechanical properties of the rigid walls that encase them. A highly ordered network of protein polymers known as the cortical microtubule cytoskeleton is employed to organize cell walls and to build specific cell shapes. The cortical microtubule cytoskeleton has been shown to (1) guide the trajectories of cellulose synthase complexes as they deposit cellulose, the major structural component of the cell wall; (2) position the delivery of these complexes to their sites of action at the cell membrane; and (3) tether trafficking organelles containing cellulose synthase in the cortex of the cell during conditions of stress. The molecular mechanisms by which the microtubule cytoskeleton carries out these activities are not known, and the precise biological functions of all these processes in cell growth and morphogenesis are not well understood. To investigate these questions the Ehrhardt lab designed a genetic screen for mutants that are defective in trafficking of cellulose synthase and its organization at the cell cortex and cell membrane. This screen revealed a class of mutants that are defective in CSC guidance and have lost cortical tethering, the lost mutants. These mutants are exciting tools to discover new molecules involved in cytoskeletal function, and to get new insight into the network of interactions that organize the cell wall. This project has three major experimental aims: (1) to investigate LOST function by characterizing these mutants at the levels of subcellular protein and organelle dynamics, cell and tissue growth, and cell wall structure; (2) to identify these genes and determine where the protein products reside in the cell and how they behave, and (3) to identify other proteins that interact with LOST proteins to build a molecular interaction and/or regulatory network from the microtubule cytoskeleton to the cell wall biosynthetic machinery. These studies will provide new insight into several central questions in plant cell biology: the function of the cortical cytoskeleton, the mechanisms of cell morphogenesis, and the mechanisms for biosynthesis and organization of cellulose and the cell wall - major targets of biofuel and biomaterials research. Broader Impacts. Integrating research and education. The research activities in this project will contribute to the training of a young post doctoral scientist and will give several high school students first hand exposure to basic research. These students will participate in the Carnegie Summer Internship Program, enhancing the student's experience through weekly research talks by Carnegie faculty and researchers and by giving them an opportunity to present their findings in a pubic forum. Materials from this research will be used in Stanford's Advanced Imaging Course. This course gives students hands on experience in building advanced instruments like laser traps and TIRF microscopes. Training of under represented groups in science. Summer research assistants will be recruited from local high schools with substantial minority representation. Other outreach activities include establishing collaborations with researchers in departments and institutions that only train undergraduates. Communication of research. Research results will be incorporated into the Ehrhardt lab website, deepgreen.stanford.edu. This site is used for class instruction at several universities around the world. Images and results from the lab have previously been incorporated into major textbooks. Infrastructure development. The Ehrhardt lab has generated a database of fluorescent protein markers for plant cells and the associated molecular resources. Materials generated by this research will be added to this database and distributed by the lab's website and by the Arabidopsis stock center. Other societal benefits. Cellulose provides the basis for mechanical support of terrestrial plant life and is the major sink for photosynthetic output. The molecular mechanisms that govern cellulose biosynthesis and cell wall organization have relevance for crop improvement, engineering of biomaterials, and biofuel development.