Eyespot Placement and Assembly in Chlamydomonas

衣藻眼点的放置和组装

• 批准号: 0843094
• 负责人: Carol Dieckmann
• 金额: $ 57.12万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843094&HistoricalAwards=false
• 关键词: Eyespot; Placement; Assembly; Chlamydomonas

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The intellectual merit of this project is to understand cellular asymmetry and membrane adhesion, two processes that are not well understood and which are crucial for eukaryotic cells. Many structures, including the mitotic spindle, nuclear pore complexes, and Golgi must be assembled asymmetrically at every cell division. Regulated adhesion between membranes is not fully understood in many cellular systems including vesicle trafficking, synaptogenesis, and fertilization. This project is focused on understanding the asymmetric arrangement of the microtubule cytoskeleton, and the asymmetric placement and complex assembly of the eyespot in the simple eukaryotic alga, Chlamydomonas. The eyespot senses light intensity and direction and sends signals to the paired flagella, which change their breast stroke wave form to turn the cell toward or away from light. As viewed by light microscopy, the eyespot is an orange-red spot at the equator of the cell with the pole of the cell marked by the location of the flagella. The eyespot is made up of a layered array of four membranes and carotenoid-filled granules. The plasma membrane contains rhodopsin photoreceptors. The envelope membranes of the single, large chloroplast are tightly apposed to the plasma membrane in the region of the eyespot. The carotenoid granules are packed against the envelope membrane in an ordered array and are subtended by thylakoid membranes. There are multiple layers of granules and thylakoids in the Chlamydomonas reinhardtii eyespot. The eyespot is completely reassembled anew at every cell division and is placed uniquely in the cell relative to the microtubule cytoskeleton. A cruciate structure of microtubule rootlets emanates from the region of the basal bodies at the base of the flagella and extends down to the equator of the cell. A two-membered and a four-membered rootlet are paired with the parental and with the daughter basal body. The eyespot is always placed adjacent to the daughter four-membered rootlet as viewed from the flagellar pole. Four genes have been identified that affect assembly and placement of the eyespot and are required for wild-type phototaxis. eye2 and eye3 mutant cells do not have eyespots, min1 mutants have miniature eyespots and mlt1mutants have two eyespots. The EYE2 protein has thioredoxin domain and LysM domains. The MIN1 protein contains a C2 domain, which, in other proteins is responsible for phospholipid binding that can be calcium-dependent, and a LysM domain. The EYE3 protein sequence encodes a homolog of a serine-threonine kinase that regulates ubiquinone biosynthesis. The MLT1 protein codes for a protein with low sequence complexity. The objectives of this project are to: 1) determine whether the microtubule rootlet system is acetylated asymmetrically, if that asymmetry is fixed relative to the cleavage furrow and mother flagellum, and how the eyespot is positioned relative to the rootlets; 2) to alter rootlet acetylation genetically or pharmaceutically and observe the affect on rootlet and eyespot positioning and function; 3) to investigate the dependence/independence of eyespot granule placement on photoreceptor and rootlet localization; and 4) to investigate the function of EYE2, MIN1, EYE3 and MLT1 proteins in eyespot assembly and placement. The broader impacts of this work are: 1) to include the participation of a small college professor, who will share her experiences with undergraduates in her laboratory during the academic year and alternate summers, 2) to participate in the NSF REU and other undergraduate research programs at the University of Arizona, and 3) to continue outreach lab tours and high school projects in K-12.

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。该项目的智力价值是了解细胞的不对称性和膜粘附，这两个过程还没有得到很好的理解，这对真核细胞至关重要。许多结构，包括有丝分裂纺锤体，核孔复合体和高尔基体必须在每次细胞分裂时不对称地组装。在许多细胞系统中，包括囊泡运输、突触发生和受精，膜之间的调节粘附尚未完全理解。该项目的重点是了解微管细胞骨架的不对称排列，以及简单的真核细胞衣原体中眼点的不对称放置和复杂组装。眼点感知光线的强度和方向，并向成对的鞭毛发送信号，鞭毛改变它们的蛙泳波形，使细胞朝向或远离光线。通过光学显微镜观察，眼点是位于细胞赤道的橙红色斑点，细胞的极点由鞭毛的位置标记。眼斑由四层膜和填充类胡萝卜素的颗粒的分层阵列组成。质膜含有视紫红质光感受器。单个大叶绿体的被膜在眼点区域与质膜紧密贴壁。类胡萝卜素颗粒以有序的阵列排列在被膜上，并被类囊体膜包着。莱茵衣藻眼斑内有多层颗粒和类囊体。眼点在每次细胞分裂时完全重新组装，并且相对于微管细胞骨架在细胞中被独特地放置。十字形结构的微管根从鞭毛基部的基体区域发出，向下延伸到细胞的赤道。一个两元和四元的小根与父母和女儿基体配对。从鞭毛极观察，眼斑总是位于子四元小根附近。四个基因已被确定，影响装配和安置的眼点和所需的野生型趋光性。Eye 2和Eye 3突变体细胞没有眼斑，Min 1突变体有小眼斑，而MLT 1突变体有两个眼斑。EYE 2蛋白具有硫氧还蛋白结构域和LysM结构域。MIN 1蛋白含有C2结构域和LysM结构域，在其他蛋白中，C2结构域负责钙依赖性的磷脂结合。EYE 3蛋白序列编码调节泛醌生物合成的丝氨酸-苏氨酸激酶的同源物。MLT 1蛋白编码具有低序列复杂性的蛋白。本研究的目的是：1）确定微管根系统是否被不对称乙酰化，这种不对称性相对于裂沟和母鞭毛是否是固定的，以及眼斑相对于根的位置如何：2）通过遗传或药物改变根的乙酰化，观察对根和眼斑的位置和功能的影响;研究EYE 2、MIN 1、EYE 3和MLT 1蛋白在眼斑组装和定位中的作用。这项工作的更广泛的影响是：1）包括一个小的大学教授的参与，谁将分享她的经验与本科生在她的实验室在学年和交替的夏天，2）参加美国国家科学基金会REU和其他本科生在亚利桑那大学的研究计划，和3）继续推广实验室图尔斯旅游和高中项目在K-12。