Eyespot Placement and Assembly in Chlamydomonas

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。这个项目的智力价值在于理解细胞不对称和膜粘附，这两个过程不是很清楚，但对真核细胞至关重要。许多结构，包括有丝分裂纺锤体、核孔复合物和高尔基体在每次细胞分裂时必须不对称地组装。在包括囊泡运输、突触发生和受精在内的许多细胞系统中，膜间的调节粘附尚未完全了解。本项目的重点是了解简单真核藻类衣藻微管细胞骨架的不对称排列，以及眼斑的不对称放置和复杂组装。眼点感知光的强度和方向，并向成对的鞭毛发送信号，鞭毛改变它们的拍打波浪的形式，使细胞朝向或远离光。在光镜下，眼斑是细胞赤道上的一个橘红色斑点，细胞的两极是鞭毛的位置。眼点由四层膜和类胡萝卜素填充的颗粒组成。质膜含有视紫红质感光体。单个大叶绿体的包膜紧紧地贴在眼斑区域的质膜上。类胡萝卜素颗粒以有序的排列排列在包膜上，并由类囊体膜包裹。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独特地放置在细胞中。微管细根的十字形结构从鞭毛基部的基部区域发出，并向下延伸到细胞的赤道。二根和四根的根与亲本和与子基体配对。从鞭毛杆上看，眼斑总是与四根子根相邻。已经鉴定出四个基因影响眼点的组装和位置，并且是野生型趋光性所必需的。Eye2和eye3突变细胞没有眼点，min1突变细胞有微型眼点，mlt1突变细胞有两个眼点。EYE2蛋白具有硫氧还蛋白结构域和LysM结构域。MIN1蛋白含有一个C2结构域和一个LysM结构域，在其他蛋白质中，C2结构域负责钙依赖性磷脂结合。EYE3蛋白序列编码一种丝氨酸-苏氨酸激酶的同源物，该激酶调节泛素的生物合成。MLT1蛋白编码一个低序列复杂度的蛋白。该项目的目的是：1)确定微管根系统乙酰化是否不对称，如果这种不对称相对于卵裂沟和母鞭毛是固定的，以及眼斑相对于根的位置如何；2)基因或药物改变小根乙酰化，观察对小根和眼点定位和功能的影响；3)研究眼斑颗粒放置对光感受器和根茎定位的依赖性/独立性；4)研究EYE2、MIN1、EYE3和MLT1蛋白在眼斑组装和定位中的功能。这项工作的更广泛的影响是：1)包括一个小大学教授的参与，她将在学年和交替的夏天与本科生分享她在实验室的经验，2)参加美国国家科学基金会REU和亚利桑那大学的其他本科生研究项目，3)继续扩展实验室参观和高中K-12项目。

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.