EYESPOT POSITIONING AND ASSEMBLY

这个建议的长期目标是了解光感应细胞器，即眼点，是如何不对称地定位在藻类细胞中的，以及它是如何在每次细胞分裂时由叶绿体和宿主细胞成分组装起来的。眼点允许单细胞藻类向光/远离光游泳，以优化光合能力。眼斑与来自基部区域的四个高度乙酰化的微管根中的一个密切相关。眼斑是由质膜、叶绿体内外膜、叶绿体基质中的类胡萝卜素颗粒和类囊体膜组成的复杂夹层。在细胞分裂时，眼点被吸收并产生两个新的眼点。眼点是如何定位的，以及它是如何在每次细胞分裂中重新构建的，这是一个谜。一种遗传方法已经确定了六个基因所需的眼点组装和定位。四个基因（EYE1-4）的突变使细胞失去了眼睛。MLT1基因的突变导致多个眼点位置不正确。MIN1基因的突变导致眼斑比正常的小，并且失去了眼斑中质膜和叶绿体包膜的紧密结合。已对EYE2基因进行了鉴定，并提出了针对该蛋白的抗血清。可以分离MIN1、MLT1和EYE3基因的插入突变已经掌握。遗传学、分子生物学和免疫荧光显微镜将用于分离基因和制备荧光抗体/蛋白质，以探测细胞分裂过程中从头合成眼斑成分的时间、位置和组装。了解细胞极性和细胞与细胞器的复杂相互作用是很重要的。细胞骨架元素的破坏可导致组织发育和功能的严重缺陷。细胞器的组装和整合与宿主细胞的结构和代谢对细胞、组织和生物体的健康至关重要。从模式生物的遗传学力量中学到的生物学基本教训将成为人类健康许多进步的基础。

The long term goal of this proposal is to understand how a light- sensing organelle, the eyespot, is asymmetrically positioned in an algal cell and how it is assembled from chloroplast and host cell components at every cell division. The eyespot allows the unicellular alga to swim toward/away from light to optimize photosynthetic capacity. The eyespot is closely associated with a specific one of four highly acetylated microtubule rootlets that emanate from the region of the basal bodies. The eyespot is a complex sandwich of plasma membrane, outer and inner chloroplast membranes, carotenoid granules in the stroma of the chloroplast and thylakoid membranes. At cytokinesis, the eyespot is resorbed and two new eyespots are generated. It is a mystery how the eyespot is positioned, and how it is built de novo at each cell division. A genetic approach has identified six genes required for eyespot assembly and positioning. Mutations in four genes (EYE1-4) render the cells eyeless. Mutations in the MLT1 gene result in multiple eyespots that are incorrectly positioned. Mutations in the MIN1 gene result in smaller than normal eyespots and the close apposition of the plasma membrane and chloroplast envelope in the eyespot is lost. The EYE2 gene has been characterized and antisera raised to the protein. Insertion mutations that will allow isolation of the genes are in-hand for MIN1, MLT1 and EYE3. Genetics, molecular biology and immunofluorescence microscopy will be used to isolate genes and prepare fluorescent antibodies/proteins to probe the timing, placement and assembly of eyespot components during de novo eyespot synthesis at cytokinesis. Understanding cell polarity and the complex interaction of cells with their organelles is important. Disruption of cytoskeletal elements can lead to serious defects in tissue development and function. Organelle assembly and integration with the host cell structure and metabolism is essential for the health of cells, tissues and organisms. Fundamental lessons of biology learned through the power of genetics in model organisms will be the foundation for many advancements in human health.