EYESPOT POSITIONING AND ASSEMBLY

这项提议的长期目标是了解感光细胞器(眼点)是如何在藻类细胞中不对称地定位的，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而成的。眼斑允许单细胞藻类游向/远离光线，以优化光合作用能力。眼斑与四条高度乙酰化的微管根中的一条密切相关，这些微管发自基体区。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。在胞质分裂时，眼点被吸收，并产生两个新的眼点。眼点是如何定位的，以及它是如何在每个细胞分裂时重新构建的，这是一个谜。一种遗传学方法已经确定了眼点组装和定位所需的六个基因。四个基因(EYE1-4)的突变使细胞失明。MLT1基因的突变会导致多个眼点定位错误。MIN1基因的突变导致比正常眼点小，并且眼点中质膜和叶绿体包膜的紧密结合丢失。已经对Eye2基因进行了鉴定，并针对该蛋白制备了抗血清。MIN1、MLT1和EYE3都有能够分离基因的插入突变。遗传学、分子生物学和免疫荧光显微镜将被用来分离基因和制备荧光抗体/蛋白，以探索眼点成分在胞质分裂时重新合成眼点的时间、位置和组装。了解细胞的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会导致组织发育和功能的严重缺陷。细胞器的组装和与宿主细胞结构和代谢的整合对细胞、组织和生物体的健康至关重要。在模式生物中通过遗传学的力量学到的生物学基本经验将是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.