Regulation of Retinal Functions by Light and a Circadian Clock

生活在昼夜环境中的动物的生理和行为-每天有节奏变化的环境-在很大程度上受到来自内部24小时（昼夜）时钟的信号的控制。 生物钟对人类的影响在轮班工作者和长途旅行者中尤为明显，因为当内部时钟与外部光暗周期不同步时，会出现“时差”。 生物钟调节生理的机制在很大程度上是未知的。该提案旨在从分子水平上了解环境信号和昼夜节律如何单独或组合影响视力。 视网膜是眼睛中含有检测光的细胞（感光细胞）的组织，它如何开始处理传入的视觉信息，并将这些信息传递到大脑进行进一步处理，将被研究。 生活在昼夜环境中的动物的视网膜通常经历结构和功能的每日节律性变化，这优化了视网膜在白天的明亮光下和在夜间的昏暗光下的功能。 这些昼夜变化对正常视力至关重要，是由生物钟和光驱动的生化反应的单独和组合效应产生的。 美洲鲎之所以被使用，是因为在它的眼中，生物钟的影响特别引人注目。 例如，当来自生物钟的信号在夜间到达眼睛时，它们的结构和功能会发生变化，从而使它们对光线的敏感度提高十万倍。 对于鲎来说，夜间视觉灵敏度的增加被认为是繁殖的关键。目前研究的两个具体目标旨在确定时钟和光驱动的生化级联对编码光检测（视蛋白）和光感受器对光的适当反应（arrestin）所需的蛋白质的基因表达的单独和组合影响。 将测定编码这些蛋白质的mRNA水平的变化。 第三个目的是研究光感受器特异性蛋白（肌球蛋白III）的功能，在鲎，是由光和时钟驱动的级联调制。 这种蛋白质是特别感兴趣的，因为：1。它对于至少一些无脊椎动物光感受器的存活是至关重要的。 2.在多种无脊椎动物和脊椎动物物种的光感受器中发现了类似的蛋白质，因此该蛋白质对于所有光感受器的功能和/或维持可能是关键的。 3.在鲎中，它可以整合来自光和时钟驱动的生化级联的信号。 鲎肌球蛋白III已在本实验室的昆虫细胞系统中表达并大量生产。 这为详细研究其结构和功能提供了独特的机会。这项研究将产生许多对感觉和细胞生物学重要的成果。 由于环境线索和昼夜节律的点击几乎影响所有细胞，因此这些结果应该进一步了解这些因素如何影响暴露于自然光照条件下的动物（包括人类轮班工人）的细胞生物学。 肌球蛋白III的这些研究将提供关于一种蛋白质的具体新信息，该蛋白质被假设为对所有光感受器的结构和功能至关重要。此外，这项研究将涉及本科生和预科少数民族学生，这是该实验室的一个悠久传统。 所产生的材料和信息也将用于惠特尼实验室运行的各种K-12外展计划。 因此，这项研究将有助于培养下一代科学家和一个有科学素养的公民在国家的一个地区，获得任何形式的科学活动是有限的。

The physiology and behavior of animals that live in diurnal environments-- environments where there is a daily rhythmic change in light and darkness-- are controlled in large part by signals that arise from an internal, 24 hour (circadian) clock. The impact of circadian clocks on humans is particularly evident in shift workers and as "jet lag" in long-distance travelers when the internal clock becomes out of phase with the external light-dark cycle. The mechanisms by which the clock regulates physiology are largely unknown. This proposal seeks to understand, at a molecular level, how environmental signals and the circadian click, separately and in combination, impact vision. How the retina, the tissue in the eye that contains the cells which detect light (photoreceptor cells), begins to process incoming visual information, and transmits this information to the brain for further processing will be studied. The retinas of animals living in diurnal environments typically undergo daily rhythmic changes in structure and function that optimize the retina for functioning in bright light during the day and in dim light during the night. These day-night changes, which are critical for normal vision, are produced by the separate and combined effects of biochemical reactions that are driven by the circadian clock and by light. The American horseshoe crab Limulus polyphemus is used because in its eyes, the effects of the circadian clock are particularly dramatic. For example, when signals from the circadian clock reach the eyes at night their structure and function change such that they become one hundred thousand times more sensitive to light. For Limulus, increased visual sensitivity at night is thought critical for reproduction. Two specific aims of the current research are designed to determine the separate and combined effects of clock- and light-driven biochemical cascades on the expression of genes encoding proteins that are required for the detection of light (opsin) and for the appropriate response of photoreceptors to light (arrestin). Changes in the levels of mRNAs encoding these proteins will be assayed. A third aim is to examine the function of a photoreceptor-specific protein (myosin III) which, in Limulus, is modulated by both light- and clock-driven cascades. This protein is of particular interest because: 1. it is critical for the survival of at least some invertebrate photoreceptors. 2. similar proteins are found in the photoreceptors of a wide variety of invertebrate and vertebrate species, and thus the protein may be critical for the function and/or maintenance of all photoreceptors. 3. in Limulus, it may integrate the signals from both light- and clock-driven biochemical cascades. Limulus myosin III has been expressed in this laboratory in an insect cell system and produced in large quantities. This provides the unique opportunity to study its structure and function in detail. This research will have a number of outcomes important for sensory and cell biology. Since environmental cues and the circadian click influence virtually all cells, the results should further a general understanding of how these factors impact the cell biology of animals exposed to natural lighting conditions, including human shift workers. These studies of myosin III will provide specific new information about a protein that is hypothesized to be critical for the structure and function of all photoreceptors. Furthermore, the research will involve undergraduates and pre-collegiate minority students, a long tradition in this laboratory. The materials and information generated also will be used in a variety of K-12 outreach programs run by the Whitney Laboratory. Thus the research will contribute toward developing the next generation of scientists and a scientifically literate citizenry in a region of the nation where access to scientific activities of any kind is limited.