Functional in vivo Studies of Algal Sensory Photoreceptors

衣原体隐花色素aCry和pCry的内源性时钟的时间设置，表面附着和滑行，以及高能量淬火的相互作用仍然是一个悬而未决的问题。我们将专注于aCRY，pCRY和一个假设的红光光受体在不同的菌株中的失活，并研究这些受体对几种生理反应的影响。我们将在不同的氧气、光照和营养条件下培养细胞，应用不同的光照程序，监测野生型和缺失型菌株的生理反应以及翻译和蛋白质丰度，并研究对光合作用和高能猝灭的影响。我们将集中在丰富的时钟蛋白，积累的光捕获蛋白LHCSR 3和其他感光蛋白的通道视紫红质，组氨酸激酶视紫红质（HKR），和phototropin（Phot）。我们将研究细胞内部的Ca ~（2+）信号，包括叶绿体和内质网的Ca ~（2+）释放。在第二种方法中，我们将研究视紫红质的体内功能，视紫红质是视紫红质与bestrophin结构域的新型杂交体，形成包含5个通道亚基和5或10个视紫红质的大型复杂光敏离子通道。我们将通过用来自Cymbomonas的光调制的视紫红质替换衣原体中的三种光不敏感的视紫红质来表征视紫红质的体内功能，分析细胞和生物体背景下的功能。我们认为这是一个理想的和互补的方法，以更好地了解在体内功能的bestrophin和bestrohodopsin在同一时间。

The interplay of the Chlamydomonas Cryptochromes aCry and pCry for time setting of the endogenous clock, surface attachment and gliding, and high energy quenching is still an open question. We will focus on the inactivation of aCRY, pCRY and a hypothetical red-light photoreceptor in different strains and study the influence of these receptors on several physiological responses. We will grow cells under different oxygen, light, and nutrient conditions, apply different illumination programs, and monitor physiological responses as well as translation and protein abundance in wt and deletion strains and study the influence on photosynthesis and high energy quenching. We will focus on the abundance of clock proteins, accumulation of the light harvesting protein LHCSR3 and of other photoreceptor proteins as channelrhodopsins, histidine-kinase-rhodopsins (HKRs), and phototropin (Phot). We will study internal Ca2+-signaling including Ca2+ release from Chloroplast and ER. In a second approach we will study the in vivo function of Bestrhodopsins, which are novel hybrids of rhodopsins with bestrophin domains forming large complex light-sensitive ion channels comprising 5 channel subunits and 5 or 10 rhodopsins. We will characterize the bestrhodopsin in vivo function by replacing in Chlamydomonas the three light-insensitive bestrophins with the light modulated bestrhodopsins from Cymbomonas analyze the function within the cellular and organismal context. We consider this as an ideal and complementary approach to better understand the in vivo function of both bestrophin and bestrhodopsin at the same time.