Characterization of biomodal light-switchable rhodopsins and tailoring for optogentic application

黑视素是一种光感受器，在哺乳动物中负责非视觉光接受和控制瞳孔收缩和昼夜节律等过程。目前已知的哺乳动物（小鼠）和一些斑马鱼的黑视素是光致变色的，在黑暗状态和信号状态之间可以双峰切换，这是光遗传学应用中最感兴趣的特性。在这个项目中，我们将在hek细胞、COPS细胞和毕赤酵母中表达几种黑视素，并在时间尺度上进行紫外/可见光谱和振动光谱的研究，时间尺度从5秒到秒不等（与阿姆斯特丹的Tilo Mathes合作）。对于光遗传学应用，我们将生产光致变色黑视素和gpcr型激素受体的杂交体，目的是用光激活激素受体信号。我们将与刘秀珍合作，以促肾上腺皮质激素释放激素（CRH）为目标，验证这一概念。为此，我们将对七鳃鳗和斑马鱼的opn4进行纯化和表征。我们将细胞内环与激素受体的互补环交换，并与Soojin Ryu一起在鱼苗中测试了其功能。

Melanopsins are Photoreceptors that are in mamals responsible for non-viual photoreception and control processes like pupil constriction and circadian rhythm. The so far known Melanopsins of mamals (mouse) and some of the zebrafish melanopsins are photochromic and bimodal switchable between the dark state and the signaling state, a property that is of highest interest for optogenetic application. In this project we will express several melanopsins in HEK-cells, COPS cells and in Pichia pastoris and carry out studies in the UV/Vis and vibrational spectroscopy on time scales ranging from fs to seconds (with Tilo Mathes in Amsterdam). For optogenetic applications we will produce hybrids of photochromic melanopsins and GPCR-type hormone receptors with the intention to activate hormone receptor signalling with light. We will validate this concept in collaboration with Soojin Ryu targeting corticotrophin releasing hormone (CRH). For this approach we will purify and characterise Parapinopsin of lamprey and opn4 of zebrafish. We exchange the intracellular loops against the complementary loops of the hormone receptor and test the function in fish larvae with Soojin Ryu.