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

黑色素是哺乳动物体内的光感受器，负责非视觉的光接收和控制过程，如瞳孔收缩和昼夜节律。到目前为止，哺乳动物(小鼠)的黑色素和斑马鱼的一些黑色素是光致变色的，可以在暗状态和信号状态之间进行双峰切换，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色素，并在从飞秒到秒的时间尺度上进行UV/VIS和振动光谱的研究(与Tilo Maths在阿姆斯特丹)。对于光遗传应用，我们将生产光致变色黑色素和GPCR型激素受体的杂交，目的是用光激活激素受体信号。我们将与Soojin Ryu合作，以促肾上腺皮质激素释放激素(CRH)为靶点，验证这一概念。对于这种方法，我们将提纯和鉴定七鳃鳗的对乙酰胆碱和斑马鱼的Opn4。我们用激素受体的互补环交换细胞内环，并用Soojin Ryu测试鱼类幼体的功能。

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.