Agonist - G protein-coupled receptor interactions, recycling and cell-surface targeting in neuroendocrine systems

我的研究计划旨在阐明神经内分泌细胞和神经元中G蛋白偶联受体(GPCRs)调节细胞表面靶向和亚细胞定位的机制。这两个过程对细胞，特别是神经细胞的正常功能至关重要，神经细胞由于其复杂性，在维持其关键成分蛋白的正确细胞内靶向和定位方面面临着特殊的挑战。为此，我们使用经典和尖端的细胞生物学技术，并与实验室合作，通过使用CRISPR/Cas9技术进行基因组编辑的新技术，创造出细胞和实验室小鼠的转基因变体。此外，基于对许多荧光团的光化学性质的巧妙利用，最近出现的新的显微技术超越了光学显微镜的分辨率极限，被称为超分辨率显微镜(SRM)，有望直接可视化调节这些过程的蛋白质相互作用。与透射电子显微镜非常相似，新的SRM技术对样品制备和数据分析有特定的要求，需要进行大量研究以建立合适的实验方案，并对HQP进行高强度的培训以执行程序。多亏了MNI显微镜核心设备提供的基于SRM的点定位、双平面原理的SRM显微镜的访问，我们最近以50 nm的分辨率可视化了例如sst2A受体在Gen簇处的亚细胞定位，Gen是中枢神经系统中抑制性突触的突触后密度的标志性结构成分。这至少比最好的标准光学显微镜好5倍，接近单个蛋白质分子的大小。因此，本申请以我在超分辨率显微镜和GPCRs细胞生物学方面的专业知识为基础，通过a)将新的SRM技术与细胞生物学、共聚焦显微镜和电子显微镜的既定方案相结合，以及b)使用所有这些方法来阐明神经内分泌和神经细胞中GPCRs的调节和靶向的公开问题，从而进一步推进我的研究和培训计划。

My research program is geared towards the elucidation of the mechanisms regulating cell-surface targeting and sub-cellular localization of G protein-coupled receptors (GPCRs) in neuroendocrine cells and neurons. Both these processes are key to the normal functioning of cells, in particular of nerve cells which due to their complexity face particular challenges in maintaining correct intracellular targeting and positioning of their key component proteins. To this end we use classical and cutting-edge cell biological techniques and collaborate with laboratories to create genetically modified variants of cells and laboratory mice by the new technique of genome editing using CRISPR/Cas9 technology. Moreover, the recent advent of new microscopic techniques surmounting the resolution limit of light microscopy based on the clever exploitation of the photo-chemical properties of many fluorophores, referred to as super-resolution microscopy (SRM), promises the direct visualization of protein interactions regulating these processes. Much like transmission electron microscopy, the new SRM techniques have specific requirements for sample preparation and data analysis requiring substantial research to establish suitable experimental protocols and intense training of HQP to perform the procedures. Thanks to the access to a SRM microscope that is based on the point-localization, bi-plane principle of SRM provided by the MNI Microscopy Core Facility, we recently visualized for instance the sub-cellular localization of sst2A receptors at clusters of gephyrin, a hallmark structural component of the post-synaptic density at inhibitory synapses in the central nervous system at 50 nm resolution. This is at least 5 times better than the best standard light microscope can do and approaches the size of individual protein molecules. Therefore, the present application builds on my expertise in super-resolution microscopy and the cell biology of GPCRs to further my research and training program by a) integrating the new SRM techniques with established protocols in cell biology, confocal and electron microscopy and b) using all of these approaches to elucidate the open questions around the regulation of intracellular trafficking and targeting of GPCRs in neuroendocrine and neuronal cells.