Deciphering the molecular function of otoferlin in synaptic vesicle replenishment in inner ear sensory hair cells

Otoferlin在内耳的感觉毛细胞中表达，属于Ferlin蛋白家族。Ferlins具有由串联的C2结构域和单一的C-末端跨膜片段组成的保守结构，在功能上参与了肌肉和上皮细胞中由钙离子触发的膜融合过程。Otoferlin是一种隐性形式的严重人类语前听力损失DFNB9，而Otoferlin缺陷小鼠的听力损伤是由于感觉毛细胞带状突触上的突触小泡对钙的依赖减少而导致的。为了破译这一过程背后的奥费林的分子功能，本研究计划的目标是基于蛋白质组学的鉴定和奥托费林相互作用伙伴的功能表征。为了达到这一目标，我将从鸡胚胎耳朵的听觉和前庭器官的毛细胞中进行奥弗林的免疫共沉淀。利用超灵敏的质谱学，免疫沉淀蛋白将被识别，并根据在钙信号、囊泡循环或膜融合中的潜在作用来选择候选蛋白。我们将分三步对候选基因如AHNAK1进行进一步分析：i)用共聚焦免疫荧光和电子显微镜测定其在毛细胞中的亚细胞定位(S)；ii)重组表达的otoferlin及其相互作用伙伴(如AHNAK1)的GST-Pull试验以证明物理相互作用并确定特定的结合位点；以及iii)使用适当的基因敲除小鼠来评估新的otoferlin相互作用伙伴在突触小泡补充中的生理相关性。我设想，解开耳铁蛋白发挥作用的分子网络，将极大地促进对突触小泡如何在毛细胞中移动和排出，以及这些过程如何构成听力和前庭功能的基础知识的基础知识。

Otoferlin is expressed in sensory hair cells of the inner ear and belongs to the ferlin protein family. Ferlins share a conserved architecture consisting of tandem C2 domains, and a single C-terminal transmembrane segment and are functionally involved in Ca2+-triggered membrane fusion processes in muscle and epithelial cells. Otoferlin is defective in a recessive form of profound human prelingual hearing loss DFNB9, and otoferlin-deficient mice have a profound hearing impairment caused by reduced Ca2+-dependent replenishment of synaptic vesicles at the ribbon synapses of sensory hair cells. To decipher the molecular function of otoferlin underlying this process, the goal of this research proposal is a proteomic based identification and functional characterization of otoferlin interaction partners. To reach this goal, I will conduct co-immunoprecipitations of otoferlin from hair cells of the auditory and vestibular organs of embryonic chicken ears. Using ultrasensitive mass spectrometry, immunoprecipitated proteins will be identified and candidate proteins selected according to a potential role in Ca2+-signaling, vesicle cycling, or membrane fusion. Candidates such as AHNAK1, which I identified in a pilot-experiment, will be further analyzed in three steps: i) determination of their subcellular localization(s) in hair cells with confocal immuno-fluorescence and electron microscopy, ii) GST-pulldown assays of recombinantly expressed otoferlin and interaction partners (such as AHNAK1) to prove a physical interaction and to determine specific binding sites, and iii) usage of appropriate knock-out mice to assess a physiological relevance of the novel otoferlin interaction partner in synaptic vesicle replenishment. I envision that unravelling the molecular network in which otoferlin functions, will substantially advance fundamental knowledge of how synaptic vesicles are moved and exocytosed in hair cells, and how these processes underlie hearing and vestibular functions.