Abstract:
Our understanding of how otoferlin, the major calcium sensor in inner hair cells (IHCs) synaptic transmission, contributes to the overall dynamics of synaptic vesicle (SV) trafficking remains limited. To address this question, we generated a knock-in mouse model expressing an otoferlin-GFP protein, where GFP was fused to its C-terminal transmembrane domain. Similar to the wild type protein, the GFP-tagged otoferlin showed normal expression and was associated with IHC SV. Surprisingly, while the heterozygote Otof+/GFP mice exhibited a normal hearing function, homozygote OtofGFP/GFP mice were profoundly deaf attributed to severe reduction in SV exocytosis. Fluorescence recovery after photobleaching revealed a markedly increased mobile fraction of the otof-GFP-associated SV in Otof GFP/GFP IHCs. Correspondingly, 3D-electron tomographic of the ribbon synapses indicated a reduced density of SV attached to the ribbon active zone. Collectively, these results indicate that otoferlin requires a free intravesicular C-terminal end for normal SV docking and fusion.
摘要:
我们对otoferlin的理解,内毛细胞(IHC)突触传递中的主要钙传感器,有助于突触小泡(SV)运输的整体动力学仍然有限。为了解决这个问题,我们建立了一个敲入小鼠模型,表达了一种Otoferlin-GFP蛋白,其中GFP融合到其C末端跨膜结构域。类似于野生型蛋白,GFP标记的otoferlin显示正常表达,并与IHCSV相关。令人惊讶的是,而杂合子Otof+/GFP小鼠表现出正常的听力功能,纯合子OtofGFP/GFP小鼠由于SV胞吐作用的严重减少而极度聋。光漂白后的荧光恢复表明,OtofGFP/GFPIHC中与Otof-GFP相关的SV的移动分数显着增加。相应地,带状突触的3D电子层析成像表明,附着在带状活动区的SV密度降低。总的来说,这些结果表明,耳铁蛋白需要一个游离的囊内C末端来进行正常的SV对接和融合。