Modes and Regulation of Endocytic Membrane Retrieval in Mouse Auditory Hair Cells
Modes and Regulation of Endocytic Membrane Retrieval in Mouse Auditory Hair Cells
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DOI：
10.1523/jneurosci.3313-13.2014
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发表时间：
2014-01-15
影响因子：
5.3
通讯作者：
Moser, Tobias
中科院分区：
文献类型：
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作者：
Neef, Jakob;Jung, SangYong;Moser, Tobias
突触小泡再循环维持了小鼠听觉内毛细胞带状活动区(AZs)的高水平神经传递，但其模式和分子调控尚不清楚。电子显微镜下可见网状蛋白介导的内吞作用(CME)和块状内吞作用。内吞蛋白Dynamin、clathrin和两栖动物蛋白在IHC中广泛表达和分布。我们使用共聚焦vher1-pHluorin成像和膜电容(C-m)测量来研究IHC胞吐和内吞的空间组织和动力学。病毒基因转移在IHCs中表达vGlu1-pHluorin，并将其靶向突触小泡。囊泡内pH与6.5相似，仅支持胞吐和pH中和过程中vGlu1-pHluorin荧光的轻微增加。Ca~(2+)内流引起AZs胞外vGlu1-pHluorin荧光增强，并持续数秒。易释放池(RRP)胞吐后，胞内C-m以恒定速率下降(线性成分)。当胞吐作用超过三到四个RRP当量时，IHC额外招募更快的C-m下降(指数分量)，该下降速度随着先前胞吐作用的量增加而增加，并可能反映大量内吞作用。动力蛋白抑制剂Dygo-4a和笼状蛋白阻滞剂Pitstop2选择性地削弱细胞内C-m下降的线性成分。动力蛋白1的错义突变(间歇性)抑制内吞作用的程度与dyngo-4a相似。我们认为IHC通过CME使用动力蛋白依赖的内吞作用来支持轻微刺激时的囊泡循环，但招募大量内吞作用来平衡大量的胞吐作用。
Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (C-m) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was similar to 6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca2+ influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic C-m decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster C-m decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic C-m decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.