Harasztosi C, Gummer A W
Section of Physiological Acoustics and Communication, Faculty of Medicine, Eberhard Karls University Tübingen, Elfriede-Aulhorn-Str. 5, 72076, Tübingen, Germany.
HNO. 2019 Jun;67(6):449-457. doi: 10.1007/s00106-019-0674-y.
Intense endocytic activity at the apex of outer hair cells (OHCs)-the electromechanical cells of the cochlea-has been demonstrated using the vital plasma-membrane marker FM1-43 and confocal laser-scanning microscopy. Vesicular traffic toward the cell nucleus to distinct locations of the endoplasmic reticulum has also been shown.
The current study characterizes the dynamics of endocytic activity, as well as apicobasal and basoapical trafficking, using a local perfusion technique that we recently developed and published to visualize bidirectional trafficking in isolated bipolar cells.
The fluorescent plasma-membrane markers FM1-43 (10 µM) and FM4-64 (10 µM), together with a fluid-phase marker, Lucifer yellow (50 µM), were used to label endocytosed vesicles in isolated OHCs of the guinea pig cochlea. Targets of endocytosed vesicles were examined with a fluorescent marker of subsurface cisternae, DiOC (0.87 µM). Single- and two-photon confocal laser-scanning microscopy was used to visualize labeled vesicles.
The plasma-membrane markers presented more intense vesicle internalization at the synaptic pole than at the apical pole of the OHC. Intracellular basoapical vesicle trafficking was faster than apicobasal trafficking. Vesicles endocytosed at the synaptic pole were transcytosed to the endoplasmic reticulum system. An intracellular Lucifer yellow signal was not detected.
The larger endocytic fluorescent signals in the synaptic pole and the faster basoapical trafficking imply that membrane internalization and vesicle trafficking are more efficient at the synaptic pole than at the apical pole of the OHC.
使用活体细胞膜标记物FM1-43和共聚焦激光扫描显微镜已证明，外毛细胞（OHCs）——耳蜗的机电细胞——顶端存在强烈的内吞活性。还显示了向细胞核至内质网不同位置的囊泡运输。
本研究使用我们最近开发并发表的局部灌注技术来表征内吞活性的动力学，以及顶-基和基-顶运输，以可视化分离的双极细胞中的双向运输。
荧光细胞膜标记物FM1-43（10µM）和FM4-64（10µM），以及液相标记物路西法黄（50µM），用于标记豚鼠耳蜗分离的OHCs中的内吞囊泡。用亚表面池的荧光标记物DiOC（0.87µM）检查内吞囊泡的靶点。使用单光子和双光子共聚焦激光扫描显微镜观察标记的囊泡。
细胞膜标记物在OHC的突触极比顶端呈现出更强的囊泡内化。细胞内基-顶囊泡运输比顶-基运输更快。在突触极内吞的囊泡被转运到内质网系统。未检测到细胞内路西法黄信号。
突触极较大的内吞荧光信号和较快的基-顶运输表明，OHC突触极的膜内化和囊泡运输比顶端更有效。
