使用质膜标记FM1-43来揭示豚鼠耳蜗外毛细胞(OHC)中不同类型的内吞和跨细胞机制的相对重要性。使用双管局部灌注系统独立标记分离的OHC的顶端或突触极,以研究极点处的囊泡摄取以及沿细胞和跨细胞的囊泡运输的机制。用巨噬细胞和吞噬作用抑制剂治疗,氧化苯砷,或网格蛋白介导的内吞活性,伴刀豆球蛋白A,显著降低了顶端和突触极的染料摄取,表明在两极都存在非网格蛋白依赖性和网格蛋白介导的过程。然而,在存在抑制剂的情况下对摄取速度的测量表明,与网格蛋白无关的过程对基底极的内吞作用的贡献比对顶极的贡献更大。用肌球蛋白VI抑制剂治疗,2,4,6-三碘苯酚,显着延迟了根尖和根尖荧光信号。然而,用驱动蛋白抑制剂治疗,monastrol,或者动力蛋白,CiliobrevinD,仅在基底顶端方向显着延迟了信号。肌球蛋白VI抑制剂,但是驱动蛋白和动力蛋白抑制剂都不是,显着延迟了到地下水箱的信号。也就是说,肌球蛋白VI在两个纵向方向以及径向地携带囊泡到地下水箱,而驱动蛋白和动力蛋白主要参与基底顶端的贩运。这些基本信息对于阐明参与建立的特定蛋白质的再循环机制至关重要,控制和维护OHCs的机电作用,因此,对于理解听觉感知至关重要。
The plasma-membrane marker FM1-43 was employed to reveal the relative significance of different types of endocytic and transcytic mechanisms in outer hair cells (OHCs) of the guinea-pig cochlea. A double-barrel local perfusion system was used to label independently the apical or synaptic pole of the isolated OHC to study mechanisms of vesicle uptake at the poles and of vesicle trafficking along and across the cell. Treatment with an inhibitor of macropino- and phagocytosis, phenylarsine oxide, or of clathrin-mediated endocytic activity, concanavalin A, significantly reduced the dye uptake at both the apical and the synaptic poles, indicating the presence of both clathrin-independent and clathrin-mediated processes at both poles. However, measurement of uptake speed in the presence of the inhibitors suggested that clathrin-independent processes contribute more extensively to endocytosis at the basal pole than the apical pole. Treatment with an inhibitor of myosin VI, 2,4,6-triiodophenol, significantly delayed both the apicobasal and the basoapical fluorescence signals. However, treatment with an inhibitor of kinesin, monastrol, or of dynein, ciliobrevin D, significantly delayed the signals only in the basoapical direction. The myosinVI inhibitor, but neither the kinesin nor dynein inhibitors, significantly delayed the signals to the subsurface cisternae. That is, myosin VI carries vesicles in both longitudinal directions as well as radially to the subsurface cisternae, whereas kinesin and dynein participate primarily in basoapical trafficking. This fundamental information is essential for elucidating recycling mechanisms of specific proteins involved in establishing, controlling and maintaining the electromechanical action of OHCs and, therefore, is vital for understanding auditory perception.