Endocytosis and transcytosis in the outer hair cell of the guinea pig cochlea

Abstract

In the present study, endocytosis and transcytosis mechanisms were investigated in the bipolar OHC. This type of epithelial cell, located in the organ of Corti, contributes to the amplification of sound. As a key feature of epithelial cells, endocytosis is responsible for altering the biochemical composition inside the cell. Upon entering the cell, cargo is transported via motor proteins along different pathways to its required intracellular destination, through the process called transcytosis. Until now, in the OHC most of the research was focused on investigating the apical pole and destinations of apically endocytosed product. The prevalent uptake mechanism at each pole as well as the traffic pathways and motor proteins involved in cargo movement, had not been investigated. Therefore, the goal of this study was to clarify the endocytosis and transcytosis processes in the OHC. For endocytosis experiments, the aim was to elucidate the different types of endocytosis present at both poles, using blockers of different types of internalization, as well as to quantify the molecular-weight limit of molecules which can be internalized. Since intracellular targets of basally endocytosed product were not yet clear, in another set of experiments the destinations of basally endocytosed vesicles were investigated. For transcytosis experiments, the aim was to compare the dynamics and pathways of apicobasal and basoapical vesicle traffic. Using blockers, another goal was to investigate the participation of motor proteins in vesicle traffic. In the last set of experiments, processes to the SSC were compared to those along the cell. To apply fluorescent markers to exclusively one pole of the cell, a newly developed double-barrel perfusion system was used. Visualizing the uptake and intracellular dye distribution was enabled through confocal laser scanning microscopy. Additional patch clamp experiments were performed to investigate whether uptake might be charge dependent. Basal uptake was more intense than apical uptake. Furthermore, at the apex the principle uptake mechanism was clathrin-mediated endocytosis, whereas at the base internalization was both non-clathrin mediated and the clathrin-mediated. Pinocytosis was restricted to molecules no larger than 500 Da. It was found that upon entering the OHC through the basal pole of the cell, vesicles traffic towards the ER and the mitochondria. The basoapical traffic was faster than the apicobasal traffic. Both basoapical and apicobasal traffic was myosin VI dependent, whereas only the traffic from apex to base was kinesin dependent. The first destinations after internalization at the apex and base of the OHC were myosin VI dependent. Endocytosed vesicles trafficked first along a longitudinal axis to the central areas of the cell and then were transported to the SSC. The SSC traffic appeared to be mainly myosin VI dependent. In summary, in this study it is demonstrated for the first time which uptake mechanisms dominate at the apical and the basal pole and which motor proteins are involved in traffic along the cell and to the SSC. The molecular identity of endocytosed and transcytosed material in vivo has yet to be identified.