Cochlear hair cells show pronounced endocytosis from their apical surface (Meyer et al. 2001; Griesinger et al. 2002). To assess the role of apical endocytosis, we used two-photon imaging of fluorescent markers to study endocytosis and protein uptake in hair cells in the in situ epithelium of adult guinea-pigs, killed by quick cervical dislocation in accordance with UK legislation.

Both inner and outer hair cells (IHCs and OHCs) showed rapid calcium-dependent internalisation of apical FM 1-43 (5 µM)-labelled membrane. The delay between signal in plasma membrane and that in internal endosomes was similar for IHCs (20 s) and OHCs (25 s). Both calcium dependency and fast rate suggest the presence of clathrin-independent endocytosis. Disruption of mechanotransduction did not change FM 1-43 uptake. Hence all internal signal was derived from endocytosis. In both cells, the fluid phase marker Lucifer Yellow (20 mM) revealed staining patterns similar to that of FM 1-43. Both IHCs and OHCs showed a functional domain dedicated to membrane internalisation at their apical membrane. IHCs internalised membrane from a ring around the cuticular plate, while OHCs showed membrane uptake from a circular patch within the cuticular plate.

IHCs and OHCs differed in patterns and kinetics of membrane trafficking. IHCs showed rapid kinesin-dependent trafficking of apically internalised membrane to about 30 basolateral hotspots at the synaptic pole. These reached 60 % of the intensity of the apical signal. In contrast, OHCs showed only weak labelling (5 % intensity compared with apex) of a disk-like structure (about 8 µm diameter) at the synaptic pole, most likely the basal cisterna. Instead, OHCs showed rapid kinesin-independent trafficking of membrane to patches along the basolateral membrane that showed strong signal (90 % of apical signal) and subsequently (66 s later) to a central strand.

To test an involvement of apical endocytosis in protein recycling, we used FITC-labelled lectin wheatgerm agglutinin (WGA), a marker for adsorptive endocytosis. WGA labels proteins containing N-acetyl-glucosamine, a signal for apical protein sorting in epithelial cells. Only OHCs internalised WGA through small vesicles about 1 µm in diameter in the apex. WGA appeared in the apical part of the basolateral membrane after a delay of about 50 min. Thus OHCs feature the ‘indirect route’ of protein uptake by transcytosis, described for various epithelial cells. However, at the apical membrane WGA signal was excluded from the domain dedicated to membrane uptake. The data suggest that there are spatially distinct pathways for protein and membrane trafficking in OHCs.

This work was supported by the MRC, Wellcome Trust and BBSRC.