Structural variability of the sub-surface cisternae in intact, isolated outer hair cells shown by fluorescent labelling of intracellular membranes and freeze-fracture

Abstract

The intracellular membrane systems in intact, isolated outer hair cells were visualised using the fluorescent membrane probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and by freeze-fracture, and f-actin distribution was examined with rhodamine-phalloidin. DiOC6 stained the sub-surface cisternal membranes in the lateral wall and revealed a membrane system running in the centre of the cell from the nucleus to the sub-cuticular region. In optical sections of the lateral wall of fluorescently labelled cells, obtained by scanning laser confocal microscopy, the sub-surface membrane appeared as a fenestrated sheet or a fine network of tubules. Freeze-fracture replicas of rapidly-frozen, unfixed outer hair cells also showed the sub-surface membrane as a fenestrated sheet in some cells or as a network of tubules in others. These combined studies indicate that the interruptions within the cisternal membranes as seen in normal thin sections of outer hair cells are not fixation artefacts but may reflect the dynamic and plastic properties of this membrane system. Double staining of cells with rhodamine-phalloidin and DiOC6 showed substantial co-localisation of intracellular membranes and f-actin. The results suggest there may be a continuous, dynamic endoplasmic reticulum system, forming a core in the centre of the cell, broadening in the subcuticular region and extending down the lateral wall, that may have a role in the turnover and distribution of cytoskeletal assemblies within the outer hair cell.