fm1-43 and 3-3--dihexyl-2-2--oxacarbocyanine

Outer hair cells (OHCs), the sensory-motor cells of the mammalian cochlea, contain an endocytic tubulovesicular compartment below their apical stereocilia. We have used two-photon imaging of FM1-43 in the intact epithelium to show that these cells take up membrane in a Ca(2+)-dependent manner from a distinct apical site. The uptake rate was 0.8 microm(2)/s and internalized membrane was trafficked rapidly to a compartment along the lateral wall and distinct intracellular compartments. Double labelling with FM1-43 and DiOC(6), an endoplasmic reticulum (ER) marker, showed that these compartments are part of the tubulovesicular endoplasmic reticulum of OHCs. Labelling with a lysosomal marker showed that OHC lysosomes are restricted to the apex. Using the protein marker wheat germ agglutinin (WGA-FITC) we demonstrate that apical protein internalization and trafficking is about eight times slower than membrane internalization. Using double labelling with FM1-43 and WGA-FITC, we show that membrane and protein internalization are apically colocalized but that patterns of protein and membrane traffic differ. Protein was targeted only to the most apical third of the lateral wall. In control conditions, OHCs displayed only weak WGA-FITC surface labelling at the site of endocytosis. Lowering the rate of apical endocytosis increased this surface signal. The results suggest that OHCs endocytose membrane and membrane proteins with a high turnover rate and that these cells may use apical endocytosis to sort proteins via an indirect pathway to the lateral membrane.

Topics: Animals; Biological Transport; Calcium; Carbocyanines; Cell Membrane; Cochlea; Diagnostic Imaging; Endocytosis; Endoplasmic Reticulum; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Guinea Pigs; Hair Cells, Auditory, Outer; In Vitro Techniques; Lysosomes; Pyridinium Compounds; Quaternary Ammonium Compounds; Time Factors; Wheat Germ Agglutinins