STED imaging of uptaken mCLING fluorescence indicates large mCLING‐positive structures (indicated by dashed lines) near AZs during K
+ depolarization in
AP‐2μ control IHCs (A) and to a lesser degree in
AP‐2μ
fl/fl
:Cre
IHCs (B). IHCs were chemically fixed immediately following stimulation by 65 mM K
+ for 1 min in the presence of 1.7 μM mCLING‐Atto647N (red) and thereafter immunolabeled for RIBEYE/CtBP2, marking the AZ (green), and otoferlin (not shown). The mCLING‐labeled structures most likely reflect ELVs, arising from bulk endocytic retrieval of membrane, which are broken down into smaller compartments during 5 min of recovery after stimulation (as above) in
AP‐2μ control IHCs (C) but persist in
AP‐2μ
fl/fl
:Cre
IHCs (D). Mice lacking the GFP reporter were used for STED experiments, as the green channel was used for RIBEYE/CtBP2 immunolabeling. Representative STED images of 200‐nm melamine sections of IHCs are shown in the left panels of (A‐D). For a more quantitative view of endocytosis across different release sites, average images (false color‐coded panels, right panels in A–D) centered on the synaptic ribbons were generated from 10 to 15 individual ribbon synapses. White lines indicate the location of the plasma membrane, which was approximated in STED images of otoferlin fluorescence in the same sections (where the cell borders are quite evident, see Fig
9) and copied onto the mCLING images. Scale bar, 1 μm. Note that mCLING, as a non‐specific membrane marker, is taken up not only by the IHCs, but by other cells as well. In addition, mCLING decorates the extracellular leaflets of all plasma membranes, including those of supporting cells and of the afferent and efferent synapses that are positioned below the AZs of the IHCs, which likely explains the fluorescence outside the IHCs.
