Figure 3. Nanoscale organization of the postsynaptic endocytic zone.

(A) SMLM image of dendrite expressing Homer1c-mCherry and GFP-CLCa labeled with CF568 and A647 and zooms of individual EZs, respectively. Scale bar upper panel: 2 µm, zooms: 250 nm. (B) Individual molecules of Homer1c (magenta) and CLCa (orange) are outlined using DBScan. Black dot and line indicate center of the EZ (dot) and distance to the border of the PSD (line). Scale bar: 500 nm. (C) Histogram of the border (Homer1c) to center (CLCa) distance in nm. (D) Scatterplot of the FWTM length (nm) and FWTM width (nm) of the EZ. (E) Histogram of the area of the EZ plotted as x10³ nm². (C–E) n = 107.

Figure 3—figure supplement 1. SMLM reveals nanoscale scale architecture of PSD-associated CLCa structures.

(A) Example plots visualizing the individual localizations obtained for Homer1c (magenta) and CLCa (orange). Two or more individual CLCa structures can be observed per PSD. Scale bar: 500 nm. (B–D) Data visualized in these plots is the same data represented in Figure 3, but here a distinction was made between PSDs containing one (blue) or two (green) CLCa structures is made. (B) Histogram of the distance from the border of the PSD to the border of CLCa structures. (C) Scatterplot showing the dimensions of larger (blue) CLCa structures, most likely representing the EZ, and smaller CLCa structures (green) (big structures: n = 92, small structures: n = 13). (D) Histogram showing circularity ratio of CLCa structures. (E) Comparison of the average area of CLCa puncta labelled with different strategies (GFP-CLCa labeled with monoclonal GFP and CF568, Halo-CLCa labeled with halo-JF646), plotted as mean ± SEM (GFP-CF568: n = 103, Halo-JF646: n = 50).