Fig 2: ClC4 is expressed in amacrine cells, Müller glia, and nNOS-positive cells. A–C, Single z plane images of chicken retinal tissue sections double-labeled with antibodies specific for ClC4 and the amacrine cell marker, HPC-1 reveal that ClC4 is expressed in the HPC-1-positive cells. D–F, Zoom images of ClC4 and HPC-1 antibody labeling of the blue box region in A–C. ClC4 is expressed in amacrine cells, predominantly at cytosolic locations (asterisks). G–I, Zoomed images of the purple box region in A–C show that ClC4 is expressed in displaced amacrine cells in the retina (asterisk). J–L, Retinal tissue double-labeled with ClC4 antibody and an antibody specific for the Müller cell marker, glutamine synthetase (GS). Arrows indicate examples of ClC4 expression in Müller glia. M–O, Retinal sections double-labeled with ClC4 antibody and a nNOS-specific antibody. The nNOS antibody labels cells in all layers of the chicken retina, with strong labeling of horizontal cells (yellow arrows), a subset of amacrine cells and processes (white asterisk) as well as putative Müller cells, efferent target cells (ETCs, yellow asterisk), IPL processes (white arrows) and bands, ganglion cells, and scattered processes in the NFL. ClC4 is expressed in almost all nNOS-expressing cells. Note co-localization of ClC4 and nNOS in an immunoreactive band near to the IPL/GCL boundary (arrowheads). P–R, Single z plane zooms of ClC4 and nNOS antibody labeling confirms co-labeling of glial cells (arrowheads) and IPL processes (white arrows). (Scale in A, 50 µm, applies to B–C, J–L, and M–O; Scale D–F, 15 µm; Scale G–I, 15 µm; Scale P–R, 15 µm.)

Fig 3: SNAP25 crowds bind to syntaxin 1A clusters via the N-terminal SNARE domain of SNAP25.Stx-full (syntaxin 1A is stained by the HPC-1 antibody visualized by an AlexaFluor594-coupled secondary antibody) is overexpressed in PC12 cells together with GFP-SNAP25 or GFP-SNAP25 lacking either the C-terminal SNARE domain (SNAP25-?C), the N-terminal SNARE domain (SNAP25-?N), or both (SNAP25-?N/C) (visualized by an anti-GFP nanobody coupled to ATTO647). Membrane sheets are generated and analyzed exactly as in Figure 4. (A) Representative images from the different channels and overlays. Please note that SNAP25-wildtype (SNAP25-WT) staining was on average 40 % dimmer than for example SNAP25-?N. Pictograms in the upper image row illustrate the SNARE domains of SNAP25 that are depicted as white bars. (B) Pearson correlation coefficient (PCC) between GFP and syntaxin 1A channel; for control see Figure 5—figure supplement 1. (C) Clustering degrees normalized to SNAP25-WT (100 % = 0.523). Values are shown as box plots (n = 4 experiments; at least 10 membrane sheets were imaged per condition and experiment), showing the median, the 25th and 75th percentile (box), and the minimum and maximum value. Two-tailed Student’s t-test compare SNAP25-WT to SNAP25-?C/SNAP25-?N/SNAP25?N/C; ns = not significant, p > 0.05, *p < 0.05, **p < 0.01. (D) From individual membrane sheets, the degree of clustering (relative standard deviation of the mean, rSDM) is plotted against the mean region of interest (ROI) intensity of the GFP staining. Black and grey lines illustrate fits of a non-linear regression line (y = (a - b)e-d*x + b) to the values of SNAP25-WT (a = 1.35, b = 0.64, d = 0.042) and SNAP25-?N/C (a = 1.47, b = 0.41, d = 0.046), respectively.

Fig 4: Co-expression of cadherin-11 and retinal cell types in adult retina.(A, B) Cadherin-11 expression co-localizes with Müller glia cell bodies (CRALBP, 100× magnification), Müller glia cell processes (glutamine synthetase, 40× magnification) and horizontal cells (160 kDa, 40× magnification) (C, D) but not with bipolar (Chx-10, 40× and 100× magnification) or amacrine (HPC-1, 40× magnification) (white arrows) cells.

Fig 5: ClC7 is expressed in amacrine cells, but not widely expressed in Müller or nNOS-positive cells. A–C, Single z plane images of chicken retinal tissue sections double-labeled with ClC7 and HPC-1 antibodies show that ClC7 is expressed in HPC-1-positive cells. D–F, Zoom of blue box region in A–C shows that ClC7 is expressed in amacrine cells (asterisks), particularly those with cell bodies close to the IPL. G–I, Zoom of purple box region in A–C shows ClC7 antibody labeling in displaced amacrine cells of the ganglion cell layer (asterisk) as well as in neighboring, HPC-1 negative ganglion cells. J–L, Sections of retinal tissue double-labeled with ClC7 and GS antibodies. GS and ClC7 co-expression is limited to Müller cell margins in the center of the INL (arrows). M–O, Retinal sections double-labeled with ClC7 and nNOS antibodies (maximum projection images) reveal that, except for in the GCL, ClC7 is minimally expressed in nNOS-expressing cells. (Scale A–C, 50 µm, applies to J–L and M–O as well; Scale D–F, 15 µm; Scale G–I, 15 µm.)