SUPPLEMENTARY FIGURES

Transcription

1 SUPPLEMENTARY FIGURES Supplementary Figure S1. Generation of a synaptobrevin2-mrfp knock-in mouse. (a) Targeting strategy of Syb2-mRFP knock-in mouse leaving the synaptobrevin2 gene locus intact except for the addition of the coding sequence for GGSGGSGGT linker, mrfp and a remaining loxp site in the final mouse line. Positions of PCR oligonucleotides and Southern blot probes are indicated. (b) PCR analyses of genotypes using the indicated oligonucleotides (see a). (c) Western blot of brain homogenates from WT (+/+), heterozygous (+/KI), homozygous Syb2-mRFP knock-in (KI/KI) and homozygous synaptobrevin2 knock-out (-/-) mice using a monoclonal anti-synaptobrevin2 antibody. GAPDH was used as internal control for protein loading (lower panel). (d) Western blot of brain homogenates from WT (+/+), heterozygous (+/KI) and homozygous Syb2-mRFP knock-in (KI/KI) using anti-rfp antibody. GAPDH was used as internal control for protein loading (lower panel).

2 Supplementary Figure S2. Localization of Syb2-mRFP in retina. Immunolabeling showing colocalization of Syb2-mRFP and anti-synaptophysin in (a) retina slices (ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer, Scale bar: 10 µm) and in (b) acutely dissociated bipolar cell neurons that were obtained from mouse retina. Scale bar: 10 µm. (c-f) Electron microscopy of photoreceptor ribbon synapses from Syb2-mRFP (c+d) and WT (e+f) mice to confirm subcellular integrity (sr: synaptic ribbon, po: postsynapse, pr: presynapse, sv: synaptic vesicles, m: mitochondrium). Scale bars: c+e: 250 nm; d+f: 200 nm.

3 Supplementary Figure S3. Localization and synaptic transmission is unaltered in neurons from Syb2-mRFP knock-in mice. (a) Colocalization of Syb2-mRFP with Synaptophysin in DIV14 cultured hippocampal neurons. Scale bar: 10 µm. Magnification in lower panel demonstrates almost perfect overlap between both presynaptic markers. (b-h) Electrophysiological characterization of EPSCs in microisland cultures of hippocampal neurons from wildtype (WT) and Syb2-mRFP mice. (b) Averaged EPSCs of autaptic hippocampal neurons prepared from WT or Syb2-mRFP animals. (c-e) Mean evoked amplitude, charge and time to peak of the evoked EPSCs (wt, black n = 20, Syb2-mRFP, red, n = 22). (f) Exemplary secretory responses to stimulation with hypertonic solution (wt, black, Syb2-mRFP, red). The RRP charge is defined as the time integral over the first 1.5 s after the onset of the sucrose response. Readily-releasable pool charge (g) and release probability (h) are unchanged in Syb2-mRFP neurons.

4 Supplementary Figure S4. LDCV secretion from Syb2-mRFP mouse chromaffin cells is unchanged. (a) Averaged intracellular calcium concentration ([Ca 2+ ] i ; top), capacitance (ΔC m ; middle) and cumulative amperometric charge (Q amp ; bottom) in response to flash photolysis of NP-EGTA in WT (n = 29, black) and Syb2-mRFP cells (n = 28, red). (b) Kinetic analysis of the capacitance responses does not exhibit any significant difference between WT and Syb2-mRFP cells. (c) Upper panels: Example traces of individual fusion events in WT and Syb2-mRFP chromaffin cells measured by carbon fibre amperometry. Individual spike parameters like amplitude, total charge, half-width, rise time and event frequency are similar in WT and Syb2-mRFP cells. In contrast, foot amplitude, foot duration and foot charge are all significantly reduced in Syb2-mRFP cells compared to WT cells (** p < 0.001, Mann-Whitney U test). Data are shown as mean ± SEM.

5 Supplementary Figure S5. Determining the purity of activated CD8 T cells. FACS analysis of activated CD8 T cells using a control IgG-PE antibody (a) and a CD8-PE antibody (b). Inset numbers indicate the percentage of cells above the indicated threshold based on the IgG control.

6 Supplementary Figure S6. CTLs from Syb2-mRFP knock-in mice have normal activation and degranulation behavior. (a) FACS based assay to test the activation capacity of CTLs derived from Syb2-mRFP (left) and C57BL/6 mice (middle), respectively, using a CD25-FITC conjugated antibody. Overlay (right) of CD25- FITC fluorescence in stimulated Syb2-mRFP and C57BL/6 CTLs demonstrates no difference in activation capacity between both groups. (b) FACS based degranulation assay of CTLs derived from Syb2-mRFP (left) and C57BL/6 mice (middle), respectively, using a CD107-FITC conjugated antibody. Overlay (right) of CD107-FITC fluorescence in stimulated Syb2-mRFP and C57BL/6 CTLs demonstrates no difference in degranulation between both groups.