Supplementary Materials for VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission Jesica Raingo, Mikhail Khvotchev, Pei Liu, Frederic Darios, Ying C. Li, Denise M.O. Ramirez, Megumi Adachi, Philippe Lemieux, Katalin Toth, Bazbek Davletov and Ege T. Kavalali Supplementary Figure 1: Limited co-localization of VAMP4 staining with Parvalbumin positive terminals at the stratum pyramidale. Supplementary Figure 2: Wide-spread distribution of syb2 in synaptic terminals in the stratum radiatum of the CA1 area. Supplementary Figure 3: Endogenous VAMP4 is present in synapses formed in culture. Supplementary Figure 4: Parvalbumin-positive inhibitory interneurons are sparsely distributed in high density hippocampal cultures. Supplementary Figure 5: IPSCs evoked in response to paired pulse stimulation (P2/P1) showed a significant increase indicating the reduced release probability of synapses expressing VAMP4. Supplementary Figure 6: Knockdown of VAMP4 in hippocampal neurons using two lentiviral short hairpin RNA constructs Supplementary Figure 7: Prominent surface expression of VAMP4 equals the size of the cytoplasmic VAMP4 pool. Supplementary Figure 8: The VAMP4 L25A mutant forms stable SNARE complexes with syntaxin1 and SNAP-25. Supplementary Figure 9: VAMP4 L25A mutant maintains evoked neurotransmission when expressed on the background of syb2-deficient neurons. Supplementary Figure 10: The model of syb2- and VAMP4-driven synaptic transmission. 1
Supplementary Figure 1: Limited co-localization of VAMP4 staining with Parvalbumin positive terminals at the stratum pyramidale. Immunofluorescence labeling for VAMP4 (green) and PV (red) in the CA1 area of the hippocampus. Colocalization occurs in the soma of PV-positive interneurons (upper row, arrow), but not in presynaptic terminals (s.p. stratum pyramidale, upper row; lower row) where PV-positive terminals surrounding somas of pyramidal cells are VAMP4-negative. However, it is difficult to exclude some low level of co-localization, as pyramidal cell soma staining for VAMP4 dominates at the stratum pyramidale. Scale bar: 15 µm 2
Supplementary Figure 2: Wide-spread distribution of syb2 in synaptic terminals in the stratum radiatum of the CA1 area. Electron micrographs showing immunoreactivity for syb2 in the str. radiatum of the CA1 area. Large panel on the left: Several presynaptic terminals are showing positive syb2-staining, dark staining is the result of the immunoperoxidase reaction, two presynaptic terminals are shown with higher magnification on the right. Bottom right panel: Immunogold labeling for syb2 of a presynaptic terminal in the str. radiatum of the CA1 area. Scale bars: 1 µm 3
Supplementary Figure 3: Endogenous VAMP4 is present in synapses formed in culture. Neurons were processed for immunocytochemistry using a tyramide signal amplification system for synaptophysin I (synaptic marker) and VAMP4. White arrowheads indicate synaptic puncta colocalized with VAMP4 immunoreactivity. Scale bar is 10 µm. 4
Supplementary Figure 4: Parvalbumin-positive inhibitory interneurons are sparsely distributed in high density hippocampal cultures. Neurons were processed for immunocytochemistry for synapsin (synaptic marker) and parvalbumin. Scale bar is 40 µm. 5
Supplementary Figure 5: IPSCs evoked in response to paired pulse stimulation (P2/P1) showed a significant increase indicating the reduced release probability of synapses expressing VAMP4. Representative traces (left) and paired pulse ratios (right) of IPSCs evoked in response to two stimuli separate by a 100 ms interpulse interval in syb2 deficient neurons infected with VAMP4 (n=31) or syb2 (n=35) (data from the same cells as in Fig. 1a). Bars represent mean ± standard error of the mean (s.e.m.) and "*" denotes statistical significance between the groups assessed by one-way ANOVA-Fisher test at p<0.05. 6
Supplementary Figure 6: Knockdown of VAMP4 in hippocampal neurons using two lentiviral short hairpin RNA constructs (A) Bar graph depicts the reduction in mrna levels (quantified with Q-PCR) after infection with VAMP4 knockdown constructs KD1 and KD2. (B) Reduction in VAMP4 protein is also detectable as a decrease in VAMP4 immunofluorescence in infected neurons stained with VAMP4 and synapsin antibodies. 7
Supplementary Figure 7: Prominent surface expression of VAMP4 equals the size of the cytoplasmic VAMP4 pool. In this experiment we modified the external and internal ph values to assess the localization of VAMP4. Bath application of an acidified (ph 4) solution onto cultured neurons infected with VAMP4 lentivirus caused a decrease in fluorescence consistent with quenching of phluorin fluorescence at the membrane surface. The figure shows averaged time courses (n= 58 boutons; VAMP4) in control conditions (ph7.2) and in presence of acidic external solution (ph 4). After wash-out of the acidic solution, we applied 50 mm NH 4 Cl containing extracellular solution. In this case, VAMP4 showed increased fluorescence (nearly equal to the surface fraction) consistent with alkalinization of internal organelles containing this protein. 8
Supplementary Figure 8: The VAMP4 L25A mutant forms stable SNARE complexes with syntaxin1 and SNAP-25. (A) VAMP4 L25A and VAMP4 ΔN (The N-terminal sequence of VAMP4 prior to SNARE motif is deleted) forms stable SDS-resistant complex with Syntaxin 1 and SNAP-25 similar to syb2 or wild type VAMP4 (Fig. 3). Experiments were performed as described in Fig. 5a. (B) VAMP4 L25A and VAMP4 ΔN containing SNARE complexes do not engage with complexins 1&2 or synaptotagmin 1. Experiments were performed as described in Fig. 5c. 9
Supplementary Figure 9: VAMP4 L25A mutant maintains evoked neurotransmission when expressed on the background of syb2-deficient neurons. Representative traces (left) and average cumulative charge transfer values (right) of IPSCs recorded in syb2 deficient neurons infected with VAMP4 L25A (n=8 cells) and VAMP4 (n=31 cells). IPSCs were evoked by 50 APs applied at 10 Hz. Data represent means ± s.e.m. (no statistically significant difference between the groups as measured by two tailed t-test). 10
Supplementary Figure 10: The model of syb2- and VAMP4-driven synaptic transmission. Presynaptic terminals contain syb2-enriched vesicles (green) that mainly drive synchronous release and VAMP4-enriched vesicles that selectively support asynchronous release (red). Stimulation-dependent trafficking of VAMP4 suggests that activity could shift the proportion of vesicles enriched in VAMP4 and modulate the kinetics of neurotransmitter release. 11