RPH3A localizes to the pre-synapses.

(A) Representative images of WT hippocampal neurons (top) with zooms (bottom) co-stained for RPH3A (blue) and Syn1 (magenta), Homer (green) or ChgB (yellow). Scale bar, 5 µm (top) and 2 µm (bottom). (B, C, D) Line plots show normalized fluorescent intensity across dotted line in example zooms. (E) Pearson’s correlation coefficient and (F) Mander’s overlap coefficient comparing the colocalization of RPH3A with Syn1, Homer and ChgB. (G) Mander’s overlap coefficient comparing the colocalization of RPH3A in either Syn1, Homer or ChgB puncta, and vice versa. RPH3A:Syn1, RPH3A:Homer and RPH3A:ChgB show the same dataset as in F. Dots represent a field of view. Boxplots represent the median (line), mean (+) and Tukey range (whiskers). Each dot represents a field of view: Syn1: 2(10); Homer 2(20); ChgB: 2(10). Kruskal-Wallis H test with Dunn’s correction: * P < 0.05, ** P < 0.01, **** P < 0.0001. ns = non-significant, P > 0.05.

RPH3A does not travel with DCVs.

(A) Domain structures of FL RPH3A and mutant RPH3A constructs lacking specific interactions: ΔRAB3A/RAB27A mutant RPH3A and truncated RPH3A that lacked its calcium and SNAP25 binding C2A and C2B domain. (B) Kymographs of EGFP-RPH3A and NPY-mCherry, (C) mCherry-truncated RPH3A and NPY-pHluorin, and (D) mCherry-ΔRAB3A/RAB27A RPH3A and NPY-pHluorin before (upper) and after (lower) photobleaching (black bar) to remove most stationary puncta and facilitate analysis. NPY-pHluorin showed more resistance for bleaching. Merged images show mCherry (pseudo-coloured magenta) and EGFP/pHluorin (green). Scale bar, 20 µm (x-axis) and 20 s (y-axis). (E) Moving fraction of NPY, FL RPH3A and truncated RPH3A puncta per kymograph. (F) Fraction of co-travel of NPY puncta with either FL- or truncated RPH3A puncta, and co-travel of FL- or truncated RPH3A with NPY puncta. Dots represent a kymograph. (G) Fluorescent recovery of the traces shown in Figure 2C after photobleaching truncated RPH3A or NPY-pHluorin. (H) Mean fluorescent recovery traces from multiple kymographs after photobleaching FL-, truncated- or ΔRAB3A/RAB27A RPH3A. Lines ± shading represents mean ± SEM. Boxplots represent median (line), mean (+) and Tukey range (whiskers).

RPH3A deficiency increases DCV exocytosis.

(A) Typical example of RPH3A WT and KO neurons immunostained for MAP2 (red) and RPH3A (white). Scale bar, 50 µm. (B) RPH3A expression in dendrites of WT and KO neurons normalized to WT per independent experiment. N numbers of individual experiments and single neuron observations in brackets: WT: 4(34); KO: 4(33). (C) Schematic representation (left) and imaging example of a WT neurite stretch (right) infected with NPY-pHluorin as optical DCV fusion reporter. NPY-pHluorin is quenched in the acidic DCV lumen before fusion (baseline) but dequenches upon fusion (stimulation). NH4+ perfusion dequenches all NPY-pHluorin labelled DCVs (remaining DCV pool). Scale bar, 5 µm. (D) Kymograph of a WT neurite stretch with the stimulation paradigm used to elicit DCV fusion (two bursts of 8 x 50 AP trains at 50 Hz interspaced by 0.5 s between each train and 30 s between each burst, blue bars) and NH4 perfusion (NH4+) used to dequench all NPY-pHluorin labelled vesicles. Arrowheads indicate fusion events. (E) Domain structure of FL RPH3A construct. (F) Cumulative median histogram of fusion events over time in WT (black), RPH3A KO (red) and KO neurons infected with FL RPH3A (cyan). Blue bars indicate the stimulation paradigm (two bursts of 8 x 50 AP bursts at 50 Hz). (G) Total number of DCV fusion events per condition (two bursts of 8 x 50 AP bursts at 50 Hz). (H) Released fraction defined as the number of fusion events normalized to the remaining pool of DCVs. N numbers of individual experiments and single neuron observations in brackets: WT: 5(51); KO: 5(43); KO + RPH3A: 5(39). (I) Cumulative median histogram of events over time in WT (black), RPH3A KO (red) and KO neurons infected with FL RPH3A (cyan). Blue bars indicate the stimulation paradigm (16 x 50 AP bursts at 50 Hz). (J) Total number of DCV fusion events per condition (16 x 50 AP bursts at 50 Hz). (K) Release fraction per cell. N numbers of individual experiments and single neuron observations in brackets: WT: 4(25); KO: 4(18); KO + RPH3A: 4(16). Boxplots represent the median (line), mean (+) and Tukey range (whiskers). Each dot represents an individual neuron. Kruskal-Wallis H test with Dunn’s correction: * P < 0.05, ** P < 0.01, **** P < 0.0001. ns = non-significant, P > 0.05.

RPH3A interaction with SNAP25, but not RAB3A, partly contributes to limiting DCV exocytosis.

This Figure shows the same dataset for WT, KO and FL RPH3A as in Figure 3. (A) Domain structures of FL RPH3A (cyan), ΔRAB3A/RAB27A- (yellow) and ΔSNAP25 mutant RPH3A (purple) with the corresponding mutant sites in red. (B) Total DCV fusion events in WT (black), KO (red) and KO neurons expressing RPH3A (cyan) or ΔRAB3A/RAB27A mutant RPH3A (yellow). (C) Released fraction of the number of fusion events normalized to the remaining DCV pool per cell. (D) Remaining NPY-pHluorin labelled DCV pool estimates derived from NH4+ perfusion after stimulation. N numbers of individual experiments and single neuron observations in brackets: WT: 4(27); KO: 4(28); KO + RPH3A: 4(24); KO + ΔRAB3A/RAB27A: 4(28). (E) Total number of DCV fusion events in WT (black), KO (red) and KO neurons expressing RPH3A (cyan) or ΔSNAP25 mutant RPH3A (purple). (F) Release fraction per cell. (G) Remaining NPY-pHluorin labelled DCV pool per cell. N numbers of individual experiments and single neuron observations in brackets: WT: 4(25); KO: 4(18); KO + RPH3A: 4(16); KO + ΔSNAP25: 4(26). Boxplots show the mean (+), median (line) and Tukey range (whiskers). Each dot represents a single neuron. Kruskal-Wallis H test with Dunn’s correction: * P < 0.05, ** P < 0.01. ns = non-significant, P > 0.05.

Increased neurite length and DCV number upon RPH3A deficiency.

(A) Typical example of a single WT and RPH3A KO hippocampal neuron (top) with zooms (bottom) stained for MAP2 (green) and the DCV marker ChgB (magenta). Scale bars, 50 µm (top) and 20 µm (bottom). (B) Total dendritic length of single hippocampal RPH3A WT or KO neurons normalized to WT per independent experiment. N numbers per condition: WT: 14(112); KO: 14(113). (C) Total number of ChgB labelled DCVs per neuron for each group. N numbers per condition: WT: 3(24); KO: 3(25). (D) Total ChgB labelled DCVs per µm for each neuron per group. (E) Mean intensity of ChgB labelled DCVs per neuron for each group. (F) Correlation between ChgB labelled DCVs and dendritic length (mm). Linear regression goodness of fit (r2) is given for each group. Boxplots represent median (line), mean (+) and Tukey range (whiskers). Each dot represents an individual neuron. Mann-Whitney U or unpaired t test: ** P < 0.01, ***P < 0.001, **** P < 0.0001.

Increased neurite length upon RPH3A deficiency partly depends on regulated secretion.

(A) Typical example of a single RPH3A KO neurons either infected with TeNT or not, showing successful VAMP2 cleavage with zooms (bottom) stained for MAP2 (green), Tau (blue) and VAMP2 (magenta). Scale bars, 50 µm (top) and 20 µm (bottom). (B) Total dendritic and (C) axonal length (mm) of WT and KO neurons - / + TeNT, and KO neurons expressing RPH3A. N numbers per condition: WT: 3(19); WT + TeNT: 3(27); KO: 3(28). KO + TeNT: 3(26), KO + RPH3A: 3(28). Boxplots represent median (line), mean (+) and Tukey range (whiskers). Each dot represents an individual neuron. Kruskal-Wallis H test with Dunn’s correction: * P < 0.05, ** P < 0.01, ***P < 0.001, **** P < 0.0001. ns = non-significant, P > 0.05.

Role of RPH3A in DCV exocytosis.

(A) RPH3A binding to RAB3 through its RAB-binding domain (RBD) ensures confined presynaptic localization of RPH3A. RPH3A binding to SNAP25 through its C2B domain inhibits DCV exocytosis, potentially by inhibiting SNAP25-binding of essential DCV proteins synaptobrevin/VAMP2 and/or synaptotagmin (not depicted). (B) In the absence of RPH3A, DCV exocytosis is not limited. (C) Upon expression of a RPH3A mutant that is unable to bind RAB3A/RAB27A, DCV exocytosis is limited. (D) When RPH3A is unable to bind SNAP25, the SNARE assembly is not restricted and therefore DCV exocytosis is not limited, while RPH3A is still recruited to synapses/release sites via RAB3.

RPH3A localization depends on RAB3A/RAB27A-binding.

(A) Representative images of WT hippocampal and striatal neurons stained for RPH3A (white), VGLUT/VGAT (not shown) and MAP2 (red). N numbers of individual experiments and single neuron observations in brackets: HC: 2(13); STR: 2(12). Scale bar, 50 µm. (B) RPH3A intensity in MAP2+ neurite mask in hippocampal and striatal neurons, normalized to batch. (C) Domain structures of FL RPH3A and mutant RPH3A constructs lacking specific interactions: ΔRAB3A/RAB27A; truncated RPH3A that lacked its calcium and SNAP25 binding C2A and C2B domains; RPH3A with two C2B domain mutations to reduce calcium binding affinity (ΔCa2+ binding); RPH3A that lacked a CaMKII dependent phosphorylation site (ΔCAMKII-dependent phosphorylation site) and ΔSNAP25. The corresponding mutation sites are indicated in red. (D, E) Representative STED images of WT neurons expressing either FL RPH3A, ΔRAB3A/RAB27A, truncated RPH3A, ΔCa2+-binding, ΔCAMKII-dependent phosphorylation site or ΔSNAP25, immunostained for mCherry (pseudo-colored blue), MAP2 (grey), Syn1 (magenta) or Homer (green). Scale bar, 5 µm. (F) Mander’s overlap coefficient analyses of mCherry with either Syn1 or Homer, in WT neurons expressing FL or mutant RPH3A constructs. (G) RPH3A expression in KO neurons expressing FL RPH3A or mutant RPH3A constructs normalized to WT per independent experiment. N numbers per condition: KO + RPH3A: 6(24); KO + ΔRAB3A/RAB27A: 3(12); truncated RPH3A: 6(28); KO + ΔCa2+ binding: 5(23); and KO + ΔCAMKII-dependent phosphorylation: 2(9). (H) RPH3A expression in KO neurons expressing FL RPH3A or mutant RPH3A unable to bind SNAP25. N numbers per condition: KO + RPH3A: 1(3); KO + ΔSNAP25: 1(6). Each dot represents a single neuron. Kruskal-Wallis H test with Dunn’s correction: ns = non-significant, P > 0.05.

RPH3A depletion increases DCV exocytosis, but does not affect remaining DCV pool size or content.

(A, B) DCV fusion events per condition for the first and second 8 x 50 APs bursts at 50 Hz. (C) Estimate of the remaining NPY-pHluorin labelled DCV pool during 2 x 8 x 50 APs at 50 Hz. (D, E) Fusion duration during first and second 2 x 8 burst stimulation protocol. (F) Fusion duration during 16 x 50 Hz stimulation protocol. (G) Estimate of the remaining NPY-pHluorin labelled DCV pool during 16 x 50 APs at 50 Hz. (H) The number of spontaneous DCV fusion events per cell during baseline. (I) Mean peak intensity of NPY-pHluorin during live imaging per independent experiment. The black dashed line represents the median. Boxplots show the mean (+), median (line) and Tukey range (whiskers). Each dot represents a single neuron. Kruskal-Wallis H test with Dunn’s correction: * P < 0.05, ** P < 0.01. ns = non-significant, P > 0.05.

Increased neurite length upon RPH3A depletion does not depend on RAB3A/RAB27A-binding, calcium binding or phosphorylation of RPH3A.

(A) Total dendritic and (B) axonal length of WT, KO and KO neurons expressing FL or mutant RPH3A (ΔRAB3A/RAB27A, truncated, ΔCa2+ binding or ΔCAMKII-dependent phosphorylation). N numbers per condition: WT 3(25); KO 3(28); KO + RPH3A: 3(29); KO + ΔRAB3A/RAB27A: 3(32); truncated RPH3A: 3(29); KO + ΔCa2+ binding: 3(30); and KO + ΔCAMKII-dependent phosphorylation: 3(30). (C) Total dendritic length of WT, KO and KO neurons expressing ΔSNAP25 mutant. N numbers per condition: WT 3(17); KO 3(21); KO + ΔSNAP25: 3(20). Each dot represents a single neuron. Kruskal-Wallis H test with Dunn’s correction: * P < 0.05. ns = non-significant, P > 0.05.