RPH3A localizes to synapses and does not travel on DCVs.

(A) Neurites co-stained for RPH3A (green) and Synapsin-1, Homer, or ChgB (all magenta). White arrows indicate co-localizing puncta whereas green and magenta arrows indicate single RPH3A or Syn1/Homer/ChgB puncta, respectively. Scale bar, 5 µm. (B) Pearson’s correlation coefficient and (C) Mander’s overlap coefficient for each condition including VGLUT1 and syn1 as positive controls. Dots represent a field of view. (D) Kymographs of EGFP-RPH3A and NPY-mCherry, and (E) mCherry-truncated RPH3A and NPY-pHluorin before (upper) and after (lower) photobleaching (black bar). Merge images show mCherry (pseudo-colored magenta) and EGFP/pHluorin (green). Scale bar, 20 µm (x-axis) and 20 s (y-axis). (F) Moving fraction of NPY and FL or truncated RPH3A puncta per kymograph. (G) Fraction of moving NPY puncta with FL or truncated RPH3A puncta, and moving FL/truncated RPH3A with NPY puncta. Dots represent a kymograph. (H) Fluorescent recovery traces after photobleaching of RPH3A, truncated RPH3A and 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 stained for MAP2 (red) and RPH3A (white). Scale bar, 50 µm. (B) RPH3A expression in neurites 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 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+ superfusion dequenches all NPY-pHluorin labeled DCVs (total DCV pool). Scale bar, 5 µm. (D) Kymograph of the 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 second between each train and 30 seconds between each burst, blue bars) and NH4 superfusion (NH4+) used to dequench all NPY-pHluorin labeled vesicles. Arrowheads indicate fusion events. (E) 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 (8 × 50 AP bursts at 50 Hz). (F) Total DCV fusion events per condition (2×8 × 50 AP bursts at 50 Hz). N numbers of individual experiments and single neuron observations in brackets: WT: 5(51); KO: 5(43); KO + RPH3A: 5(39). (G) Released fraction defined as the number of fusion events normalized to the total pool of DCVs. (H) 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 × 50 AP bursts at 50 Hz). (I) Number of DCV fusion events per condition (16 × 50 AP bursts at 50 Hz). N numbers of individual experiments and single neuron observations in brackets: WT: 4(25); KO: 4(18); KO + RPH3A: 4(16). (J) Release fraction per cell. 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. ns = non-significant, P > 0.05.

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

(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 labeled DCVs per neuron for each group. N numbers per condition: WT: 3(24); KO: 3(25). (D) Total ChgB labeled DCVs per µm for each neuron per group. (E) Mean intensity of ChgB labeled DCVs per neuron for each group. (F) Correlation between ChgB labeled DCVs and dendritic length (mm). Linear regression goodness of fit (r2) is given for each group. (G) Total dendritic and (H) axonal length (mm) of WT and KO neurons − / + TeNT, and KO neurons expressing RPH3A + TeNT. N numbers per condition: WT: 3(19); WT + TeNT: 3(27); KO: 3(28). KO + TeNT: 3(26), KO + RPH3A + TeNT: 3(28). Boxplots represent median (line), mean (+) and Tukey range (whiskers). Each dot represents an individual neuron. Mann-Whitney U or unpaired t test: * P < 0.05, ** P < 0.01, ***P < 0.001, **** P < 0.0001. ns = non-significant, P > 0.05.

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

(A) Domain structure 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 RPH3A mutant (yellow). N numbers per condition: WT: 4(27); KO: 4(28); KO + RPH3A: 4(24); KO + ΔRAB3A/RAB27A: 4(28). (C) Released fraction of the number of fusion events normalized by the total DCV pool per cell. (D) Total NPY-pHluorin labeled DCV pool estimates derived from NH4+ superfusion. (E) Total number of DCV fusion events in WT (black), KO (red) and KO neurons expressing RPH3A (cyan) or ΔSNAP25 RPH3A mutant (purple). N number per condition: WT: 4(25); KO: 4(18); KO + RPH3A: 4(16); KO + ΔSNAP25: 4(26). (F) Release fraction per cell. (G) Total NPY-pHluorin labeled DCV pool per cell. 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.

RPH3A localization depends on RAB3A-binding.

(A) 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 domain; 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. (B) Neurite stretches of WT, KO or KO neurons expressing either FL RPH3A, ΔRAB3A/RAB27A, truncated RPH3A, ΔCa2+-binding and ΔCAMKII-dependent phosphorylation site immunostained for MAP2 (green), Tau (blue) and RPH3A (magenta). Scale bar, 5 µm. (C) Neurite stretch of a KO neuron expressing ΔSNAP25 mutant RPH3A immunostained for MAP2 (green) and RPH3A (magenta). Scale bar, 10 µm. (D) Neurite stretch of KO neuron expressing RPH3A co-stained for RPH3A (magenta) and VGLUT1 (green). White arrows indicate co-localizing puncta whereas magenta and green arrows indicate single RPH3A or VLGUT1 puncta. Scale bar, 10 µm. (E) 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). (F) 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 increased DCV exocytosis, but does not affect DCV pool size or content.

(A, B) DCV fusion events per condition for the first and second 8 × 50 AP bursts at 50 Hz. (C) Estimate of the total NPY-pHluorin labeled DCV pool during 2×8 × 50 AP bursts at 50 Hz and (D) 16 × 50 AP bursts at 50 Hz. (E) 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) Typical example of a single RPH3A WT or KO neuron expressing TeNT 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 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). Each dot represents a single neuron. Kruskal-Wallis H test with Dunn’s correction: * P < 0.05. ns = non-significant, P > 0.05.