Figures and data
Rab10 is required for neuronal outgrowth but dispensable for SV exocytosis evoked by intense stimulations.
(A) Representative immunoblotting showing knockdown and rescue of Rab10 expression in cultured primary neurons infected with shRNA against Rab10 or rescue constructs (upper) and quantification of Rab10 levels (bottom).
(B) Example images of control or Rab10 KD hippocampal neurons (DIV14) stained for the dendrite marker MAP2 (blue), the synapse marker Syp1 (red), and the axonal marker SMI312 (green). Scale bar: 50 μm (upper) and 10 μm (bottom).
(C) Quantification of the dendritic length (MAP2).
(D) Quantification of the axonal length (SMI312).
(E) Quantification of Syp1 intensity per synapse per neuron.
(F) Quantification of the Syp1-positive synapse density in MAP2-positive dendrites.
(G) Sholl analysis showing the mean number of dendritic branches against the distance from the soma.
(H) Example neurons infected with the SV fusion marker SypHy (upper), typical kymographs of neurites showing SypHy intensity increase during stimulation and upon NH4Cl superfusion (bottom).
(I) The average signal SypHy from active synapses, normalized from baseline to maximum fluorescence upon NH4Cl superfusion.
(J) SV exocytosis determined as the ratio of the maximum SypHy intensity during stimulation to the maximum during NH4Cl stimulation.
(K) SV endocytosis determined as the SypHy signal decay time constant τ in the 60 s after field stimulation.
All Data are plotted as mean±s.e.m. (A) N = 4, n = 4, one sample t-test. (C-G) Control: N = 3, n = 35; ShRNA#9: N = 3, n = 32. (J, K) Control: N = 3, n = 56; ShRNA#9: N = 3, n = 56. (C-F, J, K) A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001, ** = p<0.01, ns = not significant.
Rab10 is a major regulator of DCV exocytosis.
(A) Example images of control and Rab10 KD hippocampal neurons (DIV14) stained for MAP2 (blue), Syp1 (red) and SMI312 (green). Scale bar: 30 μm.
(B) Quantification of the dendritic length (MAP2).
(C) Quantification of the axonal length (SMI312).
(D) Quantification of the Syp1-positive synapse density in MAP2-positive dendrites.
(E) Sholl analysis showing the mean number of dendritic branches against the distance from the soma.
(F) Schematic representation of DCV fusion assay. DCVs are labeled with NPY-pHluorin, and neurons are stimulated with one train of 16 bursts of 50 APs at 50 Hz (light blue bars).
(G) Representative neurons during electrical stimulation superimposed with NPY-pHluorin fusion events (green dots). Scale bar: 5 μm.
(H) Cumulative plot of DCV fusion events per cell. Light blue bars represent the stimulation trains.
(I) Summary graph of DCV fusion events per cell.
(J) The total number of DCVs (total pool) of neurons analyzed in Figure 2H, measured as the number of NPY-pHluorin puncta upon NH4Cl perfusion.
(K) Fraction of NPY-pHluorin-labeled DCV fusing during stimulation.
All Data are plotted as mean±s.e.m. (B-D) Control: N = 3, n = 31; ShRNA#9: N = 3, N = 28; ShRNA#11: N = 3, n = 31. (I-K) Control: N = 4, n = 36; shRNA#9: N = 4, N = 37; shRNA#11: N = 4, n = 30; Rescue: N = 4, n = 34. A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001, *** = p<0.001, ** = p<0.01, * = p <0.05, ns = not significant.
Rab10 depletion at DIV0 impedes DCV fusion.
(A) Schematic representation of DCV fusion assay. DCVs are labeled with NPY-pHluorin, and neurons are stimulated with one train of 16 bursts of 50 APs at 50 Hz (light blue bars).
(B) Representative neurons during electrode stimulation superimposed with NPY-pHluorin fusion events (green dots). Scale bar: 10 μm.
(C) Cumulative plot of DCV fusion events per cell.
(D) Summary graph of DCV fusion events per cell.
(E) Total number of DCVs (total pool) of neurons, measured as the number of NPY-pHluorin puncta upon NH4Cl perfusion.
(F) Fraction of NPY-pHluorin-labeled DCV fusing during stimulation.
All Data are plotted as mean±s.e.m. (D-F) Control: N = 3, n = 26; Rab10 KD: N = 3, n = 47; Rescue: N = 3, n = 22. (D-F) A one-way ANOVA tested the significance of adding experimental group as a predictor. *** = p<0.001, ** = p<0.01, * = p <0.05.
Rab10 depletion does not affect DCV transport or cargo loading.
(A) Representative kymographs illustrating the transport of NPY-mCherry labeled DCVs in control and Rab10 KD neurons.
(B) Quantification of average velocity (µm/s) of control and Rab10 KD neurons.
(C) Quantification of average distance moved from the start (µm) of control and Rab10 KD neurons.
(D) Histogram of average velocity (µm/s) of control and Rab10 KD neurons.
(E) Histogram of average distance moved from the start (µm) of control and Rab10 KD neurons.
(F) Typical neurite expressing NPY-pHluorin during baseline (b), and during stimulation (s).
(G) Average traces of NPY-pHluorin fusion events aligned at the moment of fusion (0 seconds).
(H) Quantification of NPY-pHluorin baseline fluorescence before stimulation.
(I) Quantification of average NPY-pHluorin fusion intensity per cell.
All Data are plotted as mean±s.e.m. (B, C) Control: N = 3, n = 18; Rab10 KD: N = 3, n = 17. (H, I) Control: N = 3, n = 37; Rab10 KD: N = 3, n = 35. A one-way ANOVA tested the significance of adding experimental group as a predictor. ns = not significant.
Rab10 does not typically co-transport together with DCVs.
(A) Representative kymographs of neurons co-infected with Rab10-GFP and NPY-mCherry.
(B) Percentage moving DCVs that co-transport with Rab10. Data are plotted as mean±s.e.m (N = 3, n = 22).
Data are plotted as mean±s.e.m.
Depletion of Rab10 leads to dysregulation of proteins enriched in presynaptic transmission and cytosolic translation.
(A) Volcano plots showing significantly dysregulated proteins in Rab10 depleted neurons.
(B) GO enrichment analysis of functional pathways of the significant hits with ClueGO. Shown are the Bonferroni corrected p-values.
(C) GO enrichment analysis of subcellular localization of the significant hits with ClueGO. Shown are the Bonferroni corrected p-values.
(D) Sunburst plot showing the annotation in synaptic function of the altered proteins in Rab10 depleted neurons.
(E) Sunburst plot showing the annotation in synaptic location of the altered proteins in Rab10 depleted neurons.
(F) Log2 fold changes of synaptic proteins within SynGO terms. Downregulated proteins are shown in blue and upregulated proteins are shown in black.
(G) Examples of proteins that are significantly affected by Rab10 depletion grouped by their subcellular localization. Heat maps represent the degree of up-or downregulation.
(H) Selective MS data analysis of ER-related proteins in Rab10 KD neurons. Bars show the fold change of the indicated peptides compared to the control.
Rab10 regulates synapse size and ER morphology.
(A) Representative EM pictures showing the ultrastructure of synapses. Scale bar 100 nm. Synaptic ER is indicated by red dotted lines.
(B) Representative EM pictures showing the ultrastructure of soma. Rough ER (rER) is indicated by red dotted lines. M: mitochondrion, G: Golgi. Scale bar: 100 nm.
(C) Quantification of the length of active zone and postsynaptic density (PSD).
(D) Quantification of the length of postsynaptic density (PSD).
(E) Quantification of SV number per synapse and SV diameter.
(F) Quantification of SV diameter.
(G) Quantification of DCV diameter.
(H) Frequency distribution of DCVs by diameter.
(I) Quantification of the diameter of rough ER (rER).
Data are plotted with Superplot (Figure. 4C-G, I), where averages from three independent cultures are shown as large circles and single observations are shown as dots. Horizontal lines represent the means of the averages from three weeks. Data from different cultures are grouped with different colors. (C-D) Control: N = 3, n = 184; shRNA#9: N = 3, n = 187. (E) Control: N = 3, n = 189; shRNA#9: N = 3, n = 188. (F) Control: N = 3, n = 1770; shRNA#9: N = 3, n = 1803. (G) Control: N = 3, n = 137; shRNA#9: N = 3, n = 122. (I) Control: N = 3, n = 63; shRNA#9: N = 3, n = 64. (C-G, I) linear mixed model analysis. *** = p<0.001, * = p <0.05, ns = not significant.
Altered ER morphology in Rab10 KD neurons.
(A) Example images of control or Rab10 KD hippocampal neurons (DIV14) stained for the dendrite marker MAP2 (green), two ER markers KDEL (red) and RTN4 (magenta). Scale bar: 50 μm.
(B) Quantification of RTN4 intensity in MAP2-positive dendrites.
(C) Quantification of KDEL intensity in MAP2-positive dendrites.
(D) The ratio of neuritic to somatic RTN4 intensity (N/S).
(E) The ratio of neuritic to somatic KDEL intensity (N/S).
All Data are plotted as mean±s.e.m. (B-D) Control: N = 3, n = 18; Rab10 KD: N = 3, n = 18; (B-D) A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001, *** = p<0.001, ** = p<0.01, * = p <0.05.
Impaired ER dynamics in Rab10 KD neurons.
(A) Representative time-lapse of ER-mCherry3 signal before (upper), upon (middle) and after (bottom) photobleaching.
(B) Average normalized ER-mCherry3 fluorescence recovery after photobleaching in control and Rab10 KD hippocampal neurons.
(C) Normalized ER-mCherry3 fluorescence recovery after photobleaching at T = 220 s in control and Rab10 KD hippocampal neurons.
All Data are plotted as mean±s.e.m. (B, C) Control: N = 3, n = 23; Rab10 KD: N = 3, n = 23; (B, C) A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001.
Rab10 depletion does not increase ER stress.
(A) Representative images of wild-type (WT) neurons treated with vehicle (top) or tunicamycin (TM, middle) and Rab10 KD neurons treated with vehicle (bottom). Neurons were stained for ATF4 and MAP2. Scale bar: 50 μm.
(B) Quantification of ATF4 intensity in soma from each condition. All data are presented as mean±s.e.m. WT + vehicle: N = 2, n = 25; WT + vehicle: N = 2, n = 30; Rab10 KD + vehicle: N = 2, n = 14. A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001, ns = not significant.
Reduced SERCA2 levels and impaired ER Ca2+ homeostasis in Rab10 KD neurons.
(A) Typical immunoblot showing reduced SERCA2 levels in Rab10 KD hippocampal neurons.
(B) Quantification of protein levels in Rab10 KD neurons normalized to control.
(C) Quantification of somatic ER Ca2+ concentration.
(D) Quantification of dendritic ER Ca2+ concentration.
(E) Representative image of a neuron infected with ER-GCaMP6-150 displayed with a pseudo line. Scale bar: 3 μm.
(F) Typical kymographs of the somatic intensity of ER-GCaMP6-150 showing the intensity decrease upon caffeine superfusion (red line), and the recovery in intensity after caffeine washout. Scale bar: 10 s.
(G) Average normalized ER-GCaMP6-150 fluorescence recovery after caffeine treatment.
(H) Normalized ER-GCaMP6-150 fluorescence recovery after caffeine treatment at T = 190 s. All Data are plotted as mean±s.e.m. (B) Control: N = 4, n = 4; Rab10 KD: N = 4, n = 4; (C-D) Control: N = 3, n = 17; Rab10 KD: N = 3; n = 17; Rescue: N = 3, n = 17 (H) Control: N = 3, n = 23; Rab10 KD: N = 3; n = 24; GDP-Rab10: n =3, n = 10; Rescue: N = 3, n = 24. A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001, *** = p<0.001, ** = p<0.01, ns = not significant.
Caffeine triggers less ER Ca2+ release in Rab10 KD neurons.
(A) Left: representative cytosolic Fluo-5 AM signals upon Caffeine perfusion. Right: representative kymographs of cytosolic Fluo-5 AM signals upon caffeine perfusion in somas.
(B) Average traces of Fluo-5 AM signals.
(C) Quantification of the peak values of the Fluo-5 AM fluorescence traces upon caffeine perfusion.
(D) Quantification of the area under the curve (AUC) of the Fluo-5 AM fluorescence traces upon caffeine perfusion.
All Data are plotted as mean±s.e.m. (C, D) Control: N = 3, n = 44; Rab10 KD: N = 3, n = 35; (C, D) A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001, ** = p<0.01.
Impaired neuronal Ca2+ influx triggered by electrical stimulation.
(A) Representative time-lapse of cytosolic Fluo-5 AM upon electrical stimulation (16 APs, 50Hz) in somas of hippocampal neurons. Scale bar: 10 μm.
(B) Average normalized response of somatic Fluo-5 AM fluorescence upon stimulation (16 APs, 50Hz) in hippocampal neurons.
(C) Quantification of the area under the curve (AUC) of the Fluo-5 AM fluorescence traces.
(D) Typical neurons infected with Synaptophysin-GCaMP6 (upper), typical kymograph of a neurite (bottom) showing Synaptophysin-GCaMP6 intensity increase upon electrical stimulation (16 APs, 50Hz, blue bars). Scale bar: 5μm.
(E) Average normalized response of Synaptophysin-GCaMP6 fluorescence intensity at presynaptic boutons upon stimulation (16 APs, 50Hz) in hippocampal neurons.
(F) Quantification of the area under the curve (AUC) of the Synaptophysin-GCaMP6 fluorescence traces in control and Rab10 KD neurons.
All Data are plotted as mean±s.e.m. (C) Control: N = 4, n = 24; Rab10 KD: N = 4, n = 30; Rescue: N = 4, n = 27. (F) Control: N = 3, n = 33; Rab10 KD: N = 3; n = 27. A one-way ANOVA tested the significance of adding experimental group as a predictor. ** = p<0.01, * = p <0.05, ns = not significant.
Impaired DCV fusion induced by ionomycin in Rab10 KD neurons.
(A) Representative neurons during electrical stimulation superimposed with NPY-pHluorin fusion events (green dots). Scale bar: 10 μm.
(B) Cumulative plot of DCV fusion events per cell.
(C) Fraction of NPY-pHluorin-labeled DCVs fusing during stimulation.
(D) The total number of DCVs (total pool) of neurons analyzed in Figure 7B, measured as the number of NPY-pHluorin puncta upon NH4Cl perfusion.
All Data are plotted as mean±s.e.m. (C, D) Control: N = 3, n = 20; Rab10 KD: N = 3, n = 21. (C, D) A one-way ANOVA tested the significance of adding experimental group as a predictor. * = p <0.05, ns = not significant.
Leucine supplementation ameliorates the deficits in protein synthesis and neuropeptide release in Rab10 KD neurons.
(A) Representative western blot showing puromycinilated proteins as a measure for de novo protein synthesis in each condition.
(B) Quantification of puromycin intensity in each condition.
(C) Representation of the DCV fusion assay. Leucine (5 μM) was added to the culture media and incubated for 72 h before DCV fusion assay. DMSO (1‰) was used as a control.
(D) Cumulative plot of DCV fusion events per cell.
(E) Fraction of NPY-pHluorin-labeled DCVs fusing during stimulation.
(F) The total number of DCVs (total pool) of neurons analyzed in Figure 8D, E, measured as the number of NPY-pHluorin puncta upon NH4Cl perfusion.
All Data are plotted as mean±s.e.m. (B) All: N = 3, n = 3 (E, F) Control: N = 3, n = 47; Control + leu: N = 3, n = 45; Rab10 KD: N = 3; n = 61; Rab10 + leu: N = 3, n = 54. Rab10 KD + Rab10: N = 3, n = (B) One sample t-test. (E, F) A one-way ANOVA tested the significance of adding experimental group as a predictor. ** = p<0.01, * = p <0.05, ns = not significant.
Rab10 depletion does not upregulate mTORC1 pathway
(A) Typical immunoblot showing pS6K1 levels in each condition.
(B) Quantification of relative pS6K1 levels in each condition. All Data are plotted as mean±s.e.m. (B) Control, Control + Leu: N = 2, n = 2, Rab10 KD, Rab10 KD + Leu: N = 2, n = 4.
Leucine supplementation does not rescue ER morphological deficiency in Rab10 KD neurons
(A) Typical examples showing the KDEL signals in each condition
(B) Quantification of RTN4 intensity in MAP2-positive dendrites.
(C) The ratio of neuritic to somatic RTN4 intensity (N/S).
All Data are plotted as mean±s.e.m. (B, C) Control: N = 3, n = 10; Rab10 KD: N = 3, n = 11; Rab10 KD + Leu: N = 3; n = 11. A one-way ANOVA tested the significance of adding experimental group as a predictor. **** = p<0.0001, ns = not significant.
Overexpression of SERCA2 does not resuce the DCV fusion deficits in Rab10 KD neurons.
(A) Typical examples showing the SERCA2 signals in each condition
(B) Cumulative plot of DCV fusion events per cell
(C) Summary graph of DCV fusion events per cell.
(D) Total number of DCVs (total pool) of neurons, measured as the number of NPY-pHluorin puncta upon NH4Cl perfusion.
(E) Fraction of NPY-pHluorin-labeled DCV fusing during stimulation.
All Data are plotted as mean±s.e.m. (C-E) Control: N = 2, n = 10; Rab10 KD: N = 2, n = 13; SERCA2 OE: N = 2; n = 15. A one-way ANOVA tested the significance of adding experimental group as a predictor. *** = p<0.001, ** = p<0.01, ns = not significant.
