EMBO/soda-nel huggingface dataset:
CPAP promotes timely cilium disassembly to maintain neural progenitor pool (2016)
Figure 2

Fig. 2.

CPAP plays a role in cilium disassembly

a. Experimental plan. Both Seckel and WT cells were synchronized at G0 by serum starvation for 96 hrs. This treatment causes Seckel and WT cells to have equivalent cilia length allowing us to estimate the rate of cilia disassembly (see below and Appendix Fig. S2a).

b, Dynamic CPAP localization during cilium disassembly and cell cycle re-entry. Cep152 levels remain unchanged. Arl13b (green) or acetylated α-tubulin (magenta) marks the cilium. n > 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.

c, Seckel cells show neither CPAP dynamics nor cilium disassembly upon serum stimulation. Cep152 levels remain constant. n > 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.

d, Cilium disassembly dynamics in WT and Seckel cells. Relative cilium disassembly rate shows retarded cilium disassembly in Seckel cells (Error bars are s. e. m.) (ii). Quantification and distribution of cilia length in WT and Seckel fibroblasts. Scatter plot shows mean with ± SD (i).

e, Seckel cells with retarded cilium disassembly show delayed G1-S transition assessed by nuclear Cyclin A immunoreactivity. n = 3 independent experiments, n > 200 cells for WT and Seckel. Error bars are s.e.m.

f, Seckel cells show delayed mitotic entry. Note that an unresorbed Arl13b-positive ciliary remnant (ciliary membrane, green) is present with one of the centrosomes(Paridaen et al, 2013). Bar diagram shows mitotic index of WT and Seckel cells 24 hrs after serum stimulation. n = 3 independent experiments, n > 300 cells for WT and Seckel. Error bars are s.e.m. Scale bar 1 μm.
CPAP promotes timely cilium disassembly to maintain neural progenitor pool (2016)
Figure 3

Fig. 3.

CPAP provides a scaffold for the cilium disassembly complex

a, CPAP co-immunopurifies with CDC components from HeLa extracts. Centrosomal proteins Cap350 and ODF2 do not co-purify with CPAP.

b, Levels of CDC components (CPAP, Aurora A, OFD1, and Nde-1) are differentially regulated in response to serum starvation and stimulation in WT and Seckel fibroblasts. Specifically, they are elevated 1h after serum stimulation, thus enabling cilium disassembly. Elevated CDC levels in WT cells correlate with an elevated level of CPAP as shown in Fig. 2 as well as with the expression of cyclin A, a G1-S marker. GAPDH was used as a loading control indicating an equivalent amount of WT and Seckel fibroblast total proteins were loaded.

c. WT CPAP (WT-cells IP) but not Seckel variant CPAP (Seckel cells IP) co-immunopurifies with CDC components from asynchronously grown WT and Seckel fibroblasts cell extracts. The IP samples were adjusted so that equivalent amounts of immunoprecipitated CPAP were used in samples. Seckel (5x) and WT (1x) fibroblast cell extracts were used as input controls to show the efficacy of the various antibodies. Note that CPAP abundance in Seckel cells is ~5 times lower than that of WT cells. Centrosomal proteins Cap350 and ODF2 do not co-purify with CPAP indicating the specificity of CPAP interaction with CDC components.

d, GST pull down (i) and immunopurification assays (ii) of CPAP’s CC5 domain demonstrate its sufficiency in mediating CPAP-CDC protein interactions. The CC5 fragment is expressed as a GFP fusion protein in HeLa cells and immunopurified using anti-GFP antibodies. HeLa extracts were used as input controls. CAP350 does not co-purify with CPAP. Both Cap350 and ODF2 are not pulled down by CPAP’s CC5 domain.
CPAP promotes timely cilium disassembly to maintain neural progenitor pool (2016)
Figure 4

Fig. 4.

Retarded cilium disassembly causes premature neural differentiation

a, Seckel NPCs fail to efficiently recruit CDC components. Fraction of non-ciliated and ciliated WT and Seckel NPCs. Ciliated cells are further quantified and grouped based on whether they are positive or negative for Aurora A and CPAP at their basal bodies. Scale bar 0.5 μm (i-ii). Proportion of ciliated cells is higher in Seckel NPCs (**p<0.01). Compared to WT, a higher proportion of Seckel NPCs lack Aurora A and CPAP in their basal bodies (*p<0.05 for Auroa A and ***p<0.001 for CPAP). Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel. Error bars are s. e. m. (iii) Seckel cells consistently contain Nde-1 and Ofd1 with a significantly lesser intensity. t test; error bars are s.e.m.; n = 3 independent experiments. 110 cells for WT and 100 cells for Seckel.

b, Proportion of ciliated cells is higher in Seckel NPCs. Error bars are s. e. m. ** p<0.01, t-test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel.

c, Seckel NPCs exhibit longer cilia. Error bars are ± s. e. m. *** p<0.001, t-test. NPCs were derived from two independent iPS clones. n = 3 independent experiments. 146 cells for WT, 135 cells for Seckel (clone 1) and 202 (clone 2).

d, Seckel NPCs show reduced proliferation and delayed G1-S transition. Error bars are ± s. e. m. *** p<0.001 (EdU), * p<0.05 (Cyclin-A), t-test. For cyclin A, n = 3 independent experiments. 270 cells for WT and 232 cells for Seckel cells. For EdU, 700 cells for WT and 1630 cells for Seckel.

e, Olomoucine triggers differentiation of WT NPCs into TUJ1-positive neurons under non-differentiating conditions. Error bars are s. e. m. *** p<0.001, Ordinary Two-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments. 298 cells for untreated, 151 cells for DMSO and 146 cells for olomoucine, 25 μM.

f, Seckel NPCs differentiated into TUJ1-positive neurons under non-differentiating conditions. Error bars show ±s. e. m. *** p<0.001, t-test. n = 3 independent experiments. 633 cells for WT and 275 cells for Seckel. Scale bar 5 μm.

g, Expression of CPAP-GFP or CPAP-CC5-GFP in Seckel NPCs slightly increases nestin-positive cells (i), decreases neuronal differentiation (for CC5 *p<0.05) (ii), decreases ciliary length (for CPAP ***p<0.001 and for CC5 **p<0.01) (iii), elevates mitotic index (for CC5 *p<0.05) (iv) and decreases the number of ciliated cells (for CPAP *p<0.05 and for CC5 **p<0.01) (v) compared to control (mock electroporation). Error bars show s. e. m. One-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments with >350 (nestin staining, differentiation and mitotic index), and >70 cells (cilium length) cells.
CPAP promotes timely cilium disassembly to maintain neural progenitor pool (2016)
Figure 5

Fig. 5.

Down regulation of CDC components or a CPAP natural microcephaly mutation causes neural differentiation

a-b, siRNA-mediated down regulation of CDC components in WT NPCs causes longer cilia (Ofd1 ***p<0.001, Nde1 **p<0.01), retarded cilia disassembly as shown by increased numbers of ciliated cells (Ofd1 **p<0.01, Nde1 *p<0.05) and differentiation of NPCs into TUJ1 positive cells (Ofd1 **p<0.001, Nde1 *p<0.01). In controls, a non-targeting siRNA (scrambled) is used. Western blots at right validate siRNAs-mediated Nde1 and OFD1 depletion. Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 275 cells for scrambled, 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA.

c, VX680 (0.5 μM, a concentration that will specifically allow neuronal differentiation) was used to inhibit Aurora A activity. Bar diagrams below show quantifications. Compared to DMSO-treated control NPCs, a higher proportion of VX680 treated NPCs are ciliated (***p<0.001), exhibiting longer cilia (***p<0.001) and show Tuj1 positive neurons (***p<0.001). Error bars are s. e. m. For ciliated cells and cilium length: n = 3 independent experiments. 164 cells for DMSO-treated and 77 cells for VX680-treated. For NPC differentiation: n = 4 independent experiments. 286 cells for DMSO-treated and 118 cells for VX680-treated. Scale bar 1 μm.

d, Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours. Inhibition of Ofd1 (*p<0.05), Nde1 (**p<0.01) and Aurora A (***p<0.001) caused significantly reduced incorporation of EdU.

e-g, Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP. Both low -and high -exposures are given (e). Increased cilia length and NPC differentiation upon expression of RNAi-resistant CPAP E1235V compared to WT CPAP (f-g). Error bars are s. e. m. *** p<0.001 (cilia length), * p<0.05 (differentiation), t-test. For cilia length: n = 4 independent experiments. 25 cells for CPAP, 47 cells for CPAP E1235V. For differentiation: n = 4 independent experiments. 145 for CPAP and 81 for CPAP E1235V. Scale bar 1 μm.
CPAP promotes timely cilium disassembly to maintain neural progenitor pool (2016)
Figure 6

Fig. 6.

iPS modeling of microcephaly in 3D organoids

a, iPS-derived 14-day old organoid displaying complex morphological structures of brain-like tissues, parenthesis. Scale bar 500 μm. The ventricular zone (VZ) with radial glial cells stained with nestin (magenta) and neurons stained with TUJ1 is shown at higher magnification. Scale bar 20 μm.

b, Reduced Nestin/Pax-6-positive progenitors and increased TUJ1-positive neurons in Seckel organoids. In ventricular zones of WT organoids, there are significantly more Pax6 positive cells than TUJ1 positive cells, which is not the case in Seckel organoids (*p<0.05). TUJ1/Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs. Pax6 cells in Seckel organoid ventricular zones (* p<0.05). Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 10 WT organoids (2520 cells). Seckel n = 10 organoids (2024 cells). Scale bar 20 μm.

c, (i) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen (* p<0.05). Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids. (*p<0.05). Error bars are s. e. m. t-test. n=6 for WT organoid and 6 for Seckel organoid. Scale bar 20 μm. (ii) Seckel organoid cells display longer cilia. Error bars are s. e. m. *** p<0.001, t-test. n=3 for WT organoids and 4 for Seckel organoids. Scale bar 1 μm.

d, Seckel organoids electroporated with constructs expressing GFP alone (i), GFP with CC5-GFP and (ii) GFP with CPAP-GFP (iii). Middle panel shows staining with anti-GFP antibodies (red) to show the transfected area. Note the presence of increased Pax6-positive (magenta) NPCs in CC5 and CPAP electroporated tissues compared to GFP control. Likewise, there is a larger neural epithelium with multiple RGs (arrowheads). Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone (iv) lower TUJ1/Pax6 ratio (**p<0.01 and ***p<0.001 respectively) (v) smaller lumen diameter (**p<0.01) and (vi) shorter cilia length (**p<0.01 and ***p<0.001 respectively). Error bars are s. e. m. One-way ANOVA followed by Dunnett's multiple comparisons test. n = 3 organoids with 225 cells for GFP, 537 cells for CPAP and 569 for CC5. At least 10 ventricles from 3 independent organoids were used to measure the lumen diameter. t test. Error bars are s.e.m. Scale bar 20 μm.

e, Rescue effect of CC5 and CPAP electroporation on cleavage planes of phospho-vimentin-positive RGs in Seckel organoids. Compared to GFP control, electroporation of CC5 or CPAP increases the proportion of horizontally orientated Seckel RGs (for CC5 ***p<0.001) and decreases the portion of obliquely (for CC5 *p<0.05) or vertically oriented Seckel RGs (for CPAP and CC5 **p<0.01). Error bars are s.e.m. Two-way ANOVA followed by Tukey's multiple comparisons test. n = 4 organoids. 100 cells for GFP control, 112 cells for CPAP and 99 cells for CC5.
Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. (2011)
Figure 1

The UPR is activated in SBMA muscle.

A. Relative mRNA expression in skeletal muscle from SBMA patients (white bars, n = 3) and controls (black bars, n = 3) (mean +/− SEM). * p<0.05, ** p<0.01 by Student's t test. B. Splicing of XBP1 mRNA was assessed by RT-PCR. Products from unspliced (XBP1u) and spliced (XBP1s) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green. C. Relative mRNA expression in proximal hind limb muscle (mean +/− SEM). Mice evaluated were littermate WT (n = 6), AR113Q (n = 6), castrated WT (C-WT, n = 6) and castrated AR113Q males (C-AR113Q, n = 5) on a mixed C57BL/6J-129 genetic background. *p<0.05 by ANOVA. D. Western blot of BiP and PDI expression in proximal hind limb muscle. Right panels show quantification of signal relative to loading control (mean +/− SEM). * p<0.05 by Student's t test. E. Relative mRNA expression in proximal hind limb muscle of AR21Q (n = 5) and AR113Q (n = 3) males backcrossed to C57BL/6J. ** p<0.01, ***p<0.001 by Student's t test. F. Relative mRNA expression in spinal cord of AR21Q (n = 5) and AR113Q (n = 3) males (mean +/− SEM). n. s. = not significant by Student's t test.
Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. (2011)
Figure 2

CHOP deletion accentuates muscle atrophy in AR113Q mice.

A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limb muscle of AR113Q (black) or AR113Q, CHOP −/− mice (white) at 12 wks. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test. B. Representative image of muscle fibers following NADH stain. Bar = 20 µM. C. Distribution of proximal hind limb muscle fiber size from wt (black) and CHOP −/− (white) mice at 12 wks. Difference not significant by Mann-Whitney test.
Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. (2011)
Figure 3

Autophagy is increased in AR113Q, CHOP −/− muscle.

A. Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limb muscle of 12 wk mice (n = 5–6/genotype). *p<0.05 by ANOVA, n. s. = not significant. B. LC3 and p62 expression in proximal hind limb muscle of 12 wk mice was assessed by western blot. Right panels show quantification of signal relative to GAPDH. **p<0.01 by Student's t test. C. Relative expression of mRNAs encoding autophagy regulators in proximal hind limb muscle. *p<0.05 by Student's t test. C. Androgen receptor protein expression in skeletal muscle of 12 wk mice. Hsp90 serves as a loading control. Right panel shows quantification of relative signal intensity (n = 3/genotype). D. Proximal hind limb muscles stained for the androgen receptor (in red) exhibit intranuclear inclusions. Nuclei are stained by DAPI (in blue). E. P-JNK and P-eIF2 alpha expression (top, middle) and XBP1 mRNA splicing (bottom) in proximal hind limb muscle of 12 wk mice. Right panels show quantification of signal relative to loading control. **p<0.01 by Student's t test. n. s. = not significant.
Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. (2011)
Figure 4

CHOP deficiency increases denervation-induced atrophy through autophagy.

Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice. A. Western blot shows enhanced eIF2 alpha phosphorylation (top) and RT-PCR demonstrates increased XBP1 mRNA splicing (bottom) in denervated muscle. Right panels show relative quantification of signal intensity. *p<0.05 by Student's t test. B. Relative expression of BiP, ATF4 and CHOP mRNA (n = 3). *p<0.05 by Student's t test. C. Following surgical denervation of wild type or CHOP−/− mice, LC3 and p62 expression was assessed by western blot. Right panels show quantification of signal relative to GAPDH. ***p<0.001 by ANOVA, n. s. = not significant. D. Muscle fiber size (100 fibers/mouse) was quantified from wild type (black, n = 5), CHOP −/− (white, n = 3) and Beclin-1 +/− (grey, n = 3) mice 7 days post sciatic nerve transection. Shown is relative fiber cross sectional area (mean +/− SEM). ***p<0.001 by ANOVA. E. Muscle fiber size (100 fibers/mouse) was quantified from CHOP −/− (black, n = 6) or CHOP −/−, Beclin-1 +/− mice (white, n = 6) 7 days post sciatic nerve transection. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.
Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. (2011)
Figure 5

Effects of Beclin-1 haploinsufficiency on AR113Q muscle.

A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limb muscle of AR113Q (red, n = 6) or AR113Q, Beclin-1 +/− (blue, n = 6) mice at 16 wks. Left panel shows fiber size distribution, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left panel, p<0.0001 by Mann-Whitney test. Right panel, p<0.0001 by Student's t test. B. AR expression in skeletal muscle of 16 wk mice was assessed by western blot. GAPDH controls for loading. Right panel shows quantification of signal relative to GAPDH (mean +/− SEM). Differences not significant (n. s.).
Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. (2011)
Figure 6

Beclin-1 haploinsufficiency extends lifespan in AR113Q males.

A. Left panel, Kaplan-Meyer survival curve of AR113Q males (red line, n = 12) and AR113Q, Beclin-1 +/− males (blue line, n = 15). *p<0.05 by log-rank analysis. Right panel, mean survival +/− SEM. *p<0.05 by Student's t-test. B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice. D. Age of disease onset as measured by a decrease of 5% or more in forelimb grip strength (not significant by log rank analysis).
RAB6 and Dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination (2022)
Figure 1

Figure 1. RAB6A/B double knockout causes microcephaly

A. Schematic representation of cortical neurogenesis. Apical radial glial (aRG) cells are epithelial cells and the main neuronal progenitors in mouse. Basal radial glial (bRG) cells are rare in mouse but are the most abundant progenitor population in human. They have delaminated from the neuroepithelium. I.P.: Intermediate Progenitor.

B. Western blot analysis of RAB6A/A' and RAB6B protein levels in WT, Emx1-Cre; RAB6loxP/loxP (RAB6A KO), RAB6B-/- (RAB6B KO) and Emx1-Cre; RAB6AloxP/loxP; RAB6B-/- (RAB6A/B dKO) E15.5 cortical extracts.

C. P0 WT and RAB6A/B dKO brains. A cortical hemisphere is circled (dotted lines).

D. Cortical area in WT (N=13 brains), RAB6A KO (N=3 brains), RAB6B KO (N=5 brains) and RAB6A/B dKO (N=5 brains) at P0.

E. WT and RAB6A/B dKO brains stained for layer II/III marker CDP at P0. Scale bar = 100µm.

F. Cortical thickness (μm) in WT (N=6 brains), RAB6A KO (N=3 brains), RAB6B KO (N=3 brains) and RAB6A/B dKO (N=4 brains) at P0.

G. Immunostaining for cleaved Caspase 3 (cCAS3) in WT and RAB6A/B dKO brains at P0. Scale bar = 50µm.

Data information: (D, F) Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure. All error bars indicate SD.
RAB6 and Dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination (2022)
Figure 2

Figure 2. RAB6A/B dKO leads to aRG cell delamination during interphase

A. PAX6 staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm.

B. Percentage of PAX6+ cells located in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 4282 cells from N=6 brains. RAB6A/B dKO: 1241 cells from N=3 brains. Mann-Whitney U test.

C. Phospho-Vimentin (p-Vim) staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm.

D. Percentage p-VIM+ cells dividing ectopically, in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 1713 cells from N=6 brains. RAB6A/B dKO: 506 cells from N=3 brains. Mann-Whitney U test.

E. Mitotic index (p-VIM+ PAX6+ / PAX6+ cells) of RG cells dividing apically (at the ventricular surface) or basally (upper half) in WT and RAB6A/B dKO E15.5 brains. Apical divisions: N= 1886 cells from 3 brains for WT and 1511 cells from 3 brains for RAB6A/B dKO. Basal divisions: N=643 cells from 3 brains for WT and 809 cells from 3 brains for RAB6A/B dKO. Mann-Whitney U test.

F. Electroporation of RAB6AloxP/loxP; RAB6B-/- E14.5 embryos with GFP (control) or GFP + CRE (RAB6A/B dKO) and fixation at E18.5. Localization of GFP+ cells in the ventricular zone. White arrows indicate apical processes. Scale bar = 25µm.

G. Electroporation of RAB6AloxP/loxP; RAB6B-/- E14.5 embryos with mCherry (control) or mCherry + CRE (RAB6A/B dKO) and live imaging of delamination events at E17.5.

H. Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17.5. mCherry: N=69 cells from 5 movies. mCherry + CRE: N=52 cells from 4 movies. Fisher's exact test, *p ≤ 0.05.

Data information: All error bars indicate SD.
RAB6 and Dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination (2022)
Figure 3

Figure 3. Apical transport of RAB6A+ post-Golgi vesicles is driven by dynein

A. Localization of the Golgi apparatus (ManII-GFP) and the centrosome (𝛾-tubulin) in E15.5 mCherry-electroporated radial glial cell. The Golgi apparatus is localized basally, away from the centrosome. Scale bar = 5µm.

B. Average distance between the apical-most part of the Golgi apparatus and the apical surface in aRG cells. N=224 cells from 3 independent brains.

C. Live imaging of EB3-GFP in the apical process of an aRG cell at E15.5. Center: kymograph. Left: manual tracking of EB3 comets. Orange: basally-growing. Pink: Apically-growing. Scale bar = 5µm.

D. Co-expression of GFP-RAB6A and GalNacT2-mCherry in E15.5 aRG cells reveals colocalization at the Golgi apparatus. Scale bar = 5µm. Dashed line: cell outline

E. Live imaging of GFP-RAB6A in aRG cells at E15.5 allows tracking of individual RAB6A+ vesicles in situ, from the basal Golgi apparatus towards the apical surface. Scale bar = 5µm. Distance = 5µm, time = 30 seconds.

F. RAB6A+ vesicle directionality in apical processes of aRG cells over one-minute movies.

G. Relative time spent by RAB6A+ vesicles in apical, basal or static phases.

H. Velocity of apically and basally moving RAB6A+ vesicles.

I Live imaging of GFP-RAB6A in control, dynarrestin-treated and CC1-p150-expressing aRG cells at E15.5. Scale bars = 5µm. Distance = 5µm, time = 30 seconds.

J. Number of RAB6A+ vesicles in the apical process of DMSO and dynarrestin-treated mouse aRG cells.

K. Relative time spent by RAB6A+ vesicles in apical movement phase, in DMSO and dynarrestin-treated mouse aRG cells.

L. Number of RAB6A+ vesicles in the apical process of mCherry and CC1-p150-expressing aRG cells.

M. Relative time spent by RAB6A+ vesicles in apical movement phase, in mCherry and CC1-p150-expressing aRG cells. DMSO treatment slightly affected RAB6A dynamics, as compared to mCherry control.

Data information: (F, G, H) N= 388 vesicles from 30 cells. (J, K, L, M) 216 vesicles from N=11 cells for DMSO, 145 vesicles from N=25 cells for dynarrestin, 173 vesicles from N=17 cells for mCherry control, 71 vesicles from N=15 cells for CC1-p150. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.