MEF-MCCs grown on micropatterns highlight the pericentrosomal focalization of procentriole production

(A) Scheme of the MEF-MCC differentiation process on crossbow micropatterns. (B) MCC-induced fibroblasts on micropatterns perform the typical step-wise dynamics of centriole amplification. Representative immunofluorescence images of MEF-MCC on crossbow micropatterns showing the 4 stages of differentiation. Scale bar, 10 µM. (C) Density mapping of centrosome in MEF-MCCs before amplification (precursor stage, left column), and of procentrioles during early amplification (cells with less than 10 SAS6 focis/cell, middle column) or late amplification (cells with more than 10 SAS6 focis/cell, right column) treated with DMSO (top row) or Nocodazole 1 µM (bottom row). Three independent experiments were quantified. Scale bar, 10 µM. (D) Density mapping of PCNT fluorescence signal during centrosome stage (left column) early A-stage (middle column) and late A-stage (right column) in MEF-MCCs under DMSO (top row) or Nocodazole 1 µM (bottom row). Two independent experiments were quantified. Scale bar, 10 µM. DM1A marks all MT, YL1/2 marks tyrosinated MT, SAS6 marks procentrioles, GT335 marks centrosome, mature procentrioles and cilia, PCNT marks PERICENTRIN. Dotted lines show the crossbow shape of the micropattern, white graduated lines show x and y axes. Insets on the right of each panel on (C) and (D) show one representative cell used for the density mapping.

Live imaging endogenous DEUP1 highlights the first events of centriole amplification in brain MCC

(A) Scheme of the ependymal cell culture protocol. LVW: lateral ventricular wall. (B) Early A-stage dynamics in CEN2-GFP; mRuby-DEUP1+ cells. White arrowheads point the 2 centrosomal centrioles. The red dotted line corresponds to the cloud of DEUP1+ forming around the parental centrioles during early A-stage. The red arrowhead points to DEUP1 accumulation at one centrosomal centriole as compared to the other one. The red and white arrowheads point to CEN2-GFP+ structures corresponding to centriole loaded deuterosomes. The crosses rule out the CEN2-GFP+ aggregate not to be mistaken with a centrosome; CEN2-GFP in white, DEUP1 in red; scale bar, 5 µM. See associated Video 1. (C) Correlative light and electron microscopy resolves fibrogranular aggregate concentration in a mRuby-DEUP1+ primordial cloud. Light microscopy integrates the fluorescence signal of a z-stack of 240 nm. Electron microscopy z-sections are 70 nm. dc= centrosomal daughter centriole, mc= centrosomal mother centriole. (D) Representative ultrastructural elements present in the mRuby-DEUP1 primordial cloud resolved by correlative light and electron microscopy. Red and white arrowheads indicate deuterosome structures. White arrowheads indicate procentrioles. dc= centrosomal daughter centriole. See also Video 3. (E) Representative images of live imaged mRuby-DEUP1+ foci fluctuations in the primordial cloud (dt= 5s). A single z-slice of 0,7 µM is filmed. Black circles indicate the position of centrosomal centrioles located thanks to the CEN2-GFP signal, not shown here. mRuby-DEUP1+ foci are color-coded with arrowheads to appreciate their oscillatory behaviors towards the centrosomal mRuby-DEUP1+cloud. See associated video 4. Scale bar, 5 µM. (F) Scheme depicting the observations resulting from live-imaging CEN2-GFP; mRuby-DEUP1+ cells and correlative light and electron microscopy.

Procentriole assembly begins in a pericentrosomal nest and progresses during radial migration

(A-B) Representative U-ExM images of brain MCCs during early A-stage, when only a primordial cloud is visible but no procentriole-loaded deuterosomes can be identified. Cells were immunostained with antibodies to β-TUBULIN, SAS6, DEUP1 (A), β-TUBULIN, CENTRIN, PLK4 (B). mc: mother centriole, dc: daughter centriole. (C-D) Representative U-ExM images of brain MCCs during early A-stage, when procentriole-loaded deuterosomes are visible. Cells were immunostained with antibodies to β-TUBULIN, SAS6, DEUP1 (C) or Acetylated-TUBULIN, PLK4, DEUP1 (D). Arrows mark DEUP1 foci with either SAS6 (C) or PLK4 (D). mc: mother centriole, dc: daughter centriole. (E) Representative U-ExM images of brain MCCs during late A-stage. Boxes denote zoomed in regions on right, with 1-4 labels marking increasing distance from the parent centrioles. Cells were immunostained with antibodies to β-TUBULIN, SAS6, DEUP1 (E) (F) Quantitation showing the procentriole distance from parents is shorter in procentrioles without αβ-TUBULIN compared to procentrioles with αβ-TUBULIN. SAS6 is used as a marker for procentrioles associated with deuterosomes. Graphs show mean ± SD from n=7 cells across 3 different experiments. ****p<0,0001 using unpaired t-test. (G) Representative U-ExM images of brain MCCs during late A-stage. Boxes denote zoomed in regions on right, with 1-4 labels marking increasing distance from the parent centrioles. Cells were immunostained with antibodies to Acetylated-TUBULIN, PLK4, DEUP1. (H) Quantitation comparing percent of cells showing DEUP1 enrichment at daughter centriole over mother centriole (dc>mc), equal enrichment at both parent centrioles (dc = mc), or enrichment at mother centriole over daughter centriole (mc>dc) when cloud-stage and A-stage. A total of 54 cells were analyzed across 3 experiments. (I) Table summarizing protein localization as visualized by U-ExM along A-stage progression. “Cloud only” refers to the beginning of A-stage, when a cloud is visible but no procentriole-loaded deuterosomes can be identified. (J) Scheme representing A-stage progression in time and space.

Dynein dependent MT transport organizes the pericentrosomal nest and is required for procentriole production

(A) Immunostaining profiles of procentrioles and microtubules during brain CEN2-GFP MCC differentiation. Procentrioles were counter-stained with anti-SAS6 antibodies (red), dynamic tyrosinated MTs were counter-stained with YL1-2 antibodies and the nucleus was counter-stained with Hoechst (blue). All micrographs are max projections of whole z-stacks except for the MCC-stage where max were done from apical or basal z-stacks selected from the same cell. Arrows show centrosomal centrioles, pink dotted line delineates the CEN2-GFP+ pericentrosomal cloud area, grey dotted line delineates the area occupied by deuterosomes. Scale bar, 5 µm. (B) Scheme depicting the experimental protocol. (C) Number of cells beginning centriole amplification (emergence of mRuby-DEUP1+/CEN2-GFP+ structures in the cell) in a microscope field during a 20h movie under acute DMSO (60 fields), Nocodazole (10 µM, 43 fields) or Dynapyrazole (3 µM, 30 fields) treatments. n>3 independent experiments. (D) Quantification of the percentage of early and late A-stage cells displaying at least one mRuby-DEUP1+ structure oscillation to and from the centrosome during 1-4min movies at dt5-15s, in DMSO, acute Nocodazole (10 µM) and acute Dynapyrazole (7.5 µM) treated cells. n>3 independent experiments were scored, n=81 DMSO cells, n=20 Nocodazole cells, n=56 Dynapyrazole cells analyzed. *p = 0.0108, **p=0.005, ****p<0.0001; Chi-square test with yates correction. See Videos 8-11. (E) Quantification of the area occupied by the mRuby-DEUP1+ cloud over time in control (DMSO), Nocodazole (10 µM) and Dynapyrazole (3 µM) treated cells. Black arrow indicates that DMSO and drugs were added right after the first acquisition. Three independent experiments were analyzed, n=12 DMSO cells, n=14 Noco 10 µM cells, n=14 Dynapyrazole 3 µM cells; ****p<0,0001; non-parametric Mann Whitney test; error bars represent mean ± SD. See Videos 12-14. (F) Centriole number ratio produced during A-stage under DMSO and Dynapyrazole (3 µM) treatment normalized on t0. DMSO and Dynapyrazole are added after the first time point. dt=40 min. (G) Representative images of CEN2-GFP movies used in (F). Arrowheads represent A-stage procentrioles. See also video 15. Scale bar, 1 µm. (H) Quantification of centriole number per deuterosome (using CEN2-GFP signal) in G-stage cells under DMSO, Nocodazole (1 µM) and Dynapyrazole (3 µM) chronic treatments (48h). Three independent experiments were scored, ndeut=459 in DMSO, ndeut= 215 in Nocodazole, ndeut=383 in Dynapyrazole. Error bars represent min to max ± median. **** P<0.0001; non-parametric Mann Whitney test. (I) Quantification of SAS6+ centriole number in G- and D-stage (pooled) cells under DMSO, Nocodazole (1 µM) and Dynapyrazole (3 µM) chronic treatments (48h). Three independent experiments were scored; error bars represent min to max ± median. n=42 DMSO cells, n=34 Noco 1 µM; n= 29 Noco 5 µM cells analyzed. Error bars represent min to max ± median. ***<0.001, **** P<0.0001; non-parametric Mann Whitney test. (J) Super resolution immunostaining profile of deuterosomes (DEUP1) and procentrioles (SAS6) under chronic Nocodazole (10 µM, 24h). Deuterosomes arrange in grapes and are reduced to small subunits scarcely loaded with procentrioles. Scale bar, 500 nm. (K) Super resolution fluorescence of the mRuby-DEUP1;CEN2-GFP “inverted flower” profile frequently observed under chronic Nocodazole (10 µM, 24h) along with the underlying deuterosome and procentriole arrangement revealed by correlative light and electron microscopy (CLEM). See Fig. 4 Supplementary 5 for serial CLEM sections. (L) Scheme representing the effect of Nocodazole and Dynapyrazole on A-stage. To be compared with the scheme of Fig. 4B.

Centriole biogenesis is accelerated during a 2-in-1 MCC cell cycle variant

(A-B) Scheme comparing centriole biogenesis during centriole duplication in the canonical cell cycle and during centriole amplification in the MCC cell cycle variant. DA: distal appendages, SDA: sub-distal appendages, MT: microtubules. (C) Quantification of PLK1 intensity in A-stage and G-stage brain MCC samples using a linear regression model, with stages as the main effect and replicate as a fixed covariate, to account for batch variation. Statistical significance was assessed using Type II ANOVA. The lower and upper hinges correspond to the 25th and 75th percentiles. The upper (lower) whisker extends from the hinge to the largest (smallest) value no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the 25th and 75th percentiles); three independent experiments were quantified and colored differently; n= 32 cells; ****p<0,0001. Insets are from Figure 5 Supplementary 2A. (D) Immunostaining profile of YL1/2 in CEN2-GFP;mRuby-DEUP1 expressing brain MCCs in A- and G-stage during regrowth experiments (see methods). Arrowheads indicate centrosomal centrioles. Arrows indicate the position of A- or G-stage procentrioles. Scale bar, 1 µm. (E) Quantification of microtubule aster intensity in A-stage and G-stage brain MCC samples during regrowth experiments using a linear regression model, with stages as the main effect and replicate as a fixed covariate, to account for batch variation. Statistical significance was assessed using Type II ANOVA. The lower and upper hinges correspond to the 25th and 75th percentiles. The upper (lower) whisker extends from the hinge to the largest (smallest) value no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the 25th and 75th percentiles); three independent experiments were quantified and colored differently; n= 56 cells; ****p<0,0001. (F) Quantification of the percentage of tethered deuterosomes per G-stage cell in DMSO , Nocodazole (10 µM, 4h) and Dynapyrazole (3 µM, 4h). Deuterosomes above or below the nucleus are not quantified. Four independent experiments quantified; n=15 cells in DMSO, n=26 cells in Nocodazole, n=17 cells in Dynapyrazole. Error bars represent mean ± SD; ****p<0,0001; Chi-2 test with Yates’ correction. (G) Still images from a movie of G-stage CEN2-GFP expressing cells treated with DMSO or Dynapyrazole 3 µM at t=0. While “centriole-loaded deuterosomes” keep a perinuclear distribution in DMSO treated cells, in Dynapyrazole treated cells, the “centriole-loaded deuterosomes” start untether from the nucleus at t+30 min. Shaded grey ovals outline the nucleus identified with contrasting CEN2-GFP. scale bar, 5 µM. See Video NEW17. (H) Scheme synthesizing the main findings of main and supplementary Fig. 5 on G-stage procentrioles maturation and nuclear tethering.

Disengagement of centrioles in MCC involves deuterosome splitting and dissolution

(A) Quantification of the proportion of procentrioles disengaging with a nuclear contact. Three independent experiments were quantified; n=98 deuterosomes, n=8 cells. See also Video 17. (B) CEN2-GFP; mRuby-DEUP1 dynamics in D-stage. Upper panels: in early D-stage (00:00), some procentrioles are already individualized, separated from DEUP1+ deuterosomes as shown by the white arrows. During D-stage, procentrioles disengaged from deuterosomes can keep a subunit of DEUP1 in their proximal portion, splitting them in smaller entities bearing fewer procentrioles not disengaged yet, as well as DEUP1 signal dissolution. Lower panels: grayscale showing the initial pool of 19 deuterosomes (00 :00) splitting to 32 DEUP1+ foci (4 :10). dt=50min; scale bar, 5 µM. See also Video 19. (C-D) Quantification of the size and number of deuterosomes per cell over time in D-stage relative to G-stage. n=10 cells analyzed were plotted individually to reveal the tendency to decrease in size, while increasing the number of DEUP1 foci. Each measurement in D-stage was normalized on G-stage. Three independent experiments were scored; error bars represent mean ± SD. (E) CEN2-GFP;mRuby-DEUP1 time lapse images of a single z-plane (0.7µm) of a D-stage cell showing a deuterosome which seemingly split into smaller deuterosomes migrating away from each others along the nuclear envelope. White dotted line outlines the nuclear membrane identified with contrasting CEN2-GFP signal. Left picture is the zoom-out picture of the D-stage cell, showing the position of the cropped still images. dt=5s, scale bar, 1 µm. See Video 20 to see the entire movie. (F) Time lapse images of a D-stage cell showing the deuterosome mRuby-DEUP1+ signal dissolution correlated with CEN2-GFP rosette dismantlement. dt=40min, scale bar, 1 µm. See also Video 21. (G) CEN2-GFP;mRuby-DEUP1 time lapse images of a D-stage cell showing migration of a centriole-loaded deuterosome along the nuclear membrane (line 1), change of deuterosome shape and mixing with another deuterosome (line 2), followed by consecutive unmixing (line 3). dt=40min, scale bar, 1 µm. See Video 22 to see the entire movie. (H) Scheme synthesizing the main findings of main and supplementary Fig. 6 on D-stage deuterosome dynamics and centriole disengagement.

Disengagement of centrioles is assisted by MT and dyneins

(A) Disengaged procentriole associated to a deuterosomal subunit migrating on the nuclear envelope. White arrow head indicates the centriole, the white dot line outlines the shadow of the nuclear membrane seen in CEN2-GFP. dt=5s; Single z-section of 0.5 µm, scale bar, 2 µm. See Video 23 to see zoom out and whole movie. (B) Representative picture of tyrosinated microtubules (YL1/2) and DEUP1 immuno-reactivity in brain CEN2-GFP MCC during D-stage after MT depolymerization through nocodazole treatment (10 µM, 24h) and subsequent nocodazole whashout (4 h). Scale bar, 5 µm. (C) Tyrosinated microtubules and deuterosomes immuno-reactivity profiles in brain CEN2-GFP MCC in D-stage. These pictures show one z-section (0.25 µm) in the basal part of the cell. Tyrosinated MTs were stained with (YL 1-2). CEN2-GFP signal intensity is decreased from left to right to show that centrioles are located next to MT. Scale bar, 5 µm. See Video 24 to see all z-sections and DEUP1 staining. (D) Schematic representation of brain MCC differentiation depicting the timing of drug treatments. The eye shows the amplification stage monitored. (E) Quantification of the proportion of D-stage cells per fields among amplifying cells under chronic DMSO and Nocodazole treatments (48h, 1 µM and 5 µM). At least three independent experiments were quantified, n=2626 DMSO cells, n=1483 Noco 1 µM cells, n=909 Noco 5 µM cells; error bars represent mean ± SD. ns, not significant; ****p<0,0001; non-parametric Mann Whitney test. (F) Quantification of D-stage quality phenotype under chronic DMSO and Nocodazole (48h, 1 µM). Total disengagement in black, incomplete disengagement in white. At least three independent experiments were quantified, n=35 DMSO cells analyzed, n=25 Noco 1 µM cells analyzed. ****p<0,0001; Chi-2 test with Yates’ correction. (G) Quantification of disengagement duration in CEN2-GFP brain MCCs under DMSO and acute Nocodazole (10 µM) or Dynapyrazole (3 µM) treatments. >4 independent experiments were quantified, n=32 DMSO cells, n=10 Nocodazole cells and n=22 Dynapyrazole cells. *p<0,05; ****p < 0/0001 non-parametric Mann Whitney test. (H) Representative movie of CEN2-GFP dynamics in D-stage of brain MCCs under DMSO and acute Nocodazole (10 µM) or Dynapyrazole (3 µM) treatments. (t-1) represents the timepoint before drug treatments. scale bar, 5 µm. See Video 25 for whole movies.

Disengaged centrioles finally converge with the former centrosome

(A) Quantification of the relative frequency of the centriole position regarding the centrosomal centriole (CC) at the end of centriole individualization (green) and the end of apical migration (red). Bottom: representative apical projections of segmented CEN2-GFP+ centrioles at the end of centriole individualization (green frame) and the end of apical migration (red frame). Green arrow shows the centrosome which served as the reference point; the nucleus is in dark blue. n=3 cells; scale bar, 5 µM. (B) Manual single centriole 3D tracking reveals convergence of centrioles towards the centrosome region. Top left panel: apical view of manually tracked migrating centrioles trajectories. Top right panel: apical view of the directionality vectors of the migrating centrioles. Bottom panel: corresponding profile views of the top panels. dt=5min; scale bar, 5 µM. See Video 26. (C) Evolution of individual centriole distance to centrosome over time. dt=5min. Three independent experiments were analyzed, n=33 centriole trajectories in n=5 cells. (D) Example of the single blue centriole trajectory from (B) towards the centrosome in the XY plane and in the Z plane. Yellow dots represent the steps of apical migration. The origin represents the position of the centrosome. See Video 26. (E) Evolution of the Z coordinates during the apical migration of 16 centrioles from 5 cells. Three independent experiments were analyzed. The origin represents the Z position of the centrosome. dt=5min. (F) Speed of migration plotted from all 953 steps of 33 migrating centrioles versus the 74 extracted steps of apical migration of the 16 centrioles basally located in the cells. Box and whiskers plots (2,5-97,5 percentile). See distribution in Fig. 8 Supplementary 1C. (G) Schematic representation of brain MCC differentiation depicting the timing of MT depolymerisation treatment. The eye shows the amplification stage monitored. (H) Quantification of apical migration duration in DMSO and acute nocodazole (10 µM) or dynapyrazole (3 µM) in brain MCCs. Eight independent experiments were scored for n=32 DMSO cells, 3 experiments for n=10 nocodazole cells and 5 experiments for n=8 dyanpyrazole cells. Error bars represent min to max ± median. ****p<0,0001; non-parametric Mann Whitney test. (I) Profile view of centriole apical migration after D-stage in CEN2-GFP+ DIV2 brain MCCs treated with acute DMSO and Nocodazole 10 µM. At t-1, cells display late G-stage/early D-stage procentrioles and are not treated with any drug. DMSO and Nocodazole 10 µM were added right after the first timepoint acquisition. In DMSO, 2 centriole patches are above and below (red arrow) the nucleus (t+0h). The patch below the nucleus progressively joins the apical one (t+2h) to become one apical patch (t+4h) and stabilizes (t+6h). In 10 µM Nocodazole, procentrioles start to scatter after 1h, and the group of centrioles below the nucleus travel up and down (t+2h, t+4h and t+6h) and does not succeed to become one apical group of centrioles (t+6h). Red arrowheads point to group of centrioles that we consider as basally localized. *CC corresponds to the centrosome of a neighboring cell. dt=5min; scale bar, 5 µm. See also Video 27.

The MCC cell cycle variant incorporates a 2-in-1 centriole biogenesis cycle.

Parallel between centriole biogenesis during the canonical and the MCC cell cycle variants showing that centriole elongation and maturation cycles are conserved but superimposed during the MCC cell cycle. The “daughter centriole” step is skipped and prematurely mature centrioles participate in their own dynamics of sub-cellular organization disengagement and migration. DA: distal appendages, SDA: sub-distal appendages, MTOC: microtubule organizing center.