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 (centrosome stage, left column), and 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) and Nocodazole 1µM (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) and Nocodazole 1µM (bottom row). Insets on the right of each panel is a representative cell used for the density mapping. 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 is a representative cell used for the density mapping.

Deup1 is a centrosomal protein and assembles deuterosomes in the pericentrosomal region in brain MCC

(A) Scheme of the ependymal cell culture protocol. Eye shows which stage is observed.

(B) Cen2-GFP; mRuby-Deup1 dynamics of the new mouse line during centriole amplification. The previously described stages of centriole amplification can be identified with the Cen2-GFP signal. The procentrioles are seen organizing around the endogenous mRuby3-Deup1+ deuterosomes in A-, G- and D-stage. Scale bar, 5µm.

(C) Early A-stage dynamics in Cen2-GFP; mRuby-Deup1+ cells. The horizontal blue bar illustrates the newly observed “cloud” stage where a Deup1+ cloud forms, before the A-phase (05:00), around the parental centrioles. The horizontal grey bar illustrates the previously described “Amplification” A-stage, where Cen2-GFP+/mRuby3-Deup1+ procentriole loaded deuterosomes exit the cloud (7:00 – 12:00, arrows). White dotted circles enclose parental centrioles. The white and black cross rules out the Cen2-GFP+ aggregate not to be mistaken with a centrosome; dt=1h; Cen2-GFP in green, Deup1 in red; scale bar, 5 µm. See Video 1.

(D) Cen2-GFP and mRuby-Deup1 dynamics during the cloud stage. Cen2-GFP+ and mRuby-Deup1+ foci are not always colocalized. Yellow dotted circles enclose parental centrioles. Deup1+ foci can interact persistently with one parental centriole. dt=1h; Cen2-GFP in green, Deup1 in red. Scale bar, 5µm. See Video 2.

(E) mRuby-Deup1+ signal during the cloud stage in Cen2-GFP cells. Deup1 foci are color-coded with arrowheads to appreciate their oscillatory behaviors towards the centrosomal mRuby-Deup1+cloud; green shades enclose parental centrioles that were localized using Cen2-GFP signal that is not visible here. See associated video. dt=5s; scale bar, 5µm. See Video 3.

(F) mRuby-Deup1+ intermittent interaction with one parental centriole in Cen2-GFP+ cells. Deup1+ foci indicated by white arrow head oscillates to and from one parental centriole in yellow dotted circles. dt=5s; scale bar, 5 µm. See Video 4.

(G) Early deuterosome oscillating behavior during A-stage. White arrows show deuterosomes back and forth movements towards the centrosomal cloud; blue arrows show a deuterosome moving to the cytoplasm and away from other deuterosomes. dt=5s; scale bar, 5 µm. See Video 5.

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

(A-C) Representative U-ExM images of brain MCCs during cloud stage. Cells were immunostained with antibodies to β-tubulin, Centrin, Deup1 (A), β-tubulin, SAS6, Deup1 (B), or β-tubulin, PLK4, Cen (C). mc: mother centriole, dc: daughter centriole.

(D-F) Representative U-ExM images of brain MCCs during early A-stage. Cells were immunostained with antibodies to β-tubulin, Centrin, Deup1 (D), β-tubulin, SAS6, Deup1 (E), or Acetylated-tubulin, PLK4, Deup1 (F). Arrows mark Deup1 foci with either centrin (D) or procentrioles (E, F). mc: mother centriole, dc: daughter centriole.

(G-I) 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 (G), Acetylated-tubulin, PLK4, Deup1 (H), or β-tubulin, Centrin, Acetylated-tubulin (I). mc: mother centriole, dc: daughter centriole.

(J) 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.

(K) Quantitation showing the procentriole distance from parents is shorter in procentrioles without Acetylated-tubulin compared to procentrioles with Acetylated-tubulin. PLK4 is used as a marker for procentrioles associated with deuterosomes. Graphs show mean ±SD from n=3 cells across 3 different experiments. ****p<0,0001 using unpaired t-test.

(L) Quantitation showing procentriole length measured by β-tubulin increases with distance from parents during A-stage. Graphs show mean ±SD from n=10 cells across 3 different experiments.

(M) Quantitation showing procentriole width measured by β-tubulin increases with distance from parents during A-stage. Graphs show mean ±SD from n=10 cells across 3 different experiments.

(N) 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) in cloud-stage and A-stage. A total of 54 cells were analyzed across 3 experiments.

(O) Table summarizing protein localization as visualized by U-ExM during cloud, early A-, and late A-stage. “+” indicates protein is present; “–” indicates not present.

(P) Scheme representing cloud and A-phase progression in time and space.

Microtubules organize the pericentrosomal nest, promote deuterosome growth and hinder the dispersion of deuterosomes

(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. White and red arrow heads show parental centrioles. Scale bar, 5 µm.

(B) Cen2-GFP; mRuby-Deup1 profiles in control and Nocodazole (10µM) treated A-stage cell. t0 corresponds to the first acquisition when cells were still untreated. White arrows indicate the centrosomal centrioles. Top row: at t+3h and 10h after DMSO addition, the procentriole-loaded deuterosomes stay in a restricted area around the pericentrosomal region. Plain white line outlines the border of the cell; plain blue lines enclose the area of the Cen2-GFP; mRuby-Deup1+ deuterosomes in control cells; blue dotted lines enclose the area of the Cen2-GFP; mRuby-Deup1+ cloud. Bottom row: At t+3h after Nocodazole (10µM) addition, the procentriole-loaded deuterosomes disperse and appear scattered in the cell, covering almost the whole cell surface at t+10h. Plain white line outlines the border of the cell; plain orange line encloses the area of Cen2-GFP; mRuby-Deup1+ deuterosomes; orange dot line encloses the area of the Cen2-GFP; mRuby-Deup1+ cloud; the black cross rules out the Cen2-GFP+ aggregate not to be mistaken with a centrosome. dt=40min; scale bar, 5µm. See Video 6.

(C) Quantification of the area occupied by the mRuby-Deup1 signal over time in cloud stage in control (DMSO) and Nocodazole (10µM) treated cells represented by the dotted blue and orange lines respectively in (B). Black arrow indicates that Nocodazole and DMSO were added right after the first acquisition. Three independent experiments were analyzed, n=12 DMSO cells, n=14 Noco 10µM cells. dt=40min; ****p<0,0001; non-parametric Mann Whitney test; error bars represent mean ± SD.

(D) Quantification of the area occupied by the deuterosomes over time in A-stage cells under DMSO and acute Nocodazole 10µM treated cells represented by the plain blue and orange lines respectively in (B). Black arrow indicates that Nocodazole and DMSO were added right after the first acquisition. Three independent experiments were analyzed, n=12 DMSO cells; n= 15 Noco 10µM cells. dt=40min; ****p<0,0001; non-parametric Mann Whitney test; error bars represent mean ± SD.

(E) Super resolution immunostaining profile of deuterosomes (Deup1) and procentrioles (SAS6) under Nocodazole (10µM, 24h). Deuterosomes are reduced to small subunits scarcely loaded with procentrioles. Scale bar, 5 µm.

(F) Quantification of the percentage of “cloud” and A-stage cells displaying at least one Deup1 foci oscillation to and from the centrosome during 1-4min movies at dt5-15s, in DMSO and acute Nocodazole (10µM) treated cells. Three independent experiments were scored, n=25 DMSO cells, n=20 Noco 10µM cells analyzed. ****p=0.0001; Chi-square test.

(G) Quantification of deuterosome size in A and G-stage cells (pooled) under DMSO and Nocodazole chronic treatments (48h; 1µM and 5µM). Three independent experiments were scored, n=80 DMSO cells, n=43 Noco 1µM cells, n=63 Noco 5µM cells. Error bars represent mean ± SD. **** P<0.0001; ***p=0.0002; **p=0.0025; non-parametric Mann Whitney test.

(H) Quantification of centriole number per deuterosome in G-stage cells under DMSO and Nocodazole chronic treatments (48h; 1µM and 5µM). Three independent experiments were scored, n=11 DMSO cells, n=17 Noco 1µM cells, n=21 Noco 5µM cells. Error bars represent min to max ± median. **** P<0.0001; non-parametric Mann Whitney test.

(I) Quantification of the deuterosome number per cell in A and G-stage cells (pooled) under DMSO and Nocodazole chronic treatments (48h; 1µM and 5µM). Three independent experiments were scored, n=80 DMSO cells, n=43 Noco 1µM cells, n=63 Noco 5µM cells. Error bars represent min to max ± median. **** P<0.0001; ***p=0.0002; **p=0.0025; non-parametric Mann Whitney test.

(J) Quantification of SAS6+ centriole number in G- and D-stage (pooled) brain cells under DMSO and Nocodazole chronic treatments (48h; 1µM and 5µM). Three independent experiments were scored; error bars represent min to max ± median. n=42 DMSO cells, n=34 Noco 1µM; n= 29 Noco 5µM cells analyzed. Error bars represent min to max ± median. ns, not significant; ** p=0,002; non-parametric Mann Whitney test.

Amplified procentrioles convert to multiple microtubule organizing centers (MTOC) during the amplification (A) to growth (G) transition

(A-B) Representative immuno-reactivity profile of PCNT and Plk1 in A- and G-phases in Cen2-GFP+ brain MCC progenitors. Dotted line square surrounds procentrioles shown in the magnified view. Scale bar, 5µm.

(C-D) Quantification of PCNT and Plk1 intensities on procentrioles in A- and G-stage brain MCCs. Fluorescence intensity was normalized on PCNT and Plk1 intensity of the centrosome of one non-differentiating neighboring cell. Three independent experiments were scored for both stainings; n= 19 PCNT cells, n= 32 PLK1 cells analyzed; error bars represent mean ± SD. ****p<0,0001; non-parametric Mann Whitney test.

(E-G) Immunostaining profile of YL1/2 in Cen2-GFP+ brain MCCs (E) and Sas6 stained MEF-MCCs (G) in A- and G-stage after repolymerization experiment. Dot lined square surrounds a procentriole shown in the magnified view. White arrowheads indicate centrosomal centrioles. Three independent experiments were scored for each brain or MEF-MCCs. Scale bar, 10 µm.

(F-H) Quantification of YL1/2 intensity in A-stage and G-stage brain MCCs (F) and MEF-MCCs (H). Fluorescence intensity was normalized on the centrosome of one non-differentiating neighboring cell. Three independent experiments were quantified for brain MCCs (F) n= 56 cells. Three independent experiments were quantified for MEF-MCCs (H) n= 46 cells. Error bars represent mean ± SD; ****p<0,0001; non-parametric Mann Whitney test.

Microtubules are required for procentriole attachment to the nuclear membrane and efficient disengagement

(A) Cen2-GFP+ dynamics from G-stage to migration stage. Top line: time-lapse images showing perinuclear distribution of centrioles at G-stage (00:00); perinuclear disengagement of centrioles from deuterosomes (empty flowers) and parental centrioles (plain flowers, arrows) in a Cen2-GFP+ cell (4:30); release of centrioles from the nuclear membrane (6:30) for apical migration (from 10:00). Bottom line: profile view of the Cen2-GFP+ signal dynamics. Dotted white line outlines the nucleus identified by contrasting Cen2-GFP signal. dt=30min, scale bar, 5 µm. See Videos 7.

(B) Magnified view of a Cen2-GFP; mRuby-Deup1 flower dynamics in G-stage. White arrowheads indicate the procentriole-loaded deuterosomes migrating on the nuclear envelope; dotted white line outlines the nucleus identified by contrasting Cen2-GFP signal. dt=5s, scale bar, 2 µm. See Video 9.

(C) Quantification of the duration of the interaction between procentriole-loaded deuterosome and the nuclear envelope before D-stage in Cen2-GFP brain MCCs. Four independent experiments quantified, n=75 flower, n=8 cells analyzed. Error bars represent min to max ± median.

(D) Schematic representation of brain MCC differentiation depicting the timing of MT depolymerisation treatment. The eye shows the amplification stage monitored.”

(E) Immuno-reactivity of Deup1 on Cen2-GFP brain MCCs during G-stage, treated acutely with DMSO or Nocodazole (10µM, 4h). The nucleus is counter-stained with Hoechst. Scale bar, 5µm.

(F) Quantification of the percentage of tethered deuterosomes per G-stage cell in DMSO and Nocodazole (10µM, 4h). Four independent experiments quantified; n=75 deuterosomes in DMSO, n=31 deuterosomes in Nocodazole. Error bars represent mean ± SD; ****p<0,0001; Chi-2 test with Yates’ correction.

(G) Quantification of the proportion of procentrioles disengaging with a nuclear contact. Three independent experiments were quantified; n=98 flowers, n=8cells.

(H) Schematic representation of brain MCC differentiation depicting the timing of MT depolymerisation treatments. The eye shows the amplification stage monitored.

(I) Quantification of the proportion of D-stage cells per fields among amplifying cells under chronic DMSO and Nocodazole treatments (48h, 1µM and 5µM). At least three independent experiments were quantified, n=2626 DMSO cells, n=1483 Noco 1µM cells, n=909 Noco 5µM cells; error bars represent mean ± SD. ns, not significant; ****p<0,0001; non-parametric Mann Whitney test.

(J) Quantification of D-stage quality phenotype under chronic DMSO and Nocodazole (48h, 1µM). Total disengagement in black, partial disengagement in white. At least three independent experiments were quantified, n=35 DMSO cells analyzed, n=25 Noco 1µM cells analyzed. ****p<0,0001; Chi-2 test with Yates’ correction.

(K) Quantification of disengagement duration in Cen2-GFP brain MCCs under DMSO and acute Nocodazole treatment (10µM). Three independent experiments were quantified, n=16 DMSO cells analyzed, n=11 Noco 10µM cells analyzed. *p=0,0235; non-parametric Mann Whitney test.

(L) Representative movie of Cen2-GFP dynamics in D-stage of brain MCCs under DMSO and acute Nocodazole treatment (10µM). (t-1) represents the timepoint before drug treatments. At t+3h45, the DMSO cell centrioles are almost all disengaged while in Nocodazole 10µM, some flowers are not disengaged yet. At t+11h20, the control cell displays a regrouped patch of centrioles whereas Nocodazole cell shows some scattered centrioles that cover the whole surface of the cytoplasm. dt=5min; scale bar, 5 µm. See Video 12.

Disengagement of centrioles is a combination of centriole individualization and deuterosome splitting or dissolution

(A) 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 (red arrows, 1:40), splitting them in smaller entities bearing fewer procentrioles not disengaged yet (4:10), 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 Video 13.

(B-C) 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.

(D) Splitted deuterosomes with a single centriole migrating away from each other on the nuclear envelope. White dotted line outlines shadow of the nuclear membrane identified with contrasting Cen2-GFP signal. dt=5s, scale bar, 2 µm. See Video 14.

(E) Tyrosinated microtubules and deuterosomes immuno-reactivity profiles in brain MCC in D-stage. These pictures show one z plane in the basal part of the cell. Tyrosinated MTs were stained with (YL 1-2) and deuterosomes with Deup1. Deup1 signal intensity is decreased from left to right to show that deuterosomes are located on MT nodes. Scale bar, 5 µm. See Video 16.

(F) Representative U-ExM image of brain MCCs during G-stage. Cells were immunostained with antibodies to Acetylated-tubulin, Deup1, and the nucleus was counter stained with Hoechst. Scale bar, 1µm. Right insets show magnification of a deuterosome bearing multiple procentrioles. Scale bar, 250nm.

(G) Representative U-ExM images of brain MCCs during D-stage. Cells were immunostained with antibodies to Acetylated-tubulin, Deup1, and the nucleus was counter stained with Hoechst. Scale bar, 1µm. Right insets show: (top) single centrioles disengaged from deuterosomes and empty deuterosomes; (bottom) small deuterosomes with single centrioles. Scale bar, 250nm.

Disengaged centrioles finally converge with the former centrosome in a microtubule-dependent manner

(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 17.

(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.

(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) Profile view of centriole apical migration after D-stage in Cen2-GFP+ DIV2 brain MCCs treated with acute DMSO and Nocodazole 10µM. At t-1, cells display late G-stage/early D-stage procentrioles and are not treated with any drug. DMSO and Nocodazole 10µm were added right after the first timepoint acquisition. In DMSO, 2 centriole patches are under and over the nucleus (t+0h). The patch under the nucleus progressively joins the apical one (t+2h) to become one apical patch (t+4h) and stabilizes (t+6h). In 10µM Nocodazole, procentrioles start to scatter after 1h, and the group of centrioles below the nucleus travel up and down (t+2h and t+4h) and does not succeed to become one apical group of centrioles (t+6h). Four independent experiments were scored for n=7 DMSO cells and two independent experiment were scored for n=4 Noco 10µM cells. dt=5min; scale bar, 5 µm. See Video 18.

(I) Quantification of apical migration duration in DMSO and acute Nocodazole 10µM in brain MCCs. Four independent experiments were scored for n=7 DMSO cells and two independent experiment were scored for n=4 Noco 10µM cells. Error bars represent min to max ± median. ****p<0,0001; non-parametric Mann Whitney test.

(J) Quantification of MBB patch area (µm²) in DMSO and chronic Nocodazole treatment (48h, 1 and 5µM) in brain MCCs. Three independent experiments were scored, n=22 DMSO cells analyzed, n=15 Noco 1µM cells and n=18 Noco 5µM cells. Error bars represent min to max ± median. ****p<0,0001; non-parametric Mann Whitney test.

Model proposed by this study on the role of microtubules in centriole amplification in brain MCC.

Progenitor stage: Undifferentiated brain MCC progenitor. The centrosome is close to the apical membrane, the mother centriole is docked and templates the primary cilium. At this stage, MTs are organized by the centrosome.

Amplification stage: MTs organize a Deup1, Centrin and PCNT nest around the centrosome from which new centrioles emerge. In the earliest step of amplification, a Centrin, Deup1 and PCNT cloud (pink area) appears around the centrosomal centrioles, in a MT dependent fashion. Then, early Plk4+, SAS6+, Cen2-GFP+ procentrioles form on small Deup1+ structures emerge. Procentrioles don’t have a MT wall yet (small blue procentrioles). Once they accumulate, they start exiting the cloud, and are maintained by the MTs in a restricted area around this pericentrosomal nest. As procentriole loaded deuterosomes migrate away from the cloud, a growing number of procentrioles recruit tubulin, building MT centriolar walls. These microtubules are progressively acetylating (pink centriole walls).

Growth stage: Procentrioles become MT nucleators and MTs anchor procentrioles to the nuclear membrane. In this stage, procentrioles mature on deuterosomes and parental centrioles by acquisition of polyglutamylated tubulin (blue centriole walls). Concomitantly, they migrate towards the nuclear membrane through a MT dependent mechanism where they distribute almost isotropically. They stay associated to the nuclear membrane along which they are able to migrate thanks to MT related mechanisms.

Disengagement stage: MTs contribute to the centrioles’ disengagement. Upon APC/C, Plk1 and MT actions, centrioles disengage from the deuterosomes and parental centrioles. Disengagement from deuterosomes consists of centriole detachment from deuterosomes, deuterosome splitting and Deup1 disintegration. Some Deup1 agregates (centriole free) can remain in the cytoplasm until ciliation.

Migration stage: MTs help centrioles reach their docking site. Once all centrioles are individualized, they start migrating collectively from the basal pole and periphery of the cell, with the help of MTs, towards the apical membrane where the former parental centrioles stand and dock.