M–metaphase to anaphase transitions and multiciliated A to G–G to D transitions.
To validate the dormant mitotic capacity of
differentiating progenitors, we live imaged centriole and chromosome dynamics with APC/C
inhibition. Differentiating cells entered mitosis after the G to D transition, as suggested by
immunostainings (Fig. 2E; fig. S8, A and B; and
movie S2). Mitosis was characterized by cyclin B1
accumulation and degradation; nuclear envelope
breakdown and reformation; and unduplicated
chromosome condensation and segregation (figs.
S8, B to F; S9; and S10, A and B). Mitosis duration
depended on pro TAME concentration (fig. S10C),
as in cycling cells (21). Exit from the metaphase-like state and karyokinesis completion suggested
that APC/C inhibition was transient, as in cycling
cells (21, 22), and that CDK1 hyperactivation
eventually reactivated APC/C. Co-incubating
cells with pro TAME and the synergistic APC/
CCDC20 inhibitor, apcin (22), increased mitotic entry and blocked mitotic exit (fig. S10, C to F). Thus,
CDK1-APC/C–dependent molecular switches,
which drive each stage of mitosis forward, are
conserved in postmitotic progenitors of multiciliated cells. However, physiological CDK1 activity is actively restrained, consequently preventing
To test if CDK1 controls centriole amplification
dynamics, we live imaged centrin 2–green fluores-
cent protein (CEN2-GFP+) progenitors and pharma-
cologically targeted mitosis regulators. Inhibiting
CDK1 or PLK1 (polo-like kinase 1) (17 ) hindered A
to G and G to D transitions (Fig. 3A and fig. S11, A
and B) and impeded multiciliated-cell formation
(fig. S12A). The A to G transition was delayed and
the A phase prolonged (Fig. 3A and movie S3).
Because procentrioles are generated exclusively
during the A phase (9), we investigated if displac-
ing the A to G transition affects centriole number.
Inhibiting CDK1 or PLK1 increased deuterosome
number per cell and subsequent centriole number
(Fig. 3B and fig. S12, B to D). It also prevented
centrosomal centriole distancing and deuterosome
dispersion around the nucleus (fig. S13). The G to D
transition was also delayed and failure to initiate
D phase led to deuterosome regrouping, suggest-
ing an arrest of the centriole dynamic (Fig. 3A
and fig. S14, A and B). In cells passing the G to D
transition, D phase decelerated and centriole
disengagement and migration were incomplete
(Fig. 3A, fig. S14C, and movie S4), leading to
partial motile ciliation (fig. S14, C to E). Con-
versely, inhibiting WEE1-MYT1 with PD 166285
accelerated A to G and G to D transitions and
multiciliated-cell formation (Fig. 3A, figs. S11B
and S12A, and movie S5). Because PD 166285 can
affect other kinases, we inhibited WEE1 with MK
1775 and observed comparable tendencies (fig.
S12A). Acceleration of the A to G transition de-
creased deuterosome, centriole, and motile cilia
number (Fig. 3B and fig. S12, D to H). Centrosomal
centriole distancing, deuterosome dispersion, and
motile ciliation were comparable to controls (figs.
S13 and S14, C to E), suggesting that accelerated
transitions led to efficient centriole growth, dis-
engagement, and docking. CDK1 activity is there-
fore fine-tuned to control centriole number, growth,
and disengagement for accurate motile ciliation
while avoiding mitotic commitment.
Finally, we monitored centriole dynamics
with disrupted APC/C activity (fig. S11, C and D).
Pro TAME with or without apcin treatments did
not affect the A to G transition (A-phase length,
deuterosome number, and centriole dynamics;
Fig. 3C; figs. S12, B and C, and S13; and movie S6).
The G to D transition was significantly affected.
It was slightly delayed, and cells that failed to
initiate D phase regrouped their deuterosomes
(Fig. 3C and fig. S14B). As expected, some cells
passing the G to D transition underwent mitosis
(fig. S11, C and D, and movie S6). In cells with a D
phase spared from mitotic entry, D-phase dura-
tion increased in a dose-dependent and syner-
gistic manner (Fig. 3C and movie S6) and led to
partial motile ciliation (fig. S14, D and E), as with
CDK1 and PLK1 inhibition. This suggests that
CDK1 activates APC/CCDC20. Together with PLK1,
APC/C triggers the G to D transition and controls
synchronous centriole disengagement required
for functional migration, docking, and ciliation.
In return, APC/C dampens CDK1 activity, thus
preventing mitosis in differentiating progenitors.
This study reveals that the CDK1-APC/C mitotic
oscillator is summoned after progenitor cell di-
vision to drive terminal differentiation instead
of proliferation (Fig. 3D). Postmitotic progenitors
redeploy the robust mitotic clocklike regulatory
circuit to drive the orderly progression of cen-
triole production—number control, growth, and
disengagement—and provide multiciliated cells
with a sized patch of centrioles competent for
motile ciliation. This finding aids understanding
of the development of multiciliated cells and
motile cilia-powered flows crucial for organism
Although centrosome duplication in cycling
cells is coupled to cell division (10–16), multiciliated progenitors dampen the mitosis machinery to drive centriole dynamics but avoid nuclear
division (Fig. 3E). This is consistent with studies
in mammalian cycling cells showing that CDK1
couples nuclear and cytoplasmic events at mitotic
entry (23, 24) and with studies in Drosophila
showing the uncoupling of nuclear-cytoplasmic
events by experimentally dampening CDK1 (25–27).
Thus, calibration of the mitosis machinery to uncouple cytoplasmic from nuclear processes exists
physiologically in mammalian cells. This mechanism can be used by postmitotic progenitors to
impose timing and directionality in the control
of cytoplasmic events such as organelle remodeling associated with differentiation. By contrast,
this kind of calibration could allow cycling cells to
undergo pathological centriole amplification linked
to cancer and microcephaly (5, 28–30).
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We thank all members of the Spassky laboratory for
comments and discussions. We thank A. Aguilar, P. Bastin,
M. Bornens, B. Durand, O. Gavet, and M. Piel for their
comments on the project. We thank X. Morin for the
pCAAGS-H2B-RFP, CMV-CEN2-TagRFP, and pCAAGS-CEN2-
GFP-mCherry plasmids and T. Caspary for the Arl13b-GFP
plasmid. We thank N. Menezes for her contribution on the
image analysis bioinformatic scripts; F. Delestro Matos from the
IBENS Bioinformatics Platform for collaborating on the
conception and design of illustrations; A.-K. Konate and
R. Nagalingum for administrative support; the IBENS Animal
Facility for animal care; and Le Service des Animaux
Transgéniques (SEAT) (UMS3655, PFEP, Institut Gustave
Roussy, Villejuif, France) for generating the Arl13b-GFP
mouse strain. We thank the IBENS Imaging Facility, with
grants from Région Ile-de-France (NERF 2011-45), Fondation
pour la Recherche Médicale (FRM) (DGE 20111123023), and
Fédération pour la Recherche sur le Cerveau–Rotary
International France (2011). The IBENS Imaging Facility
and the team received support from Agence Nationale
de la Recherche (ANR) Investissements d’Avenir
(ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02
PSL* Research University). A.K. and J.S.-T. are supported by
UPMC, INSERM, and GEFLUC. The Spassky laboratory is
supported by INSERM, CNRS, l’École Normale Supérieure
(ENS), ANR (ANR-12-BSV4-0006), European Research
Council (ERC Consolidator grant 647466), FRM
(FRM20140329547), Cancéropôle Ile-de-France (2014-1-PL
BIO-11-INSERM 12–1), and Fondation Pierre-Gilles de Gennes
(FPGG03). A.M. is funded by Association pour la Recherche sur
le Cancer (ARC PJA-20131200184) and ANR (ANRJC JC-15-
CE13-0005-01). A.A.J. received fellowships from the French
Ministry of Higher Education and Research, FRM
(FDT20150531994), and Labex MEMOLIFE. All data are in the
manuscript and the supplementary materials. The authors
declare no competing financial interests.
Materials and Methods
Figs. S1 to S14
Movies S1 to S6
8 June 2017; accepted 27 September 2017
Published online 5 October 2017
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