Cep44 Functions in Centrosome Cohesion by Stabilizing Rootletin Delowar Hossain1,2, Sunny Y.-P

SHORT REPORT Cep44 functions in centrosome cohesion by stabilizing rootletin Delowar Hossain1,2, Sunny Y.-P. Shih1,*, Xintong Xiao1,*, Julia White1,* and William Y. Tsang1,2,3,‡

ABSTRACT The centrosome linker is a proteinaceous structure whose The centrosome linker serves to hold the duplicated centrosomes molecular composition, architecture and assembly mechanism are together until they separate in late G2/early mitosis. Precisely how the not fully understood. A handful of proteins known to be involved in linker is assembled remains an open question. In this study, we linker biology include C-Nap1 (Cep250) (Fry et al., 1998a; Mayor identify Cep44 as a novel component of the linker in human cells. et al., 2000), rootletin (CROCC) (Bahe et al., 2005; Yang et al., Cep44 localizes to the proximal end of centrioles, including mother 2006), LRRC45 (He et al., 2013), Cep68 (Graser et al., 2007), and daughter centrioles, and its ablation leads to loss of centrosome CCDC102B (Xia et al., 2018), centlein (Fang et al., 2014), Cep215 cohesion. Cep44 does not impinge on the stability of C-Nap1 (also (Cdk5rap2) (Barrera et al., 2010; Graser et al., 2007) and pericentrin known as CEP250), LRRC45 or Cep215 (also known as (Jurczyk et al., 2004). C-Nap1 localizes to the proximal end of CDK5RAP2), and vice versa, and these proteins are independently mother and daughter centrioles, where it docks LRRC45 and recruited to the centrosome. Rather, Cep44 associates with rootletin rootletin. LRRC45 can self assemble into filaments that link the and regulates its stability and localization to the centrosome. Our proximal end of mother and daughter centrioles together (He et al., findings reveal a role of the previously uncharacterized protein Cep44 2013). Likewise, rootletin is an integral component of the linker for centrosome cohesion and linker assembly. capable of self assembling into thin filaments (Vlijm et al., 2018). Cep68 bundles rootletin thin filaments into thick filaments and is KEY WORDS: Centrosome, Cohesion, Splitting, Cep44, Rootletin periodically present along the filaments (Vlijm et al., 2018). It is suspected that CCDC10B may serve a similar role to Cep68. INTRODUCTION Furthermore, centlein is proposed to act as a molecular bridge between The centrosome is the major microtubule-organizing center in C-Nap1 and Cep68. Finally, Cep215 and pericentrin are PCM higher eukaryotes (Bornens, 2012). It is composed of two components that form toroids around mother centrioles and contribute centrioles, the mother and daughter centrioles, surrounded by the to centrosome cohesion through poorly defined mechanisms. pericentriolar material (PCM), which nucleates and extends Centrosome disjunction is mainly promoted by the protein kinase microtubules. During G1 phase, the mother and daughter Nek2A (Fry et al., 1998b). At late G2/early mitosis, Nek2A is centrioles are held together by the centrosome linker (or G1–G2 phosphorylated and activated by Mst2 (also known as STK3), tether) (Nigg and Stearns, 2011). Following G1, this linker connects which in turn is phosphorylated and activated by Plk1 (Mardin et al., the duplicated centrosomes in S and G2 phases to ensure organelle 2010). Upon activation, Nek2A phosphorylates several proteins cohesion. At late G2/early mitosis, the linker is dissolved through a including C-Nap1 (Fry et al., 1998a), rootletin (Bahe et al., 2005), process called centrosome disjunction, which allows the duplicated LRRC45 (He et al., 2013), Cep68 (Fang et al., 2014), CCDC102B organelles to separate and migrate towards opposite poles for the (Xia et al., 2018) and centlein (Fang et al., 2014), leading to their establishment of the mitotic spindle. The timing of linker delocalization from the centrosome and subsequently linker dissolution is critical for the duration and fidelity of mitosis dissolution. In addition to delocalization, Cep68 is also known to (Kaseda et al., 2012; Nam and van Deursen, 2014; Silkworth et al., undergo ubiquitylation and proteasomal degradation in mitosis 2012; Zhang et al., 2012). Accelerated or delayed centrosome (Pagan et al., 2015). However, for most other linker proteins, it is disjunction is thought to induce chromosome lagging, resulting in unclear whether and how their protein abundances are regulated. aneuploidy and cancer. It can also cause spindle misorientation, Here, we found that Cep44 is a novel linker component that leading to alteration of balance between symmetrical and maintains centrosome cohesion by specifically controlling the asymmetrical division and hence, cell renewal and differentiation stability of rootletin. (Nam et al., 2015). RESULTS AND DISCUSSION Cep44 is a novel protein localized to the proximal end of centrioles 1Institut de Recherches Cliniques de Montréal, 110 avenue des Pins Ouest, Recent proteomic analyses of human centrosomes have led to the 2 Montréal, Québec H2W 1R7, Canada. Division of Experimental Medicine, McGill identification of a large number of proteins, some of which remain University, Montréal, Québec H3A 1A3, Canada. 3Facultéde Médecine, Département de pathologie et Biologie Cellulaire, Universitéde Montréal, Montréal, uncharacterized (Andersen et al., 2003; Jakobsen et al., 2011). Among Québec H3C 3J7, Canada. these, Cep44 is highly conserved among eukaryotes. Full-length *These authors contributed equally to this work human protein contains 390 amino acids (aa) and is predicted to have a ‡Author for correspondence ([email protected]) coiled-coil domain (aa 235–268) and a low complexity region (aa 335–345). To characterize Cep44, we obtained a polyclonal antibody W.Y.T., 0000-0002-1584-616X against Cep44 from a commercial source. Western blot studies This is an Open Access article distributed under the terms of the Creative Commons Attribution revealed that this antibody recognizes a single band of ∼44 kDa in License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. U2OS cell extracts (Fig. S1A). This band was markedly reduced upon depletion of Cep44 with two individual siRNA oligonucleotides, thus

Received 25 September 2019; Accepted 13 January 2020 confirming antibody specificity (Fig. S1A). Journal of Cell Science

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To examine the localization of Cep44, we conducted rootletin, Cep215 and Cep68, and vice versa. Ablation of Cep44 led immunofluorescence experiments with antibodies against Cep44 to a severe loss of centrosomal rootletin (Figs 2A and 3C,D), which in U2OS or RPE-1 cells and detected a signal that colocalized with could be rescued by GFP–Cep44 expression (Fig. 3E,F), and a the proximal centriolar markers C-Nap1 and glutamylated tubulin significant reduction of Cep68 signal (Figs 2A and 3G,H) without (Fig. 1A). This signal did not colocalize with the distal centriolar affecting C-Nap1, LRRC45 or Cep215 (Fig. 2A). On the other marker centrin, the mother centriole-specific distal appendage hand, reciprocal depletion of C-Nap1, LRRC45, rootletin, Cep215 marker Cep164 or the PCM marker γ-tubulin (Fig. 1A). Moreover, or Cep68 did not impinge on the localization of Cep44 (Fig. 4A). the signal was notably absent upon depletion of Cep44 (Fig. S1B), These results indicate that Cep44 and C-Nap1, LRRC45 or Cep215 further validating antibody specificity. 3D-SIM studies showed a are independently recruited to the centrosome. Moreover, they dot-like pattern of Cep44. In the top view, one Cep44 dot could be suggest that Cep44 is required for proper centrosomal localization of found inside the cylinder of Cep164 and overlapped with one rootletin and to a lesser extent, Cep68. glutamylated tubulin dot, while the other Cep44 dot was close to Next, we investigated the effects of depleting Cep44 on the protein another glutamylated tubulin dot (Fig. 1B). In the side view, Cep44 levels of C-Nap1, LRRC45, rootletin, Cep215 or Cep68, and vice was located in close proximity to glutamylated tubulin but far away versa. The level of rootletin, but not C-Nap1, LRRC45, Cep215 or from Cep164 (Fig. 1B). Unlike rootletin or LRRC45, Cep44 did not Cep68, was dramatically reduced upon Cep44 loss (Fig. 4B). In exhibit a fiber-like appearance (Fig. 1A,B). In addition to contrast, ablation of C-Nap1, LRRC45, rootletin, Cep215 or Cep68 endogenous Cep44, we found that recombinant Cep44 expressed had no effect on the level of Cep44 (Fig. 4C). Furthermore, depletion in RPE-1 cells also colocalized with glutamylated tubulin (Fig. 1C). of C-Nap1, LRRC45, Cep215 or Cep68 did not alter the level of These results together suggest that Cep44 localizes to the proximal rootletin (Fig. 4C). Collectively, these data support the notion that end of mother and daughter centrioles. Cep44 specifically regulates the stability of rootletin, which in turn Next, we studied the localization and protein level of Cep44 across impinges on its localization to the centrosome. the cell cycle in RPE-1 cells. Cep44 antibodies stained two dots in G0 Finally, we asked whether Cep44 physically interacts with rootletin and G1, and four dots in S and G2 phase cells (Fig. 1D), suggesting and C-Nap1. By conducting immunoprecipitation experiments, we that the protein localizes to procentrioles in addition to mother and demonstrated co-immunoprecipitation of endogenous Cep44 and daughter centrioles. During mitosis and, in particular, prophase, rootletin (Fig. 4D). In contrast, Cep44 did not bind to C-Nap1 metaphase and anaphase, Cep44 dots were less intense and staining (Fig. 4D). Thus, it seems likely that Cep44 associates with rootletin to became more diffuse (Fig. 1D). Quantification of centrosomal Cep44 control its stability. intensity showed a moderate signal in G0 and G1, a bright signal in S Because the centrosome linker is a highly dynamic structure, we and G2, and a weak signal in prophase, metaphase and anaphase speculate that linker proteins might turn over quickly. One linker (Fig. 1E). Likewise, the protein level of Cep44 peaked in S and G2, component Cep68 has been shown to be ubiquitylated by two and dropped in M phase (Fig. 1F,G). Given that the protein levels of ubiquitin ligases, βTrCP and VHL (Pagan et al., 2015; Yin et al., C-Nap1 and rootletin were also reduced in mitosis (Fig. 1F), as 2017). Although it is known that VHL-mediated ubiquitylation and reported previously for C-Nap1 (Mardin et al., 2010; Mayor et al., degradation of Cep68 is inhibited by rootletin and enhanced by 2002), the decrease in Cep44 abundance might be related to the MCM7 (Kong et al., 2017; Yin et al., 2017), it remains unclear dissolution of the centrosome linker. whether the level of rootletin itself is regulated. Based on our findings, it appears that none of the previously known linker Cep44 is required for centrosome cohesion proteins, namely C-Nap1, LRRC45, Cep215 and Cep68, is able to To explore whether Cep44 plays a role in centrosome cohesion, we impinge on rootletin protein level. More importantly, we discovered depleted Cep44 by using two individual siRNAs. We found that the that a novel protein Cep44 is the major player responsible for percentage of Cep44-depleted U2OS cells with premature controlling the abundance of rootletin. Cep44 might stabilize separation of γ-tubulin dots is much higher than that of control rootletin by preventing rootletin ubiquitylation and/or degradation. cells (Fig. 2A,B). This phenotype could be rescued by expression of Addition of MG132, but not chloroquine, stabilized rootletin upon GFP–Cep44 (Fig. 2C), and did not arise from perturbation of the Cep44 ablation (Fig. 4E), suggesting that rootletin is degraded by cell cycle (Fig. 2D). Premature separation of γ-tubulin dots is the ubiquitin-proteasome system rather than through autophagy. indicative of loss of centrosome cohesion since we observed this in Since Cep44 controls the stability of rootletin, which influences cells depleted of C-Nap1, LRRC45, rootletin, Cep215 or Cep68 the stability of Cep68 (Yin et al., 2017), ablation of Cep44 might (Fig. 2B). Besides γ-tubulin dots, centrin, C-Nap1, LRRC45, compromise Cep68 localization and protein level. We indeed found Cep215 and Cep68 dots were also found to be prematurely separated that Cep68 is delocalized from the centrosome upon Cep44 loss; (Fig. 2A). Centrosome cohesion was deemed to be lost when the however, the protein level of Cep68 remained unchanged. Thus, the distance between the mother and daughter centriole, which were effect of Cep68 provoked by Cep44 depletion is likely indirect and marked by centrin, exceeded >2 µm (Graser et al., 2007). The mean therefore, less severe in comparison with rootletin depletion. distance between the two centrin dots was 12.0 µm (oligo 1) or In comparison with Cep44, other linker proteins might employ 12.7 µm (oligo 2) in Cep44-depleted cells, compared to 0.6 µm in different mechanisms to control centrosome cohesion. C-Nap1 is control cells (Fig. 3A). Moreover, an increased distance between the known to regulate the localization of rootletin by organizing two centrin dots was found in another cell line, RPE-1, depleted of rootletin into a ring structure with protruding filaments at the Cep44 (Fig. 3B). These data indicate that Cep44 plays a role in proximal end of mother and daughter centrioles (Vlijm et al., 2018). centrosome cohesion. Cep215, on the other hand, might be crucial for rootletin filament formation but dispensable for ring formation (Barrera et al., 2010). Cep44 controls the stability of rootletin In contrast, LRRC45 does not affect the localization of rootletin, and To address the functional relationship between Cep44 and proteins vice versa, and these proteins could presumably form independent known to participate in centrosome cohesion, we investigated the filaments between the proximal end of mother and daughter effects of depleting Cep44 on the localization of C-Nap1, LRRC45, centrioles (He et al., 2013). When cells were individually depleted Journal of Cell Science

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Fig. 1. Cep44 is a proximal end centriolar protein whose level changes during the cell cycle. (A) U2OS cells or RPE-1 G0 cells were stained with the indicated antibodies with or without DAPI. GT335, glutamylated tubulin. Scale bar: 10 μm. (B) U2OS cells were stained with the indicated antibodies and images were acquired with 3D-SIM. M, mother centriole; D, daughter centriole. Scale bar: 200 µm. (C) RPE-1 G0 cells expressing GFP–Cep44 were stained with the indicated antibodies. Scale bar: 10 μm. (D) RPE-1 cells were stained with DAPI and the indicated antibodies. Scale bar: 10 μm. (E) Quantification of Cep44 fluorescence intensity at the centrosome. Results are mean±s.e.m. based on three independent experiments with 20 cells per experiment (n=60). **P<0.01 (one-way ANOVA). (F) RPE-1 cell lysates were western blotted with antibodies against Cep44, C-Nap1 or rootletin. α-tubulin was used as loading control. AS, asynchronous. A representative experiment is shown. (G) Quantification of Cep44 level relative to α-tubulin. Results are mean±s.e.m. based on five independent experiments (n=5). *P<0.05 (one-way ANOVA). Journal of Cell Science

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Fig. 2. Cep44 is required for centrosome cohesion. (A) U2OS cells transfected with non-specific (NS) or Cep44 siRNAs (oligo 1 or 2) were stained with the indicated antibodies. Scale bar: 10 µm. (B) The percentage of U2OS cells with separated γ-tubulin dots is presented. (C) The percentage of GFP-positive U2OS cells with separated γ-tubulin dots is presented. (D) FACS analysis of the cell cycle states of U2OS cells transfected with NS or Cep44 siRNAs. For B,C, three independent experiments were performed, and each experiment involved counting >100 cells for each condition. Journal of Cell Science

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Fig. 3. Cep44 is required for the localization of rootletin to the centrosome. (A,B) Quantification of the distance between two centrin dots in U2OS or RPE-1 cells. G1 cells with the best knockdown were chosen for quantification. (C,D) Quantification of rootletin fluorescence intensity at the centrosome in U2OS or RPE-1 cells. (E,F) Quantification of rootletin fluorescence intensity at the centrosome in U2OS or RPE-1 cells. GFP-positive cells were chosen for quantification. (G,H) Quantification of Cep68 fluorescence intensity at the centrosome in U2OS or RPE-1 cells. For A–H, results are mean+s.e.m. based on three independent experiments with 20 cells per experiment (n=60). **P<0.01 (two-tailed Student’s t-test). Journal of Cell Science

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Fig. 4. Cep44 specifically binds to and stabilizes rootletin, and its localization is independent of known linker proteins. (A) U2OS cells transfected with non-specific (NS), C-Nap1, LRRC45, rootletin, Cep215 or Cep68 siRNAs were stained with the indicated antibodies. Scale bar: 10 µm. (B) U2OS cells were transfected with NS or Cep44 siRNAs (oligo 1 or 2). Lysates were western blotted with the indicated antibodies. α-tubulin was used as loading control. (C) U2OS cells were transfected with NS, C-Nap1, LRRC45, rootletin, Cep215 or Cep68 siRNAs. Lysates were western blotted with the indicated antibodies. α-tubulin was used as loading control. (D) Western blotting of endogenous Cep44, rootletin and C-Nap1 after immunoprecipitation from U2OS lysates with control, anti-Cep44 or anti-rootletin antibodies. IN, input (5 %); IP, immunoprecipitation. (E) U2OS cells were transfected with NS or Cep44 siRNAs and treated with vehicle, MG132 or chloroquine. Lysates were western blotted with the indicated antibodies. α-tubulin was used as loading control. Journal of Cell Science

6 SHORT REPORT Journal of Cell Science (2020) 133, jcs239616. doi:10.1242/jcs.239616 of Cep44, C-Nap1, LRRC45, rootletin, Cep215 and Cep68, we Immunoblotting and immunofluorescence observed an increase in the percentage of cells with separated Immunoblotting and immunofluorescence were performed as described γ-tubulin dots (Fig. 2B) and an increase in the distance between the previously (Barbelanne et al., 2016). Briefly, cells were lysed in a lysis two centrin dots (Fig. 3A), suggesting that these proteins support buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, μ μ centrosome cohesion to varying degrees. Nevertheless, in light of a 1 mM DTT, 0.1 M AEBSF, 2 g/ml leupeptin, 2 g/ml aprotinin, 10 mM NaF, 50 mM β-glycerophosphate and 10% glycerol) at 4°C for 30 min. previous report showing that the percentage of cells with split Extracted proteins were recovered in the supernatant after centrifugation at centrosomes upon ablation of rootletin and LRRC45 is comparable 16,000 g for 5 min. For immunoblotting, 100 μg of extract was used and to that when rootletin alone is not present (He et al., 2013), there are proteins were analyzed by SDS-PAGE and immunoblotted with primary likely non-redundant and redundant mechanisms regulating antibodies, followed by horseradish peroxidase-conjugated secondary centrosome cohesion. antibodies (Rockland Inc., Mississauga, ON, Canada, 610-703-002 and Recently, mutations in C-Nap1, CCDC102B and Cep215 have 611-7302). For immunofluorescence staining, cells were fixed with cold been shown to be associated with various human disorders methanol or 4% paraformaldehyde and permeabilized with 1% Triton including Usher syndrome, Seckel syndrome, high myopia, X-100 in PBS. Slides were blocked with 3% BSA in 0.1% Triton X-100 in primary microcephaly and agenesis of the corpus callosum (Bond PBS and subsequently incubated with primary antibodies and secondary et al., 2005; Fuster-Garcia et al., 2018; Hosoda et al., 2018; Jouan antibodies. Secondary antibodies used were Cy3- (Jackson Immunolabs, Burlington, ON, Canada, 711-165-151 and 715-165-152), DyLight649- et al., 2016; Khateb et al., 2014; Kubota et al., 2018; Yigit et al., (Jackson Immunolabs, 715-495-151) or Alexa Fluor 488- (Thermo Fisher 2015). It would be interesting to determine whether deficiency in Scientific, Saint-Laurent, QC, Canada, A11008, A11055 and A11001) Cep44 or any other linker protein is linked to disease. conjugated donkey anti-mouse, anti-goat or anti-rabbit IgG. DAPI (Molecular Probes, Saint-Laurent, QC, Canada, D3571) stained DNA and MATERIALS AND METHODS slides were mounted, observed and photographed using a Leitz DMRB Cell culture and plasmids (Leica, Concord, ON, Canada) microscope (100×, NA 1.3) equipped with a RPE-1 and U2OS obtained from ATCC (Manassas, VA) were regularly Retiga EXi cooled camera. Super-resolution 3D-SIM imaging was tested for mycoplasma contamination. Cells were grown in DMEM (Wisent performed by using an ELYRA PS.1 microscope (Carl Zeiss, Toronto, Inc., Montreal, QC, Canada, 319-005-CL) and supplemented with 10% FBS ON, Canada) equipped with an alpha Plan-Apochromat 100×/1.46 NA oil (Wisent Inc., 080150) at 37°C in a humidified 5% CO2 atmosphere. Human DIC M27 immersion objective and 488 nm, 561 nm and 640 nm lasers Cep44 cDNA (Dharmacon Inc, Lafayette, CO, MHS6278-202829609) was (Hossain et al., 2019). Image stacks of 2 µm in height with a z-distance sub-cloned into mammalian vector pEGFP-C1 to generate pEGFP-C1- of 0.116 µm were acquired with an Andor iXon 885 EMCCD camera. Each Cep44. The construct was verified by DNA sequencing. z-section was recorded with five grating rotation and five phase changes.

Antibodies Quantification of fluorescence intensity and protein band Antibodies used in this study included mouse anti-C-Nap1 A region of interest was drawn around a fluorescent spot in the vicinity of the [immunofluorescence (IF) 1:250 and western blotting (WB) 1:100; centrosome (Hossain et al., 2017). The area of the region of interest was used sc-390540, Santa Cruz Biotechnology, Dallas, TX], mouse anti-rootletin to determine the fluorescence intensity by using Volocity6 (PerkinElmer, (IF 1:1000 and WB 1:100; sc-374056, Santa Cruz Biotechnology), rabbit Woodbridge, ON, Canada). Image conditions were identical in all cases and anti-LRRC45 (IF 1:500 and WB 1:100; HPA024768, Sigma-Aldrich, no areas were saturated as confirmed by the pixel intensity range. Protein St Louis, MO), rabbit anti-Cep68 (IF 1:1000 and WB 1:100, 15147-1-AP; bands from western blot films were quantified with ImageJ (NIH, Bethesda, Proteintech, Burlington, ON, Canada), rabbit anti-Cep215 (IF 1:1000 and MD). Different film exposure lengths were used to prevent saturation. WB 1:500, A300-554A, Bethyl Laboratories, Burlington, ON, Canada), rabbit anti-Cep44 (IF and WB 1:1000; 24457-1-AP, Proteintech), RNA interference anti-GFP, goat anti-Cep164 (IF 1:500; sc-240226, Santa Cruz Synthetic siRNA oligonucleotides were purchased from Dharmacon and the Biotechnology), mouse anti-α-tubulin (WB 1:1000; T5168, Sigma- sequences were: Aldrich), rabbit anti-γ-tubulin (IF: 1:1000, Sigma-Aldrich, T3559), NS (non-specific), 5′-AATTCTCCGAACGTGTCACGT-3′; C-Nap1, 5′- mouse anti-γ-tubulin (IF 1:1000; T6557, Sigma-Aldrich), mouse anti- GAGCAGAGCTACAGCGAAT-3′; rootletin, 5′-AAGCCAGTCTAGAC- glutamylated tubulin (GT335) (IF 1:1000; AG-20B-0020, AdipoGen Life AAGGA-3′; LRRC45, 5′-CCAACAGAACAAGTCCATT-3′; Cep68, 5′- Sciences, Burlington, ON, Canada) and mouse anti-centrin (IF 1:1000; CGAAGATGATCCATCCCTA-3′; Cep215, 5′-GCAAGGATCTGAATT- 04-1624, Sigma-Aldrich). TGTT-3′; Cep44 oligo 1, 5′-GAGGTGGACTGTGTAGGTTTG-3′; and Cep44 oligo 2 (3′-UTR), 5′-GAGCAATGATTATACTGCTTT-3′. siRNA Cell cycle synchronization and fluorescence-activated cell transfection was performed using siIMPORTER (Millipore, Etobicoke, ON, sorting analysis Canada, 64-101) according to manufacturer’s instructions. Cells were RPE-1 cells were brought to G0 by serum starvation for 72 h. For harvested 72 h after siRNA transfection. synchronization in the G1, S, G2 and M phases, cells were treated with 0.4 mM mimosine for 24 h, with 2 mM hydroxyurea (HU) for 24 h and Statistical analysis released for 4 h, with 2 mM HU for 24 h and released for 9 h, and with Data analysis was performed using a one-way ANOVA or two-tailed 40 ng/ml nocodazole for 24 h, respectively. Cell cycle distribution was Student’s t-test on Prism 8 (GraphPad, San Diego, CA) and are indicated as confirmed by fluorescence-activated cell sorting analysis (Barbelanne et al., *P<0.05 and **P<0.01. 2013). Briefly, cells were fixed in 95% ethanol at 4°C for 1 h and subsequently incubated with 0.1 mg/ml RNase A in PBS at 37°C for Acknowledgements 30 min. After centrifugation and removal of supernatant, cells were stained We thank all members of the Tsang laboratory for constructive advice and with 50 µg/ml propidium iodide at 4°C for 30 min. Samples of 100,000 cells P. Prabhala for her effort on the project. were analyzed with a FACScalibur flow cytometer (BD Biosciences, San Jose, CA), CellQuest (BD Biosciences) and ModFit (ModFit LT, Topsham, Competing interests ME) cell cycle analysis software. The authors declare no competing or financial interests.

Treatment with proteasome and autophagy inhibitors Author contributions Cells were treated with 10 µM MG132 for 6 h or 75 µM chloroquine for 24 h Conceptualization: W.Y.T.; Methodology: W.Y.T.; Software: W.Y.T.; Validation: D.H., prior to collection. S.Y.-P.S., X.X., J.W., W.Y.T.; Formal analysis: D.H., S.Y.-P.S., X.X., J.W., W.Y.T.; Journal of Cell Science

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Investigation: D.H., S.Y.-P.S., X.X., J.W., W.Y.T.; Resources: W.Y.T.; Data curation: Jouan, L., Ouled Amar Bencheikh, B., Daoud, H., Dionne-Laporte, A., D.H., S.Y.-P.S., X.X., J.W.; Writing - original draft: W.Y.T.; Writing - review & editing: Dobrzeniecka, S., Spiegelman, D., Rochefort, D., Hince, P., Szuto, A., D.H., W.Y.T.; Visualization: D.H.; Supervision: D.H., W.Y.T.; Project administration: Lassonde, M. et al. (2016). Exome sequencing identifies recessive CDK5RAP2 W.Y.T.; Funding acquisition: W.Y.T. variants in patients with isolated agenesis of corpus callosum. Eur. J. Hum. Genet. 24, 607-610. doi:10.1038/ejhg.2015.156 Funding Jurczyk, A., Gromley, A., Redick, S., San Agustin, J., Witman, G., Pazour, G. J., W.Y.T. was a Fonds de recherche SantéJunior 2 Research Scholar. This work was Peters, D. J. and Doxsey, S. (2004). 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