Cell Cycle and Development

LIONEL PINTARD

The Cell Cycle and Development team aims to acquire new knowledge to decipher the mechanisms of cell division to understand the mechanisms of cancer, a disease resulting from uncontrolled cell division.

The team mainly uses the nematode C. elegans as a model system and employs a multidisciplinary approach combining various approaches (biochemistry, genetics, imaging, proteomics) to ask questions at different scales, from the molecule to the organism. As the mechanisms of regulation of cell division are conserved between species, the team is also studying the emerging paradigms in C. elegans in human cells.

Keywords: Cell division – Mitosis – Meiosis –Kinases – Microtubule-severing enzymes – Nuclear envelope breakdown

+33 (0)157278089 lionel.pintard(at)ijm.fr @LabPintard https://sites.google.com/site/pintardlab/

Research

OUR VISION

Acquiring new knowledge to decipher how cells divide, which is key to better understand the mechanisms of cancer, a disease caused by uncontrolled cell division.

CONTEXT

Humans are roughly built from 10^13 cells corresponding to 200 different cell types. All these cells are generated through cell divisions, starting from a single cell, the fertilized egg. To generate these large number of cells and maintain tissue homeostasis, the human body experiences as many as 10^16 cell divisions in a lifetime. During each cell division, the genome must be faithfully replicated and equally segregated between the daughter cells during mitosis. Defects in these processes can have drastic consequences, leading to outcomes such as cell death, genome instability or deregulated growth, typical of cancer. Despite considerable progress, the mechanisms regulating cell division are incompletely understood. This lack of knowledge has considerably limited the development of innovative therapeutic approaches.

RESEARCH PROGRAM

I- Mechanisms regulating mitotic entry in space and time

Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. Consistently, unscheduled mitosis may contribute to genetic instability. Entry into mitosis is controlled by evolutionarily conserved serine/threonine kinases (Aurora A, Polo-like kinase, Plk1) as well as counteracting phosphatases (PPases). How these kinase activities are regulated in space and time, and how they work in concert to trigger mitosis at the right time remain ill defined. In this context, we are investigating the

Activation mechanism of key mitotic kinases (Aurora A, Polo-like kinase 1)
Role of mitotic kinases in nuclear envelope breakdown (NEBD)
Role and regulation of Mitotic kinases during asynchronous cell division

Figure 1: Bora-Aurora A (Aurka)-Plk1 axis during mitotic entry

II- Role and regulation of the microtubule-severing enzyme Katanin

Microtubules (MTs) are dynamic cytoskeleton polymers, which play a central role in cell division, morphogenesis, motility and signaling. Most MT regulatory proteins interact with the plus or minus end of microtubules and thereby control their polymerization and depolymerization rates. However, one class of MT regulator interacts with the MT lattice and severs MTs throughout their length, thereby controlling their size and density in the cell. Three evolutionarily conserved MT-severing enzymes have been identified: Fidgetin, Spastin and Katanin. Mutations of these enzymes have been linked to various defects and pathologies including developmental defects and neurodegenerative disorders. However, little is known about the precise mechanisms by which these enzymes sever MTs. Likewise, how these enzymes are regulated in space and time is poorly understood. We are currently focusing on deciphering the mode of action and regulation of Katanin, which is essential for female meiotic spindle assembly in C. elegans. We are focusing our work on the

role of MT-severing in meiotic spindle assembly
Mechanism of MT-severing by Katanin
Regulation of Katanin-mediated MT-severing in space and time during development

Figure 2: Role and regulation of Katanin during C. elegans development (From Joly et al. JCB 2020).

III- Cullin-RING E3-Ligases in cell division

Cullin-RING E3-ligases (CRL) represent the largest family of E3 ubiquitin-ligases targeting the degradation of key cell cycle regulators in space and time, contributing to the orderly progression of the cell division cycle. We are interested in understanding how these enzymes regulate cell cycle progression in a developmental context.

CRL in the regulation of the Bora-Aurora-Plk1 pathway
CRL in the regulation of Katanin activity
CRL in the maintenance of DNA replication integrity

METHODS

We use a multidisciplinary approach including biochemistry (reconstitution of enzymatic activities from purified components to dissect molecular mechanisms), genetics, live cell imaging, proteomics approaches using both human cells and the nematode C. elegans. Mechanisms regulating cell division are conserved between species such that emerging paradigm from C. elegans can be immediately investigated in human cells. Furthermore, C. elegans offers a number of practical advantages to study conserved pathways regulating cell division (Pintard & Bowerman, Wormbook, Genetics 2019).

Members

Group Leader:

Lionel PINTARD
Téléphone : +33 (0)157278089, +33 (0)157278087
Email : lionel.pintard (at) ijm.fr

Members:

Eva	BEAUMALE	PhD Student
Nicolas	JOLY	Researcher
Sylvia	NKOMBO NKOULA	PhD Student
Batool	OSSAREH-NAZARI	Biological engineer
Anais	PILLAN	PhD Student
Lucie	VAN HOVE	Assistant biology engineer
Griselda	VELEZ AGUILERA	Postdoct

June, 2021 (c) Pintard Lab

From left to right: Anais, Griselda, Batool, Lucie, NicoT, Sylvia, Lionel, Eva, Lola, Anaelle, Emma, NicoJ

Selected Publications

Velez-Aguilera, G., Ossareh-Nazari, B., Van Hove, L., Joly, N., and Pintard, L*. (2022). Cortical microtubule pulling forces contribute to the union of the parental genomes in the. Elife Mar 8;11:e75382. doi: 10.7554/eLife.75382.

Tavernier, N., Thomas, Y., Vigneron, S., Maisonneuve, P., Orlicky, S., Mader, P., Regmi, S. G., Van Hove, L., Levinson, N. M., Gasmi-Seabrook, G., Joly, N., Poteau, M., Velez-Aguilera, G., Gavet, O., Castro, A., Dasso, M., Lorca, T., Sicheri, F.*, and Pintard, L*. (2021). Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry. Nat Commun 12, 1899. doi: 10.1038/s41467-021-21922-w

Velez-Aguilera, G., Nkombo Nkoula, S., Ossareh-Nazari, B., Link, J., Paouneskou, D., Van Hove, L., Joly, N., Tavernier, N., Verbavatz, J. M., Jantsch, V., and Pintard, L. (2020). PLK-1 promotes the merger of the parental genome into a single nucleus by triggering lamina disassembly. Elife 9, 9:e59510. doi: 10.7554/eLife.59510.

Joly, N.*, Beaumale, E., Van Hove, L., Martino, L., and Pintard, L*. (2020). Phosphorylation of the microtubule-severing AAA+ enzyme Katanin regulates C. elegans embryo development. J Cell Biol 219, (6):e201912037. doi: 10.1083/jcb.201912037.

Martino, L., Morchoisne-Bolhy, S., Cheerambathur, D. K., Van Hove, L., Dumont, J., Joly, N., Desai, A., Doye, V., and Pintard, L*. (2017). Channel Nucleoporins Recruit PLK-1 to Nuclear Pore Complexes to Direct Nuclear Envelope Breakdown in C. elegans. Dev Cell 43(2):157-171.e7. doi: 10.1016/j.devcel.2017.09.019.

* Corresponding authors

All the publications

Knox, J., Joly, N., Linossi, E. M., Carmona-Negrón, J. A., Jura, N., Pintard, L., Zuercher, W., and Roy, P. J. (2021). A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics. Sci Rep 11(1), 9161.

Gutnik, S., Thomas, Y., Guo, Y., Stoecklin, J., Neagu, A., Pintard, L., Merlet, J., and Ciosk, R. (2018). PRP-19, a conserved pre-mRNA processing factor and E3 ubiquitin ligase, inhibits the nuclear accumulation of GLP-1/Notch intracellular domain. Biol Open 7,16;7(7):bio034066. doi: 10.1242/bio.034066.

Vigneron, S., Sundermann, L., Labbé, J. C., Pintard, L., Radulescu, O., Castro, A., and Lorca, T. (2018). Cyclin A-cdk1-Dependent Phosphorylation of Bora Is the Triggering Factor Promoting Mitotic Entry. Dev Cell 45(5), 637-650.e7.

Dickinson, D. J., Schwager, F., Pintard, L., Gotta, M., and Goldstein, B. (2017). A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization. Dev Cell 42(4), 416-434.e11.

Joly, N.*, Martino, L., Gigant, E., Dumont, J., and Pintard, L.* (2016). Microtubule-severing activity of the AAA+ ATPase Katanin is essential for female meiotic spindle assembly. Development 143(19), 3604-3614.

Thomas, Y., Cirillo, L., Panbianco, C., Martino, L., Tavernier, N., Schwager, F., Van Hove, L., Joly, N., Santamaria, A., Pintard, L.*, and Gotta, M.* (2016). Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells. Cell Rep 15(3), 510-518.

Ossareh-Nazari, B., Katsiarimpa, A., Merlet, J., and Pintard, L.* (2016). RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans. G3 (Bethesda) 6(10), 3431-3442.

Parrilla, A., Cirillo, L., Thomas, Y., Gotta, M., Pintard, L., and Santamaria, A. (2016). Mitotic entry: The interplay between Cdk1, Plk1 and Bora. Cell Cycle 15(23), 3177-3182.

Tavernier, N., Noatynska, A., Panbianco, C., Martino, L., Van Hove, L., Schwager, F., Léger, T., Gotta, M.*, and Pintard, L.* (2015). Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos. J Cell Biol 208(6), 661-669.

Tavernier, N., Panbianco, C., Gotta, M., and Pintard, L.* (2015). Cdk1 plays matchmaker for the Polo-like kinase and its activator SPAT-1/Bora. Cell Cycle 14(15), 2394-2398.

Benkemoun, L., Descoteaux, C., Chartier, N. T., Pintard, L., and Labbe, J. C. (2014). PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo. J Cell Biol 205(4), 447-455.

Burger, J., Merlet, J., Tavernier, N., Richaudeau, B., Arnold, A., Ciosk, R., Bowerman, B., and Pintard, L.* (2013). CRL2^LRR-1 E3-ligase regulates proliferation and progression through meiosis in the Caenorhabditis elegans germline. PLoS Genet 9(3), e1003375.

Echalier, A., Pan, Y., Birol, M., Tavernier, N., Pintard, L., Hoh, F., Ebel, C., Galophe, N., Claret, F. X., and Dumas, C. (2013). Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1. Proc Natl Acad Sci U S A 110(4), 1273-1278.

Gomes, J. E., Tavernier, N., Richaudeau, B., Formstecher, E., Boulin, T., Mains, P. E., Dumont, J., and Pintard, L.* (2013). Microtubule severing by the katanin complex is activated by PPFR-1-dependent MEI-1 dephosphorylation. J Cell Biol 202(3), 431-439.

Merlet, J., Burger, J., Tavernier, N., Richaudeau, B., Gomes, J. E., and Pintard, L.* (2010). The CRL2^LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development. Development 137(22), 3857-3866.

Han, X., Gomes, J. E., Birmingham, C. L., Pintard, L., Sugimoto, A., and Mains, P. E. (2009). The role of protein phosphatase 4 in regulating microtubule severing in the Caenorhabditis elegans embryo. Genetics 181(3), 933-943.

Olma, M. H., Roy, M., Le Bihan, T., Sumara, I., Maerki, S., Larsen, B., Quadroni, M., Peter, M.*, Tyers, M.*, and Pintard, L.* (2009). An interaction network of the mammalian COP9 signalosome identifies Dda1 as a core subunit of multiple Cul4-based E3 ligases. J Cell Sci 122(Pt 7), 1035-1044.

Luke-Glaser, S., Roy, M., Larsen, B., Le Bihan, T., Metalnikov, P., Tyers, M., Peter, M., and Pintard, L.* (2007). CIF-1, a shared subunit of the COP9/signalosome and eukaryotic initiation factor 3 complexes, regulates MEL-26 levels in the Caenorhabditis elegans embryo. Mol Cell Biol 27(12), 4526-4540.

Luke-Glaser, S., Pintard, L., Tyers, M., and Peter, M. (2007). The AAA-ATPase FIGL-1 controls mitotic progression, and its levels are regulated by the CUL-3MEL-26 E3 ligase in the C. elegans germ line. J Cell Sci 120(Pt 18), 3179-3187.

Luke, B., Versini, G., Jaquenoud, M., Zaidi, I. W., Kurz, T., Pintard, L., Pasero, P., and Peter, M. (2006). The cullin Rtt101p promotes replication fork progression through damaged DNA and natural pause sites. Curr Biol 16(8), 786-792.

Luke-Glaser, S., Pintard, L., Lu, C., Mains, P. E., and Peter, M. (2005). The BTB Protein MEL-26 Promotes Cytokinesis in C. elegans by a CUL-3-Independent Mechanism. Curr Biol 15(18), 1605-1615.

Pintard, L., Kurz, T., Glaser, S., Willis, J. H., Peter, M., and Bowerman, B. (2003). Neddylation and Deneddylation of CUL-3 Is Required to Target MEI-1/Katanin for Degradation at the Meiosis-to-Mitosis Transition in C. elegans. Curr Biol 13(11), 911-921.

Pintard, L., Willis, J. H., Willems, A., Johnson, J. L., Srayko, M., Kurz, T., Glaser, S., Mains, P. E., Tyers, M., Bowerman, B., and Peter, M. (2003). The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425(6955), 311-316.

Kurz, T., Pintard, L., Willis, J. H., Hamill, D. R., Gonczy, P., Peter, M., and Bowerman, B. (2002). Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway. Science 295(5558), 1294-1298.

Reviews, book chapters:

Tavernier, N., Sicheri, F.*, and Pintard, L.* (2021). Aurora A kinase activation: Different means to different ends. J Cell Biol Sep 6;220(9):e202106128. doi: 10.1083/jcb.202106128.

Pintard, L.*, and Bowerman, B.* (2019). Mitotic Cell Division in Caenorhabditis elegans. Genetics 211(1), 35-73. doi: 10.1534/genetics.118.301367.

Pintard, L.*, and Archambault, V.* (2018). A unified view of spatio-temporal control of mitotic entry: Polo kinase as the key. Open Biol Aug;8(8):180114

Cirillo, L., Thomas, Y., Pintard, L., and Gotta, M. (2016). BORA-dependent PLK1 regulation: A new weapon for cancer therapy. Mol Cell Oncol 3(5), e1199265.

Tavernier, N., Labbé, J. C., and Pintard, L. (2015). Cell cycle timing regulation during asynchronous divisions of the early C. elegans embryo. Exp Cell Res 337(2), 243-248.

Merlet, J., and Pintard, L. (2013). Role of the CRL2(LRR-1) E3 ubiquitin-ligase in the development of the germline in C. elegans. Worm 2(3), e25716.

Noatynska, A., Tavernier, N., Gotta, M., and Pintard, L. (2013). Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms? Open Biol 3(8), 130083.

Burger, J., Merlet, J., and Pintard, L. (2011). [A bacterial attack on the ubiquitinproteolytic system.]. Med Sci (Paris) 27(4), 354-356.

Merlet, J., and Pintard, L.* (2011). Protéasome, ubiquitine et protéines apparentées à l’ubiquitine. Chapitre 6. Les complexes Cullin-RING E3-ligases (CRL) : fonction, architecture et régulation. Editions Lavoisier

Gomes, J. E., Merlet, J., B. J., and Pintard, L.* (2010). Remodelling the Occyte Into a Totipotent Zygote: Degradation of Maternal Products. Book Chapter – Oogenesis- The Universal Process

Verlhac, M. H.*, Terret, M. E., and Pintard, L.* (2010). Control of the oocyte-to-embryo transition by the ubiquitin-proteolytic system in mouse and C. elegans. Curr Opin Cell Biol 22(6), 758-763.

Merlet, J., Burger, J., Gomes, J. E., and Pintard, L.* (2009). Regulation of cullin-RING E3 ubiquitin-ligases by neddylation and dimerization. Cell Mol Life Sci 66(11-12), 1924-1938.

Pick, E.,* and Pintard, L.* (2009). In the land of the rising sun with the COP9 signalosome and related Zomes. Symposium on the COP9 signalosome, Proteasome and eIF3. EMBO Rep 10(4), 343-348.

Pintard, L., Willems, A., and Peter, M. (2004). Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family. EMBO J 23(8), 1681-1687.

Pintard, L., and Peter, M. (2003). Cdc34: cycling on and off the SCF. Nat Cell Biol 5(10), 856-857.

* Corresponding authors

Thesis

Collaborations

Frank SICHERI (University of Toronto, Canada)

Thierry LORCA (CRBM, Montpellier, France)

Anna CASTRO (CRBM, Montpellier, France)

Olivier GAVET (Institut Gustave Roussy, Paris Villejuif)

Mary DASSO (NIH, Bethesda USA)

Peter ASKJAER (CABD, Sevilla, Spain)

Verena JANTSCH (Max Perutz Labs Vienna, Austria)

Antoine JEGOU (Institut Jacques Monod, Paris, France)

Guillaume ROMET-LEMONNE (Institut Jacques Monod, Paris, France)

Denis CHRETIEN (IGDR, Rennes, France)

Julien DUMONT (Institut Jacques Monod, Paris, France)

Valérie DOYE (Institut Jacques Monod, Paris, France)

Bruce BOWERMAN (IMB, University of Oregon, USA)

Arshad DESAI (USCD, San Diego, USA)

Monica GOTTA (University of Geneva, Switzerland)

GRANTS – FOUNDING

ANR AMBRE

ANR REPLIGREAT

ARC

Idex « AAP Dynamique » Université de Paris

Equipe Labellisée Ligue contre le Cancer

Labex « WHO AM I »

News

25/10/2021 : Nicolas JOLY (Institut Jacques Monod), winner of the Maurice Nicloux Prize 2021!

Job

Master and PhD students

Our laboratory offers a wide range of interesting and challenging research projects for motivated master or PhD candidates aimed at understanding the mechanisms of cell cycle control during animal development.

We employ a unique combination of genetics, biochemistry, cell biology, live imaging, quantitative proteomics, and functional genomics approaches.

PostDoc candidates

We are looking for highly motivated and team-oriented scientists with a strong background in biochemistry, genetics and cell biology.

Candidates are welcome to apply with CV, publication list, motivation letter, and names of 2 referees to:

Lionel PINTARD – Université Paris cité, CNRS, Institut Jacques Monod, 15 rue Hélène Brion – 75013 PARIS cedex 13 – France

lionel.pintard (at) ijm.fr