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Non-Conventional Functions of Nuclear Pores

Valérie DOYE

Nuclear Pore Complexes (NPCs) are elaborate structures embedded in the nuclear envelope and composed of multiple copies of about 30 different proteins termed nucleoporins (Nups). Our team aims at characterizing, beyond their well-established function in transport between the cytoplasm and the nucleus, non-conventional functions of nucleoporins, notably in cell differentiation, gene regulation, and mitosis. By elucidating the role of these nucleoporins, our work should contribute to understanding the mechanisms by which mutations or misregulation of these nucleoporins lead to various pathologies.

Keywords: nuclear pores, nuclear envelope, chromatin, differentiation, cell cycle

 

+33 (0)1572780060    valerie.doye(at)ijm.fr    @DoyeValerie

Nuclear Pore Complexes (NPCs) are elaborate structures embedded in the nuclear envelope and composed of multiple copies of about 30 different proteins termed nucleoporins (Nups). Our previous work contributed to the characterization of a major structural sub-complex of NPCs, the Nup107-160 complex (also called “Y-complex“, Figure 1), and to highlight its role in NPC assembly, its contribution to various stages of mitotic progression, and its involvement in embryonic stem cell differentiation.

Figure 1: Schematic representation of a nuclear pore complex and the components of the Y-complex.

We are currently pursuing the study of the functions, regulation and direct partners of these nucleoporins in these “unconventional” activities. To do so, we use imaging, biochemical and genetic approaches (CRISPR/Cas9 strategies), mainly in pluripotent stem cells (mESCs and hiPSCs) and their 2D and 3D-differentiated derivatives.

 

 

Projet 1: Implication of Y-complex Nups in mouse embryonic stem cell (mESC) differentiation

Our team has previously shown that several Y-complex Nups, namely Nup133, Seh1 and Nup43, are dispensable for cell viability in pluripotent mouse embryonic stem cells (mESCs) but required for their survival during differentiation (Lupu et al., 2008; Gonzalez-Estevez, Verrico et al., 2021). In addition, we demonstrated that in pluripotent mESCs, Nup133 is necessary for proper assembly of the nuclear pore basket (Souquet et al., 2018) whereas mutations affecting the short arm of the Y-complex (Nup85, Seh1 and Nup43) negatively impact NPC density (Gonzalez-Estevez, Verrico et al., 2021).

Figure 2: Nuclear pore complexes in neuronal progenitors derived from mESCs expressing various levels of GFP-Nup133 (represented in blue), and labeled with antibodies directed against Nup153 (red) and TPR (green). Scale bar, 10 µm.

Additionally, an RNA-seq analysis enabled the identification of potential target genes regulated by Nup133 during early steps of neuronal differentiation (unpublished results from our team, collaboration with the Genomics facility from IBENS, Paris, France).  We are currently validating these target genes and characterizing the mechanisms by which the Y-complex contributes to their regulation.

 

 

Project 2: Molecular mechanisms leading to microcephaly and/or hereditary nephrotic syndromes upon nucleoporin defects

In recent years, several teams have identified mutations in genes encoding structural Nups, notably Y-complex Nups, in patients with an early-onset and severe kidney disease (steroid-resistant nephrotic syndrome, SRNS), and/or neurodevelopmental disorders, mainly microcephaly. However, the reason why mutations in these ubiquitously expressed Nups lead to pathologies mainly affecting the kidney and/or the brain remains a mystery.

To mimic the ”disease” condition associated with distinct nucleoporin mutations, notably those causing SRNS and/or microcephaly, we are using human induced pluripotent stem cells (hiPSCs) generated either from peripheral blood cells from patients with NUP mutations (collaboration with C. Antignac and the iPSCs facility from Imagine Institute, Paris) or from healthy individuals in which mutations are introduced by CRISPR/Cas9-mediated gene editing (with help of the enSCORE plateform – labex who am I?).

To investigate organ- and cell type-specific and thus pathologically relevant phenotypes, the iPSCs are differentiated towards 3D brain organoids (Figure 3). (with help from the enSCORE plateform) or kidney organoids (in collaboration with C. Antignac and G. Mollet, Imagine Institute).

In both pluripotent cells and their derived organoids, we then study the impact of Y-Nups mutations on NPC assembly, nuclear transport, cell division and migration, and gene regulation. We also aim to characterize Y-Nups partners that may explain the tissue-specific alterations observed in patients.

By elucidating the role of these nucleoporins, our work should contribute to understanding the mechanisms by which mutations or misregulation of these nucleoporins lead to various pathologies.

 

Figure 3: Cryosection of a cerebral organoids at day 38 of differentiation.

 

Models: pluripotent stem cells (mESCs and hiPSCs), organoids, primary and cancer cell lines

Methods: genome editing, cell differentiation, cell imaging, transcriptomics, biochemistry, yeast-two-hybrid

Group Leader:

Valérie DOYE
Téléphone : +33 (0)157278060
valerie.doye(at)ijm.fr

Members:

Stéphanie BOLHY – Biological engineer

Roger KARESS – Researcher Emeritus

Clarisse ORNIACKI – PhD Student

Natacha RAICH – Researcher

Annalisa VERRICO – Post-doc

Integrity of the short arm of the nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation.  Gonzalez-Estevez A, Verrico A, Orniacki C, Reina-San-Martin B, Doye V. J Cell Sci. 2021 May 15;134(10):jcs258340. doi: 10.1242/jcs.258340.  PMID: 34037234

Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.  Cianciolo Cosentino C, Berto A, Pelletier S, Hari M, Loffing J, Neuhauss SCF, Doye V. Sci Rep. 2019 Mar 18;9(1):4750. doi: 10.1038/s41598-019-41202-4. PMID: 30894603

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells.  Souquet B, Freed E, Berto A, Andric V, Audugé N, Reina-San-Martin B, Lacy E, Doye V. Cell Rep. 2018 May 22;23(8):2443-2454. doi: 10.1016/j.celrep.2018.04.070. PMID: 29791854

Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores.  Berto A, Yu J, Morchoisne-Bolhy S, Bertipaglia C, Vallee R, Dumont J, Ochsenbein F, Guerois R, Doye V. EMBO Rep. 2018 May;19(5):e44742. doi: 10.15252/embr.201744742.  PMID: 29632243

Probing nuclear pore complex architecture with proximity-dependent biotinylation.  Kim DI, Birendra KC, Zhu W, Motamedchaboki K, Doye V, Roux KJ. Proc Natl Acad Sci U S A. 2014 Jun 17;111(24):E2453-61. doi: 10.1073/pnas.1406459111.  PMID: 24927568

Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells.  Hu DJ, Baffet AD, Nayak T, Akhmanova A, Doye V, Vallee RB. Cell. 2013 Sep 12;154(6):1300-13. doi: 10.1016/j.cell.2013.08.024. PMID: 24034252

A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase.  Bolhy S, Bouhlel I, Dultz E, Nayak T, Zuccolo M, Gatti X, Vallee R, Ellenberg J, Doye V. J Cell Biol. 2011 Mar 7;192(5):855-71. doi: 10.1083/jcb.201007118. PMID: 21383080

Nuclear pore composition regulates neural stem/progenitor cell differentiation in the mouse embryo.  Lupu F, Alves A, Anderson K, Doye V, Lacy E. Dev Cell. 2008 Jun;14(6):831-42. doi: 10.1016/j.devcel.2008.03.011. PMID: 18539113

The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions.  Zuccolo M, Alves A, Galy V, Bolhy S, Formstecher E, Racine V, Sibarita JB, Fukagawa T, Shiekhattar R, Yen T, Doye V. EMBO J. 2007 Apr 4;26(7):1853-64. doi: 10.1038/sj.emboj.7601642. PMID: 17363900

The conserved Nup107-160 complex is critical for nuclear pore complex assembly.  Walther TC, Alves A, Pickersgill H, Loïodice I, Hetzer M, Galy V, Hülsmann BB, Köcher T, Wilm M, Allen T, Mattaj IW, Doye V. Cell. 2003 Apr 18;113(2):195-206. doi: 10.1016/s0092-8674(03)00235-6. PMID: 12705868

Since 2015 :

Integrity of the short arm of the nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation.  Gonzalez-Estevez A, Verrico A, Orniacki C, Reina-San-Martin B, Doye V. J Cell Sci. 2021 May 15;134(10):jcs258340. doi: 10.1242/jcs.258340.  PMID: 34037234

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes.  Raich N, Contremoulins V, Karess RE. J Vis Exp. 2020 Aug 27;(162). doi: 10.3791/61061. PMID: 32925873

Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function.  Menant A, Karess RE. Biol Cell. 2020 Oct;112(10):300-315. doi: 10.1111/boc.201900105.  PMID: 32602944

Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.  Cianciolo Cosentino C, Berto A, Pelletier S, Hari M, Loffing J, Neuhauss SCF, Doye V. Sci Rep. 2019 Mar 18;9(1):4750. doi: 10.1038/s41598-019-41202-4. PMID: 30894603

Mad1 influences interphase nucleoplasm organization and chromatin regulation in Drosophila.  Raich N, Mahmoudi S, Emre D, Karess RE. Open Biol. 2018 Oct 17;8(10):180166. doi: 10.1098/rsob.180166. PMID: 30333236

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells.  Souquet B, Freed E, Berto A, Andric V, Audugé N, Reina-San-Martin B, Lacy E, Doye V. Cell Rep. 2018 May 22;23(8):2443-2454. doi: 10.1016/j.celrep.2018.04.070. PMID: 29791854

Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores.  Berto A, Yu J, Morchoisne-Bolhy S, Bertipaglia C, Vallee R, Dumont J, Ochsenbein F, Guerois R, Doye V. EMBO Rep. 2018 May;19(5):e44742. doi: 10.15252/embr.201744742.  PMID: 29632243

Regulation of Cenp-F localization to nuclear pores and kinetochores.  Berto A, Doye V. Cell Cycle. 2018;17(17):2122-2133. doi: 10.1080/15384101.2018.1520569. PMID: 30198378

Channel Nucleoporins Recruit PLK-1 to Nuclear Pore Complexes to Direct Nuclear Envelope Breakdown in C. elegans.  Martino L, Morchoisne-Bolhy S, Cheerambathur DK, Van Hove L, Dumont J, Joly N, Desai A, Doye V, Pintard L. Dev Cell. 2017 Oct 23;43(2):157-171.e7. doi: 10.1016/j.devcel.2017.09.019. PMID: 29065307

Formation of Nup98-containing nuclear bodies in HeLa sublines is linked to genomic rearrangements affecting chromosome 11.  Romana S, Radford-Weiss I, Lapierre JM, Doye V, Geoffroy MC. Chromosoma. 2016 Sep;125(4):789-805. doi: 10.1007/s00412-015-0567-0.  PMID: 26685999

Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila.  Caous R, Pascal A, Romé P, Richard-Parpaillon L, Karess R, Giet R. Nat Commun. 2015 Nov 16;6:8879. doi: 10.1038/ncomms9879. PMID: 26568519

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells. Souquet B, Freed E, Berto A, Andric V, Audugé N, Reina-San-Martin B, Lacy E, Doye V. Cell Rep. 2018 May 22;23(8):2443-2454. doi: 10.1016/j.celrep.2018.04.070. PMID: 29791854

Intranuclear dynamics of the Nup107-160 complex.  Morchoisne-Bolhy S, Geoffroy MC, Bouhlel IB, Alves A, Audugé N, Baudin X, Van Bortle K, Powers MA, Doye V. Mol Biol Cell. 2015 Jun 15;26(12):2343-56. doi: 10.1091/mbc.E15-02-0060.  PMID: 25904327

RZZ and Mad1 dynamics in Drosophila mitosis.  Défachelles L, Raich N, Terracol R, Baudin X, Williams B, Goldberg M, Karess RE. Chromosome Res. 2015 Jun;23(2):333-42. doi: 10.1007/s10577-015-9472-x.  PMID: 25772408

A maternal effect rough deal mutation suggests that multiple pathways regulate Drosophila RZZ kinetochore recruitment. Défachelles L, Hainline SG, Menant A, Lee LA, Karess RE. J Cell Sci. 2015 Mar 15;128(6):1204-16. doi: 10.1242/jcs.165712.  PMID: 25616898

Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding.  Floch AG, Tareste D, Fuchs PF, Chadrin A, Naciri I, Léger T, Schlenstedt G, Palancade B, Doye V. J Cell Sci. 2015 Jan 15;128(2):305-16. doi: 10.1242/jcs.158915.  PMID: 25413348

Clarisse Orniacki – (01/10/2018 – ongoing) –  Université Paris Cité

Nuclear pores in development and disease: role of Nup133 in gene regulation upon neuronal differentiation

Alba Gonzalez-Estevez    01/10/2016 19/12/2019 – Université Paris Diderot

On the edge between cell division and cell differentiation: role of Seh1 in mouse embryonic stem cells

Alessandro Berto (01/10/2013 – 11/12/2017)- Université Paris-Saclay

Implication of the Nup133 subunit of nuclear pores in cell division and differentiation: partners and mechanisms

Aurélie Floch (01/10/2010- 26/09/2014) – Université Paris-Sud

[Targeting mechanisms of Pom33, a transmembrane protein associated with nuclear pores in the yeast Saccharomyces cerevisiae]

Analyses fonctionnelles d’une nouvelle protéine de l’enveloppe nucléaire chez la levure Saccharomyces cerevisiae

Anne CHADRIN Université (01/10/2007 – 02/07/2010) – Université Paris XI

[Functional analyses of a new nuclear envelope protein in the yeast Saccharomyces cerevisiae]

Analyses fonctionnelles d’une nouvelle protéine de l’enveloppe nucléaire chez la levure Saccharomyces cerevisiae

Michela ZUCCOLO (01/09/2005- 19/07/2008) – Université Paris XI

[Involvement of the Nup107-160 nuclear pore complex in mitosis entry, progression and exit]

Implication du complexe Nup107-160 des pores nucléaires dans l’entrée, la progression et la sortie de mitose.

Annabelle Alves (01/09/2001- 01/07/2005) – Université Paris XI- Orsay

[Functional analysis of the NUP107-160 nuclear pore complex during the vertebrate cell cycle]

Analyse fonctionnelle du complexe NUP107-160 des pores nucléaires au cours du cycle cellulaire des vertébrés

Siau Wei BAï (01/09/2000 – 10/12/2004) – Université Paris XI- Orsay

[Functional characterization of the Nup107 complex: a nuclear pore protein subcomplex in the yeast Schizoszaccharomyces pombe]

Caractérisation fonctionnele du complexe Nup107: un sous-complexe protéique du pore nucléaire chez la levure Schizoszaccharomyces pombe

Naïma BELGAREH (01/09/ 1995- 27/09/1999) – Université Paris VI

[Study nuclear pores assembly and dynamics]

Etude de l’assemblage et de la dynamique des pores nucléaires

Ongoing collaborations :

Martin BECK,  Max Planck Insitute of Biophysics, Frankfurt, Germany

Corinne ANTIGNAC et Géraldine MOLLET, Institut Image, Paris, France;

Brian BURKE, IMB, A*Star, Singapore;

Andreas SCHEDL, Institut de Biologie Valrose, Nice, France;

Fanny COULPIER and Laurent JOURDREN, Genomics facility, IBENS, Paris, France

 

Former collaborations:

Johannes LOFFING and Stephan C. F. NEUHAUSS, University of Zurich, Switzerland;

Bernardo REINA−SAN−MARTIN, IGBMC, Illkirch, France;

Elizabeth LACY, Memorial Sloan Kettering Cancer Center, New York, USA;

Raphael GUEROIS, Institut de Biologie et de Technologies de Saclay (iBiTec-S), Gif-sur-Yvette, France;

Richard VALLEE – Department of Pathology and Cell Biology, Columbia University, New York, USA;

Julien DUMONT, Institut Jacques-Monod IJM, Paris, France;

Lionel PINTARD, Institut Jacques-Monod IJM, Paris, France

The team has been recognized as “équipe FRM 2020 ” by the Fondation pour la Recherche Médicale and was supported in 2019 by a grant form IDEX Université de Paris. The team has been supported by the Labex “Who am I?” and ANR, and  currently hosts in its lab-space the members of the enSCORE facility, founded by the Labex “Who am I?”and dedicated to the development of neuronal organoids.