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the identification of potential target genes regulated by Nup133 during early steps of neuronal differentiation (unpublished results from our team, collaboration with the Genomics facility from IBENS, Paris, France).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:

Publications

Review

Clarisse Orniacki – (01/10/2018 – 12/12/2022) –  Université Paris Cité

Y-complex nucleoporins in embryonic stem cell differentiation; impact on nuclear pore integrity and gene regulation

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.