Molecular determinants and membrane mechanics involved in Nuclear Pore Complex neogenesis – NeoPore

- Cellular biology: cell culture, transient or stable transfection with fluorescent proteins and / or siRNAs, immunofluorescence.
- Fluorescence microscopy: confocal microscopy, STED, TIRF, dSTORM
- AFM
- Analysis softwares: Huygens (deconvolution), ImageJ, MatLab, Gwyddion (AFM analysis)

1) Establishment of AFM-Fluo microscopy on isolated NEs from cultured cells: Nuclei are isolated from cultured cells, loaded on glass slides, opened, fixed and labeled with specific NPC antibodies. Nuclei are then imaged by AFM with lateral and axial resolutions in the nanometer range.

2) Identification of NPC intermediates and analysis of their topography: thanks to our correlative TIRF/AFM setup, NPCs intermediates are localized by fluorescence and imaged by AFM. Different structures, likely corresponding to different stages of NPC assembly, have been observed by AFM.

3) Quantification of NPC intermediates by confocal imaging, deconvolution, automatic detection of NPCs. Differential labeling allows quantification of NPC intermediates in cells where pore formation is inhibited by different siRNAs. This makes it possible, as a first approximation, to distinguish the factors involved in the early or late phases of NPC assembly.

4) STED microscopy on cells labeled with specific NPC antibodies. STED allows to measure structural differences between mature pores and formation intermediates.

5) Role of SUN1 in NPC assembly, related to its role in maintaining the NE structure. SUN1 is essential for interphase NPC assembly by an unknown mechanism. We explore how its function in maintaining the NE integrity relates to its role in pore formation. For this, we use a series of mutants of SUN1 affecting the NE properties . We study the effect of these mutants in parallel on pore assembly and on the NE structure.

- Implementation of dSTORM on NPCs to image nuclei by correlative AFM / dSTORM. The setup allowing this correlative imaging is already operational. But specific imaging conditions will be optimized for NPCs.
- Fusion of NPC components with HALO or SNAP tags, allowing fluorescent labeling for dSTORM. Indeed, most NPC subunits cannot be labeled by immunofluorescence in conditions compatible with single molecule microscopy. We will establish stable cell lines expressing combinations of nucleoporins fused with HALO or SNAP tags. Cells will then be conjugated with SNAP- or HALO-ligands coupled to Janelia Fluor probes, compatible with single molecule microscopy.
- Analyze by STED the structure of NPC intermediates in cells upon treatment by siRNAs. This study will allow us to refine the most promising siRNAs for AFM / dSTORM. Indeed, STED allows a much faster and more systematic screening than AFM and/or dSTORM. Moreover, large amounts of cells are required for nuclei isolation for AFM.
- AFM analysis of NPC intermediates structure upon treatment of cells with siRNAs selected during the STED study.
- AFM analysis of NPC intermediate structure, correlated with the localization within these structures of some components of NPCs by dSTORM.

Participation in scientific meetings:
1. Molecular basis for membrane remodeling and organization (Jacques Monod conference, Roscoff, 2017) Poster
2. 7th GRISBI meeting «Biophysics, today and beyond« (March 2018, Montpellier) Oral presentation

A characteristic feature of eukaryotic cells is the physical isolation of the genetic material by the nuclear envelope (NE), a double lipid bilayer composed of an inner (INM) and outer nuclear membrane (ONM) separated by a lumen called the perinuclear space (PNS). Across this barrier are the nuclear pore complexes (NPCs), the only gateways between the nucleus and cytoplasm. NPCs are giant protein complexes of over 1MDa. They are made of multiple copies of 30 components called nucleoporins (nups). During interphase, proliferating cells have to duplicate their genetic material, organelles, membranes and enzymatic tools, to split them between daughter cells. While DNA replication is widely studied, duplication of other materials is largely unexplored. In most cases, this is just a matter of protein and lipid synthesis, yet NPC replication is a complex process involving macromolecular assembly combined with membrane remodeling. Moreover, the nuclear permeability barrier must remain intact during the whole process, underlining that protein assembly and membrane fusion must be tightly coordinated.
Interphase pore assembly occurs by de novo insertion of new pore units in the NE, which requires the local fusion of the ONM and INM. The fusion process must involve the close apposition of the two membranes, generating locally a complex combination of positive and negative curvature in the bilayers. Then a hemifusion step most likely occurs, followed by complete fusion and opening of a channel between the nucleus and cytoplasm.
Generally speaking, membrane deformation can be produced by four mechanisms: (i) mechanical forces pulling on membranes; (ii) membrane moulding by molecular scaffolds with a curved cationic surface; (iii) hydrophobic insertions within a leaflet of the lipid bilayer, acting as a wedge and (iv) molecular crowding at the membrane surface: high protein density generates lateral pressure due to intermolecular collisions, which can be alleviated by an increase in membrane area. According to the latter model, intensive recruitment of nups or NPC co-factors to a given site may be sufficient to locally induce membrane curvature. Importantly, most bending mechanisms trigger convex deformations, i.e. an increased surface of the membrane leaflet in contact with the bending agents. A few candidate proteins suspected to play a role in INM and ONM bending and fusion induce convex deformations on synthetic liposomes. Yet the deformation required for pore formation is concave; this discrepancy is not understood.
The difficulty to understand the mechanics and molecular regulators involved in INM/ONM deformation and fusion lies in the lack of molecular markers for early fusion intermediates. So far, these intermediates have only been assessed by Electron Microscopy, which does not provide information on dynamics.
Atomic force microscopy (AFM) can render surface topography of biological samples in physiological conditions with a resolution in the range of nm. We propose to develop AFM as a tool to render the topography of the NE and detect the hollows formed when the INM and ONM get close to each other. This will fill a technological gap to study the early steps of NPC neogenesis. Combining AFM and super-resolution microscopy, we will correlate the recruitment of NPC components and co-factors with nuclear membranes bending steps. This will tell us the sequence of partners recruitment at nascent pore sites. Candidates identified through this analysis will be applied to purified NEs and compared to other bending machineries to understand the peculiarity of NE plasticity. The role of intra-PNS bridges will be explored. Lastly, all the information gathered will be integrated to find the minimal combination of factors required to induce the fusion of two parallel bilayers and the recruitment and assembly of NPC components to heterologous fusion sites.