Le noyau cellulaire est le siège de réactions chimiques dont le but est l’expression de gènes, la duplication du génome et du maintien et l’intégrité de l’information génétique. Ces réactions sont régulées au cours du cycle cellulaire ou en réponse à un stress. Parmi elles, la transcription permet qu’une séquence d’ADN soit reproduite sous forme d’ARN messager. La transcription est un exemple frappant de processus fondamental pour la cellule impliquant parfois un nombre très faible de molécules. En effet, il n’y a souvent dans un même génome que quelques copies d’un même gène. Le but de cette thèse est d’imager les processus nucléaires dans des cellules humaines à l’échelle de la molécule unique et d’en extraire les grandeurs caractéristiques. Depuis les années 90, des inventeurs de génie ont développé des méthodes simples à partir de microscopes inversés ordinaires pour observer des molécules individuelles jusque dans le noyau des cellules. Nous avons utilisé ces méthodes pour suivre des facteurs de transcription qui régulent la transcription d’un gène. Nos mesures montrent que, bien que hiératique, l’exploration du noyau par les facteurs de transcriptions est régulée par leurs propriétés chimiques. L’agencement des composants du noyau guide les facteurs de transcription dans la recherche d’un gène. Comme exemple de cet agencement, nous nous sommes ensuite intéressés à l’organisation de l’ADN dans le noyau pour montrer qu’elle présentait les caractéristiques d’une structure auto-organisée, une structure fractale. Cette structure change en réponse aux aléas de la vie de la cellule. Dans une dernière étude, nous avons suivi un locus dans le noyau d’une levure. La structure du noyau, qui est révélée par notre méthode, contraint la diffusion du locus à un régime de reptation. Tous ces résultats montrent combien la structure du noyau et les réactions chimiques qui y ont lieu sont interdépendantes. Cette thèse a également permis le développement de méthodes de quantification précises des réactions cellulaires à l’échelle de la molécule unique. / The cell nucleus is a chemical reactor. Nuclear components interact with each other to express genes, duplicate the chromosomes for cell division, and protect DNA from alteration. These reactions are regulated along the cell cycle and in response to stress. One of the fundamental nuclear processes, transcription, enables the production of a messenger RNA from a template DNA sequence. While mandatory for the cell, transcription nevertheless may involve a very small number of molecules. Indeed, a single gene would have only few copies in the genome. During my PhD, I studied nuclear processes in human cells nuclei at the single molecule level with novel imaging techniques. I developed new statistical tools to quantify nuclear components movement that revealed a dynamic nuclear architecture. Since the 90s, simple methods have been developed for the observation of single molecules in the cell. These experiments can be conducted in an ordinary inverted microscope. We used these methods to monitor nuclear molecules called transcription factors (TF) that regulate transcription. From TF dynamics, we concluded that nuclear exploration by transcription factors is regulated by their chemical interactions with partners. The organization of the components of the nucleus guide transcription factors in their search of a gene. As an example of this organization, we then studied chromatin, the de-condensed form of nuclear DNA, proving that it displays the characteristics of a self-organized fractal structure. This structure changes in response to cellular fate and stress. In yeast, we showed that the interminglement of chromatin constrained DNA locus movement in a reptation regime. All these results show the interdependence of the structure of the nucleus and of its chemical reactions. With combination of realistic modeling and high resolution microscopy, we have enlightened the specificity of the nucleus as a chemical reactor. This thesis has also enabled the development of accurate methods for the statistical analysis of single molecule data.

Neisseria meningitidis, ou méningocoque, est une bactérie responsable de méningites et de septicémies, dont la forme la plus grave, purpura fulminans, est souvent fatale. Cette bactérie, qui réside naturellement dans le rhinopharynx de l’Homme, est pathogène lorsqu’elle atteint la circulation sanguine et entre en contact avec les cellules endothéliales. L’établissement d’une interaction étroite entre le méningocoque et les cellules endothéliales est essentiel à la résistance des bactéries au flux sanguin et à la colonisation vasculaire. Cette interaction peut conduire à une désorganisation massive des endothéliums périphériques et cérébraux permettant la dissémination de la bactérie. Ces processus dépendent de la primo-interaction des pili de type IV du méningocoque avec le récepteur endothélial CD147 et de l’activation du récepteur β2-adrénergique (β2AR). L’activation de voies de signalisation en aval du β2AR dans les cellules hôtes permet l’adhérence efficace et intime des bactéries à la surface des cellules endothéliales. Toutefois, comment les récepteurs CD147 et β2AR coopèrent pour promouvoir une interaction initiale efficace et rapide n’était pas connu. Au cours de ma thèse, j’ai donc analysé les éventuelles interactions et liens fonctionnels existants entre les récepteurs CD147 et β2AR et suivi, à l’aide de nouvelles approches d’imagerie à haute résolution, leur organisation moléculaire aux sites de contact bactéries/cellule. Mes travaux ont permis de révéler l’existence d’une interaction fonctionnelle entre les récepteurs CD147 et β2AR, et d’identifier un nouveau partenaire cytosolique interagissant directement avec ces récepteurs, l’α-actinine4 (Actn4). L’expression de l’Actn4 est requise pour l’assemblage organisé de ces récepteurs en complexes multimoléculaires aux sites de contact bactéries/cellule endothéliale. Cette organisation est déterminante pour générer une force suffisante à l’interaction initiale du méningocoque aux cellules endothéliales, et promouvoir l’activation rapide des voies de signalisation nécessaires à la consolidation de cette interaction. L’infection des cellules endothéliales par le méningocoque s’accompagne de la désorganisation des jonctions intercellulaires et l’ouverture d’une voie paracellulaire favorisant la dissémination tissulaire des bactéries. Ces évènements dépendent de l’activation de la petite GTPase Cdc42 et, en aval, de la relocalisation du complexe de polarité Par3/Par6/aPKC au site d’adhérence bactérien. Ce complexe moléculaire très conservé est impliqué dans la mise en place de la polarité baso-apicale des cellules endothéliales. La perte de la polarité cellulaire constituant un élément déterminant de la perte de l’intégrité vasculaire, dans une seconde partie de ma thèse, j’ai donc entrepris une analyse des événements de signalisation précoces conduisant au remodelage de l’organisation apico-basale des cellules endothéliales par N. meningitidis. Mes travaux montrent que rapidement après adhésion aux cellules endothéliales, le méningocoque induit la ré-orientation de l’axe de polarité noyau-centrosome des cellules en direction des bactéries et mettant en jeu un mécanisme original indépendant de l’activation de Cdc42 par le β2AR. Le recrutement des ERM (Ezrine et Moesin) et la polymérisation d’actine corticale au site d’infection semblent constituer des facteurs clé de cette étape précoce de modification de la polarité endothéliale induite par le méningocoque. Ainsi, ces études ont permis une avancée majeure dans notre compréhension du mécanisme d’adhésion du méningocoque aux cellules endothéliales et des événements moléculaires précoces conduisant à l’altération de l’intégrité vasculaire, deux étapes clés au cœur de la pathogénèse des infections invasives à méningocoque. / Neisseria meningitidis or meningococcus is a commensal bacterium of the human nasopharynx responsible for septicemia and meningitis. Establishment of a close interaction between meningococcus and endothelial cells is an important step in meningococcal pathogenesis as it promotes bacterial resistance to blood flow and vascular colonization, leading to major endothelial dysfunctions and bacterial dissemination into perivascular tissues. This process depends on the interaction of meningococcal type IV pili with the endothelial receptor CD147 and the activation of the β2 adrenergic receptor (β2AR). Activation of a cellular response downstream of β2AR activation is important to allow the efficient adhesion of meningococci at the endothelial cell surface. However, how CD147 and the β2AR cooperate to promote a rapid and efficient initial adhesion remained to be explored. During my thesis, I have analyzed the interaction and the functional link between these two receptors and, using super resolution microscopy, I have investigated their molecular organization at sites of bacterial adhesion. My work revealed a functional interaction in cis between CD147 and the β2AR and binding of these receptors complexes to the molecular scaffold protein α-actinin 4 (Actn4). Actn4 expression is required for the organized assembly of these receptors in highly-ordered complexes at bacterial adhesion sites. This specific organization is decisive to provide a sufficient binding strength of meningococcal type IV pili with endothelial receptors and to promote a rapid activation of downstream signaling events in a short time frame. Endothelial cell infection by N. meningitidis is associated with the disruption of intercellular junctions and the opening of a paracellular route favoring bacterial dissemination into tissues. These events are dependent on Cdc42 activation and on the relocalization of the Par3/Par6/aPKC polarity complex at bacterial adhesion sites. This molecular complex is conserved and involved in baso-apical polarity establishment in endothelial cells. Since endothelial polarity is essential in maintaining junction integrity, in a second part of my thesis work I have analyzed the early signaling events triggered by meningococcal infection with a particular emphasis on endothelial cell polarity modifications. I observed that bacterial adhesion rapidly induced a re-orientation of the nucleus-centrosome axis toward bacterial adhesion sites. Unexpectedly, this re-orientation was independent of Cdc42 activation downstream of the β2AR. In place, the ERM proteins (Ezrin and Moesin), along with cortical actin polymerization seem to be key factors in this process. This work contributes to the understanding of the meningococcal adhesion mechanism to endothelial cells and the early molecular events leading to the loss of vascular integrity. These two key steps are very important in the meningococcal pathogenesis.

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The nuclear pore complex (NPC) is a pivotal subcellular structure governing nucleocytoplasmic transport through a selectively permeable barrier. Comprising approximately 30 distinct proteins, it includes FG-Nups with phenylalanine-glycine (FG) motifs and non-FG Nups forming the pore's scaffold. The selectively permeable barrier formed by FG-Nups enables the passive diffusion of small molecules and facilitates the transport of larger ones recognized by nuclear transport receptors (NTRs). Their roles are critical in regulating mRNA and pre-ribosome nuclear export and the nuclear import of transcription factors, underscoring their significance in cellular processes. However, studying NPCs remains challenging due to their structural complexity, heterogeneity, dynamic interactions, and inaccessibility within live cells. In this dissertation, three core questions were investigated to elucidate the structure and function of the NPC. First, the nuclear export dynamics of pre-ribosomal subunits revealed significantly higher transport efficiency compared to other large cargos. Through inhibition of nuclear transport receptor (NTR), CRM1, by small-molecule inhibitor, leptomycin B, we found a dose-dependent inhibition of CRM1s played a crucial role in pre-ribosome export efficiency. We confirmed these results through a series of controlled environments with both import and export NTRs. Our results suggest that cooperative NTR mechanisms may enhance the nucleocytoplasmic transport of not only pre-ribosomal subunits but other protein complexes as well. Second, we investigated the dynamic properties of the NPC’s selectivity barrier by altering the concentration of O-linked β-N-acetylglucosamine (O-GlcNAc) sites on nuclear pore proteins. Using small-molecule inhibitors of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) to decrease or increase NPC O-GlcNAcylation, respectively, we found a significant change in the overall 3D spatial density of NPC O-GlcNAc sites. Then, by applying the same OGT- and OGA-inhibited conditions, we found that NPC O-GlcNAcylation significantly impacted the nuclear export of mRNA, suggesting that NPC O-GlcNAcylation regulates mRNA’s passage through the NPC’s selective permeability barrier. Third, we examined the nuclear transport mechanism for intrinsically disordered proteins (IDPs). Our findings revealed that IDPs, unlike large folded proteins, can passively diffuse through NPCs independent of size, and their diffusion behaviors are differentiated by the content ratio of charged (Ch) and hydrophobic (Hy) amino acids. Thus, we proposed a Ch/Hy-ratio mechanism for IDP nucleocytoplasmic transport. In summary, comprehending the dynamic behavior of the NPC selectivity barrier and its involvement in mediating large transiting complexes and IDPs has provided valuable insights into the fundamental nucleocytoplasmic transport mechanism, emphasizing the NPC's crucial role in cellular health and function. / Biology

The primary cilium is an immotile, microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Critically, the transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, this work employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. Using SPEED microscopy and single molecule tracking, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins. While the 3D transport pathways in this work are always replicated multiple times with a high degree of consistency, several experimental parameters directly affect the 3D transport routes’ error, such as single molecule localization precision and the number of single molecule localizations. In fact, if these experimental parameters do not meet a minimum threshold, the resultant 3D transport pathways may not have significant resolution to determine any biological details. To estimate the 3D transport routes’ error, this work will explain in detail the component of SPEED microscopy that estimates 3D sub-diffraction-limited structural or dynamic information in rotationally symmetric bio-structures, such as the primary cilium. This component is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability distributions based on prior known structural knowledge. This analysis is ideal in cases where the ultrastructure of a cellular structure is known but the sub-structural localization of a particular protein is not. This work will demonstrate how the 2D-to-3D component of SPEED microscopy can be successfully applied to achieve 3D structural and functional sub-diffraction-limited information for 25-300 nm subcellular organelles that meet the rotational symmetry requirement, such as the primary cilium and microtubules. Furthermore, this work will provide comprehensive analyses of this method by using computational simulations which investigate the role of various experimental parameters on the 3D transport pathway error. Lastly, this work will demonstrate that this method can distinguish different types of 3D transport pathway distributions in addition to their locations. / Biology

The nucleus of eukaryotic cells is a vitally important organelle that sequesters the genetic information of the cell, and protects it with the help of two highly evolved structures, the nuclear envelope (NE) and nuclear pore complexes (NPCs). Together, these two structures mediate the bidirectional trafficking of molecules between the nucleus and cytoplasm by forming a barrier. NE transmembrane proteins (NETs) embedded in either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM) play crucial roles in both nuclear structure and functions, including: genome architecture, epigenetics, transcription, splicing, DNA replication, nuclear structure, organization and positioning. Furthermore, numerous human diseases are associated with mutations and mislocalization of NETs on the NE. There are still many fundamental questions that are unresolved with NETs, but we focused on two major questions: First, the localization and transport rate of NETs, and second, the transport route taken by NETs to reach the INM. Since NETs are involved with many of the mechanisms used to maintain cellular homeostasis, it is important to quantitatively determine the spatial locations of NETs along the NE to fully understand their role in these vital processes. However, there are limited available approaches for this task, and moreover, these methods provide no information about the translocation rates of NETs between the two membranes. Furthermore, while the trafficking of soluble proteins between the cytoplasm and the nucleus has been well studied over the years, the path taken by NETs into the nucleus remains in dispute. At least four distinct models have been proposed to suggest how transmembrane proteins destined for the INM cross the NE through NPC-dependent or NPC-independent mechanisms, based on specific features found on the soluble domains of INM proteins. In order to resolve these two major questions, it is necessary to employ techniques with the capabilities to observe these dynamics at the nanoscale. Current experimental techniques are unable to break the temporal and spatial resolution barriers required to study these phenomena. Therefore, we developed and modified single-molecule techniques to answer these questions. First, to study the distribution of NETs on the NE, we developed a new single-molecule microscopy method called single-point single-molecule fluorescence recovery after photobleaching (smFRAP), which is able to provide spatial resolution <10 nm and, furthermore, provide previously unattainable information about NET translocation rates from the ONM to INM. Secondly, to examine the transport route used by NETs destined for the INM, we used a single-molecule microscopy technique previously developed in our lab called single-point edge-excitation sub-diffraction (SPEED) microscopy, which provides spatio-temporal resolution of <10 nm precision and 0.4 ms detection time. The major findings from my doctoral research work can be classified into two categories: (i) Technical developments to study NETs in vivo, and (ii) biological findings from employing these microscopy techniques. In regards to technical contributions, we created and validated of a new single-molecule microscopy method, smFRAP, to accurately determine the localization and distribution ratios of NETs on both the ONM and INM in live cells. Second, we adapted SPEED microscopy to study transmembrane protein translocation in vivo. My work has also contributed four main biological findings to the field: first, we determined the in vivo translocation rates for lamin-B receptor (LBR), a major INM protein found in the nucleus of cells. Second, we verified the existence of peripheral channels in the scaffolding of NPCs and, for the first time, directly observed the transit of INM proteins through these channels in live cells. Third, our research has elucidated the roles that both the nuclear localization signal (NLS) and intrinsically disordered (ID) domains play in INM protein transport. Finally, my work has elucidated which transport routes are used by NETs destined to localize in the INM. / Biology

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Le cerveau est composé de neurones et de cellules gliales qui jouent un rôle de soutien et de protection du réseau cellulaire. L’espace extra-cellulaire (ECS) correspond à l’espace qui existe entre ces cellules. Les modifications de sa structure peuvent dépendre de plusieurs paramètres comme l’âge, l’apprentissage ou les maladies neuro-dégénératives. Le volume de l’ECS correspond à environ 20$%$ du volume total du cerveau et les neurotransmetteurs et autres molécules circulent dans cet espace pour assurer une communication neuronale optimale. Cependant, les dimensions et la viscosité locale de cet espace restent encore mal-connues. L’ECS est composé entre autres de protéoglycans, de glycoaminoglycans (acide hyaluronique…) et de fluide cérébrospinal. Nous avons proposé dans cette thèse une stratégie pour mesurer les dimensions et les propriétés rhéologiques de l’espace extra-cellulaire de tranches de cerveaux de rats maintenue en vie à l’aide du suivi de nanotubes de carbone individuels luminescents. Pour ces applications, nous avons étudier la biocompatibilité et le rapport signal sur bruit de nos échantillons de nanotubes afin de les détecter en profondeur dans les tranches de cerveaux et de pouvoir mesurer leurs propriétés de diffusion. / The brain is mainly composed of neurons which ensure neuronal communication and glialcells which play a role in supporting and protecting the neural network. The extracellular space corresponds to the space that exists between all these cells and represents around 20 %of the whole brain volume. In this space, neurotransmitters and other molecules circulate into ensure optimal neuronal functioning and communication. Its complex organization whichis important to ensure proper functioning of the brain changes during aging, learning or neurodegenerative diseases. However, its local dimensions and viscosity are still poorly known.To understand these key parameters, in this thesis, we developed a strategy based on the tracking of single luminescent carbon nanotubes. We applied this strategy to measure the structural and viscous properties of the extracellular space of living rodent brains slices at the nanoscale. The organization of the manuscript is as follows. After an introduction of the photoluminescence properties of carbon nanotubes, we present the study that allowed us to select the optimal nanotube encapsulation protocol to achieve our biological applications. We also present a quantitative study describing the temperature increase of the sample when laser irradiations at different wavelengths are used to detect single nanotubes in a brain slice.Thanks to a fine analysis of the singular diffusion properties of carbon nanotubes in complex environments, we then present the strategy set up to reconstruct super-resolved maps (i.e. with resolution below the diffraction limit) of the brain extracellular space morphology.We also show that two local properties of this space can be extracted : a structural complexity parameter (tortuosity) and the fluid’s in situ viscosity seen by the nanotubes. This led us to propose a methodology allowing to model the viscosity in situ that would be seen, not by the nanotubes,but by any molecule of arbitrary sizes to simulate those intrinsically present or administered in the brain for pharmacological treatments. Finally, we present a strategy to make luminescent ultra-short carbon nanotubes that are not intrinsically luminescent and whose use could be a complementary approach to measure the local viscosity of the extracellular space of the brain.

The mammalian hippo signalling pathway controls cell proliferation and apoptosis via transcriptional co-activators YAP and TAZ, and as such is a key regulator of organ and tissue growth. Multiple cellular components converge in this pathway, including the actin cytoskeleton, which is required for YAP/TAZ activity. The precise mechanism by which the mechanical actomyosin network regulates Hippo signalling, however, is unknown. Optical methods provide a non-invasive way to image and study the biomechanics of cells. In the past two decades, super-resolution fluorescence microscopy techniques that break the diffraction limit of light have come to the fore, enabling visualisation of intracellular detail at the nanoscale level. Optical trapping, on the other hand, allows precise control of micron-sized objects such as cells. Here, super resolution structured illumination microscopy (SIM) and elastic resonator interference stress microscopy (ERISM) were used to investigate a potential role for the FERM-domain protein FRMD6, or Willin, in the mechanical control of the Hippo pathway in a neuronal cell model. A double optical trap was also integrated with the Nikon-SIM with the aim of cell stretching. Willin expression was shown to modify the morphology, neuronal differentiation, actin cytoskeleton and forces of SH-SY5Y cells. Optical trapping from above the SIM objective, however, was demonstrated to be ineffective for manipulation of adherent cells. The results presented here indicate a function for Willin in the assembly of actin stress fibres that may be the result of an interaction with the Hippo pathway regulator AMOT. Further investigation, for example by direct cell stretching, is required to elucidate the exact role of Willin in the mechanical control of YAP/TAZ.