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No description available.

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

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La rétinogésèse des vertébrés est la culmination de processus biologiques complexes parfaitement exécutés. Cette délicate orchestration est principalement contrôlée par les facteurs de transcription qui permettent aux progéniteurs rétiniens de proliférer, de s’auto-renouveler et de se différencier de façon appropriée. Les facteurs de transcription à homéodomaine sont les protéines qui sont responsables de la démarcation du site du primordium optique et participeront même à la différenciation tardive des différents types cellulaires de la rétine. Le contrôle génétique concernant l‘activation de l’expression de facteurs de transcription est peu connu. Nous avons étudié les séquences génomique avoisinant le gène Six6 afin d’identifier et mieux comprendre son promoteur. Des expériences d’immunoprécipitation de chromatine et des essais luciférases ont confirmé la liaison et la transactivation synergique du promoteur potentiel de Six6 par Lhx2 et Pax6 in vitro. Cette présente étude confirme et précise également le rôle de Lhx2 au niveau du développement précoce de l’oeil. La compréhension détaillée des réseaux génétiques régulant les progéniteurs rétiniens à former une rétine fonctionnelle est essentielle. En effet, lorsque ces connaissances seront acquises, nous serons en mesure d’appliquer les thérapies cellulaires pour rétablir les fonctions rétiniennes lors de pathologies dégénératives. / Vertebrate eye developement is the result of multiple perfectly executed biological process. This tight orchestration is principaly controled by transcription factors. Homeobox-containing transcription factors are expressed in the presumptive eye field and are required to initiate eye development and for final retinal cell differenciation. The genetic control of these transcription factors are poorly understood. We analysed Six6’s nearby genomic sequence to caracterise potential promoter regions. Chromatin immunoprecipitations and luciferase assays confirmed the binding and the in vitro synergic trans-activation of Six6 potential promoter by Lhx2 and Pax6. This study also demonstrate the contribution of Lhx2 for the establishment of presumptive retina field at the neural plate stage. The detailed knowledge of genetic networks regulating the formation of a fonctional retina by retinal progenitor is crucial. Indeed, when these mecanisms will be eluciated, we will be able to establish regenerative retinal cell therapy.

Les vésicules synaptiques (VSs) sont essentielles pour la neurotransmission. Les recherches actuelles se focalisent sur la caractérisation de leur contenu en neurotransmetteurs, leur cinétique de libération, leur distribution et leur mobilité. Les VS ne sont pas présentes exclusievement en paquet dans les boutons présynaptiques mais sont echangées de façon dynamique avec le reste de l’axone dans un super-contingent (super-pool). Notre laboratoire a précédement montré que le transporteur vésiculaire de glutamate de type 1 (VGLUT1) jouerait un rôle dans la régulation du super-pool. Mon projet de thèse se focalise sur la mobilité des VS dans les axones. En premier lieu, j’ai généré une souris gain de fonction VGLUT1mEos2 afin d'étudier la mobilité des VSs et de mieux caractériser le super-pool. Ensuite j’ai engagé une étude des relation entre la structure de VGLUT1 et ses fonctions afin d’identifier les signatures moléculaires responsable de la régulation de la taille du super-pool. J’ai identifié le second motif poly-proline à l’extremité C-terminale de VGLUT1 comme étant nécessaire et suffisante pour induire une diminution de la taille du super-pool des VSs. Pour conclure mes travaux de thèse ont contribué à la compréhension du rôle de VGLUT1 dans la régulation de la mobilité des VSs et à fournir les outils nécessaires pour de futures investigations concernant la physiologie du super-pool. / Synaptic vesicles (SVs) are essential for neurotransmission, and more efforts are needed for better understanding their neurotransmitter content, release kinetics, distribution and mobility. SVs are not only clustered in presynaptic boutons, but also dynamically shared among multiple en passant presynaptic boutons, a phenomenon named SV super‐pool. Previous work from our laboratory suggested that the Vesicular GLUtamate Transporter 1 (VGLUT1) may play a role in regulating SV super-­pool size beyond loading glutamate into SV. My Ph.D project is focused on SVs mobility in axons. Firstly, I generated a VGLUT1mEos2 knock-in (KI) mouse line, which provides extended possibilities to study the SV trafficking and characterize SV super‐pool. Secondly, I engaged in a thorough VGLUT1 structure‐function analysis. I identified that VGLUT1 tends to cluster SVs in the presynaptic boutons and reduce SVs exchange with the super‐pool via the second poly‐proline motif of its C­‐terminus. Overall, my Ph.D work contributes to the knowledge of the role of VGLUT1 in regulating SVs mobility and provides new tools for the further investigations on SV super-­pool physiology.

A investigação dos mecanismos de interação dos vírus com as células do hospedeiro trazem informações relevantes para a identificação de alvos no desenvolvimento de drogas para impedir a penetração e/ou desenvolvimento dos vírus. Peptídeos desenhados a partir de proteínas virais foram desenvolvidos e testados quanto as suas capacidades de inibir o processo de fusão do vírus com a célula do hospedeiro. Alguns se encontram em fase de avaliação clínica. Anticorpos contra a proteína p24 do HIV-1 foram detectados no soro de pacientes HIV-positivos, e estes reconhecem pequenas seqüências peptídicas desta proteína. Neste trabalho foi analisada a interação entre uma seqüência peptídica correspondente aos aminoácidos 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) da proteína p24, denominado p24- 1, com sistemas biomiméticos. Os sistemas utilizados foram filmes de Langmuir (monocamadas) de dipalmitoil fosfatidil colina (DPPC) e dipalmitoil fosfatidil glicerol (DPPG) e vesículas multilamelares (MLVs) de DPPC. O p24-1 encontra-se desorganizado em solução aquosa, mas com a interação com as MLVs de DPPC teve induzido uma conformação hélice ?, de acordo com o espectro de dicroísmo circular (CD). Esta característica foi confirmada pela predição de hélice a seguida por uma estrutura não ordenada contendo 11 resíduos do p24-1. As isotermas de pressão e potencial de superfície das monocamadas de DPPC foram afetadas com a presença de 0,05% mo1 de p24-1, com uma expansão de aproximadamente 5%. Para concentrações acima de 0,5% mo1 de p24-1 a expansão foi de 20%, com saturação do efeito da concentração. O efeito de expansão foi acompanhado por uma alteração na morfologia das monocamadas, estudados com microscopia no ângulo de Brewster (BAM). A incorporação do p24-1 impede a formação de grandes domínios de DPPC. O efeito cooperativo causado na monocamada de fosfolipídios pelo p24-1 sugere que esse tem um potencial na atividade antiviral por participar da expansão da membrana da célula hospedeira. / The investigation of the interaction mechanisms between the viruses and the host cells brings relevant information for the identification of targets on the development of drugs to prevent the penetration and/or development of the viruses. Peptides designed from viral proteins have been developed and tested on its capacities of inhibiting the merging process of the virus with the host cell. Some of them are in clinical evaluation. Antibodies against the protein p24 of the HIV-1 have been detected in the serum of HIV-positive patients, and they are able to recognize short peptide sequences of this protein. In this work, it was analyzed the interaction between a peptide sequence corresponding to amino acids 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) of the protein p24, called p24- 1, and biomimetic systems. The systems used were Langmuir films (monolayers) of dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG) and multilamelar vesicles (MLVs) of DPPC. p24-1 is found disorganized in watery solution, but with the interaction with the MLVs of DPPC it had induced a conformation ?-helix, according to the circular dichoism spectra (CD). This characteristic was confirmed by the prediction of ?-helix followed by an unordered structure with 11 residues of p24-1. The isotherms of pressure and potential of surface of the DPPC monolayers were affected by the presence of 0,05% mo1 of p24-1, with an expansion of approximately 5%. For concentrations above 0,5% mol of p24-1 the expansion was 20%, with saturation of the concentration effect. The expansion effect was followed by a morphologic alteration of the monolayers, studied with microscopy of the Brewster angle (BAM). The incorporation of p24-1 prevents the formation of large domains of DPPC. The cooperative effect caused in the phospholipid monolayer by p24-1 suggests that this peptide has a potential in the antiviral activity, once its participates on the expansion of the host cell membrane.

La circulation sanguine joue un rôle vital en microcirculation, et ce pour le transport de l'oxygène, le dioxide de carbone et d'autres nutriments. Les globules rouges (GR) constituent la majorité des cellules du sang, c'est pourquoi par "écoulement sanguin", nous entendrons "écoulement d'une suspension de GR". Pendant longtemps l'écoulement sanguin était vu comme un phénomène passif où les GR sont considérés comme des cargos d'oxygène. La vision moderne est tout autre: l'écoulement sanguin est bel et bien un phénomène actif. Les GR ainsi que les cellules endothéliales (qui tapissent les faces internes des vaisseaux sanguins) sont impliquées dans un grand nombre de signalisations biochimiques induites par les contraintes hydrodynamiques, la route vers des régulations vasomotrices sans l'intervention du système nerveux. Par exemple, les GR ne transportent pas que l'oxygène, mais également de l'ATP (adenosine triphosphate), qui est libérée suite à des changements de conformation de protéines membranaires induite par les contraintes hydrodynamiques. Cette thèse est dédiée à la circulation sanguine et son couplage avec la signalisation biochimique ayant lieu en microcirculation. Plus précisément, les questions traités dans cette thèse sont i) la dynamique des GR, ii) le problème de la diffiusion-advection d'espèces chimiques au sein des écoulements sanguins, et iii) le rôle de la géométrie des réseaux vasculaires dans le processus de la signalisation biochimique mentionnés plus haut. Dans un premier temps nous analysons la dynamique de GR dans un écoulement de Poiseuille en présence de valeurs réalistes de contraste de viscosité. Dans un deuxième temps nous développons un modèle de diffusion-advection et le couplons aux écoulements sanguins en adoptant la méthode de Boltzmann sur réseaux; nous exploitons ensuite formulation en l'appliquant au problème de la libération de l'ATP par les GR sous écoulement. Enfin nous présentons des résultats préliminaires pour la problématique générale de l'écoulement sanguin mettant en jeu l'ATP libéré par les GR et la signalisation de calcium par les cellules endothéliales. Cette étude constitue un premier pas vers le problème général et ambitieux de la régulation locale mechano-biochimique impliquée dans la microcirculation. / Blood flow in microcirculation is vital for oxygen, carbon dioxide and nutrients transport. Most of blood cells are red blood cells (RBCs), so that by blood flow we mean flow of a suspension of RBCs. For long time blood flow has been mainly considered as a passive phenomenon, in which RBCs are viewed as passive carriers of oxygen. The modern view is completely different: blood flow is more active than we thought. The RBCs as well as vascular endothelial cells covering the internal walls of blood vessels are involved in a number of biochemical signaling processes that are triggered by shear stress eliciting a number of biochemical events, and ultimately resulting into vasomotor regulation without participation of the nerve system. For example, RBCs do not only carry oxygen but also ATP (adenosine triphosphate) , the release of which occurs thanks to changes of RBC membrane protein conformations caused by shear stress. Released ATP reacts with some endothelial membrane receptors leading to vasodilation. This thesis is devoted to blood flow and its coupling to biochemical signaling. More precisely, we investigate i) the dynamics of RBCs, ii) the advection diffusion of chemicals in blood flow and the role of iii) the geometry of vessel networks, in the mentioned signaling processes in microcirculations. Firstly, we study the RBC dynamics in a pipe flow with realistic viscosity contrast values, where a link between shape dynamics and rheology is established. Secondly, we develop an advection-diffusion solver that can handle general moving curved boundaries based on lattice-Boltzmann method (LBM); we then implement it for the study of the problem of ATP release from RBCs under shear flow. Membrane tension and deformation induced by shear stress together with vessel network geometry contribute to ATP release. Finally we demonstrate the capability of applying our model and our numerical tool to the complete problem of blood under flow involving ATP release from RBCs and endothelial calcium signaling as a preliminary step to the ambitious task of mechano-involved local regulation events in microcirculation.

Kanalrhodpsine (ChRs) sind lichtaktivierbare Kationenkanäle, welche als primäre Fotorezeptoren in Grünalgen dienen. In der Optogenetik werden ChRs verwendet um neuronale Membranen zu depolarisieren und mit Licht Aktionspotentiale auszulösen. Das mit blauem Licht aktivierte Chlamydomonas Kanalrhodopsin 2 (C2) und effiziente Mutanten wie C2 H134R stellen die am häufigsten genutzten depolarisierenden, optogenetischen Werkzeuge dar. Komplementär zu ChRs werden Protonen- und Chloridpumpen aus Archaebakterien zur neuronalen Inhibierung durch lichtinduzierte Hyperpolarisation verwendet. In der vorliegenden Arbeit untersuchten wir die ChR-Chimäre C1V1, ein grünlichtaktiviertes ChR, das sich durch hervorragende Membranlokalisierung und hohe Fotoströme in HEK-Zellen auszeichnet. C1V1 und C1V1-Mutanten mit feinabgestimmten spektralen und kinetischen Eigenschaften ermöglichen die neuronale Aktivierung mit Wellenlängen bis 620 nm sowie die unabhängige Aktivierung zweier Zellpopulationen in Kombination mit C2. Um die strukturelle Basis von Kanalöffnung und Ionentransport in ChRs zu verstehen, wurden gezielt Mutationen in C2 und C1V1 eingeführt. Die Fotoströme der entsprechenden Mutanten wurden auf Kationenselektivität und kinetische Veränderungen untersucht. Während Aminosäuren, die den Kanal an der zytosolischen Seite begrenzen, die Kationenfreisetzung und Einwärtsgleichrichtung der ChRs bestimmen, spielen zentral im Kanal gelegende Aminosäuren ein entscheidende Rolle für Kationenselektivität und -kompetition. Ein enzymkinetisches Modell ermöglichte außerdem die Zerlegung der Fotoströme in Beiträge der verschiedenen, konkurrierenden Kationen. Im letzten Teil der Arbeit wurde pHoenix, ein optogenetisches Werkzeug zur Ansäuerung synaptischer Vesikel, entwickelt. In Neuronen des Hippocampus wurde pHoenix verwendet, um die treibenden Kräfte für die vesikuläre Neurotransmitteraufnahme sowie den Zusammenhang zwischen Vesikelfüllstand und Freisetzungswahrscheinlichkeit zu analysieren. / Channelrhodopsins (ChRs) are light-activated cation channels functioning as primary photoreceptors in green algae. In the emerging field of optogenetics, ChRs are used to depolarize neuronal membranes, thus allowing for light-induced action-potential firing. The blue light-activated Chlamydomonas channelrhodopsin 2 (C2) and high-efficiency mutants such as C2 H134R represent the most commonly used depolarizing optogenetic tools. Complementary to ChRs, green to yellow light-activated proton and chloride pumps originating from archea enable neuronal inhibition by membrane hyperpolarization. In the present work, we developed the chimeric ChR C1V1, a green-light activated ChR with excellent membrane targeting and high photocurrents in HEK cells. Action spectrum and kinetic properties of C1V1 were further fine-tuned by site-directed mutagenesis. The ensemble of C1V1 variants allows for neuronal activation with wavelengths up to 620 nm and can be used in two-color optogenetic experiments in combination with C2 derivatives. In order to understand the structural motifs involved in channel gating and ion transport, conserved residues in C2 and C1V1 were mutated and photocurrents of the respective mutants were analyzed for kinetic characteristics and cation selectivity. In these experiments, residues of the inner gate region were shown to alter cytosolic cation release and inward rectification, whereas central gate residues determine cation competition and selectivity, as well as the equilibrium between the two open channel conformations. Moreover, an enzyme-kinetic model was used to quantitatively dissect ChR photocurrents into the contribution of different competing cations. Finally, we designed pHoenix, an optogenetic tool enabling green-light induced acidification of synaptic vesicles. In hippocampal neurons, pHoenix was used to study both the energetics of vesicular neurotransmitter uptake and the impact of the vesicular contents on synaptic vesicle release.

Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.