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La microscopie de super-résolution PALM (microscopie de localisation après photo-activation) est un outil performant pour l'étude des cellules à l'échelle nanométrique. Dans ses applications avancées, la microscopie PALM permet l'étude quantitative et dynamique des objets et événements biologiques. Ces applications sont cependant limitées par le comportement photophysique complexe des protéines fluorescentes photoconvertibles vert à rouge (PCFPs) utilisées comme marqueurs. En particulier, les transitions répétées et stochastiques des PCFPs entre un état sombre et un état fluorescent (scintillement) ainsi que l'incomplétude de photoconversion compliquent l'extraction d'informations quantitatives.Nos travaux combinant cristallographie aux rayons X des protéines et microscopie de localisation ont permis de mettre en évidence le rôle central d'un acide aminé conservé au sein des PCFPs, l'arginine 66, dans le contrôle du scintillement et du photoblanchiment de la forme rouge de deux PCFPs populaires: mEos2 et Dendra2.D'autre part, des résultats préliminaires suggèrent que dans leur formes vertes et dans les conditions d'illumination classiques PALM, les PCFPs entrent dans un état sombre de long temps de vie ce qui ralentit la photoconversion.Nos résultats ouvrent la porte à la conception raisonnée de nouvelles PCFPs optimisées pour les applications quantitatives et dynamiques du PALM. / Super-resolution PALM microscopy (photoactivated localization microscopy) is a powerful tool to investigate the cells with nanoscopic accuracy. Advanced PALM microscopy allows to quantitatively and dynamically study biological objects and events. These applications are nevertheless limited by the complex photophysical behavior of the green-to-red photoconvertible fluorescent proteins (PCFPs) used as markers. In particular, PCFPs red forms repeated and stochastic transitions between a fluorescent and a dark state (blinking) as well as photoconversion uncompleteness complicate the extraction of quantitative information.Our study, by combining X-ray crystallography and localization microscopy, evidences that a single aminoacid well conserved among PCFPs, the arginine 66, controls the blinking and photobleaching behavior of two popular PCFPs: mEos2 and Dendra2.Preliminary results suggest that in their green forms and under PALM classical illumination conditions, PCFPs switch to a long-lived dark state resulting in a photoconversion slowing down.Our results open the door to future rational engineering of enhanced PCFPs for quantitative and dynamic PALM.

Standard optical microscopes cannot resolve images below 200 nm within the visible wavelengths due to optical diffraction limit. This Thesis reports an investigation into super-resolution imaging beyond the optical diffraction limit by microsphere optical nano-scopy (MONS) and submerged microsphere optical nano-scopy (SMON). The effect of microsphere size, material and the liquid type as well as light illumination conditions and focal plane positions on imaging resolution and magnification have been studied for imaging both biological (viruses and cells) and non-biological (Blu-ray disk patterns and nano-pores of anodised aluminium oxide) samples. In particular, sub-surface imaging of nano-structures (data-recorded Blu-ray) that cannot even be seen by a scanning electron microscope (SEM) has been demonstrated using the SMON technique. Adenoviruses of 75 nm in size have been observed with white light optical microscopy for the first time. High refractive index microsphere materials such as BaTiO3 (refractive index n = 1.9) and TiO2-BaO-ZnO (refractive index n = 2.2) were investigated for the first time for the imaging. The super-resolution imaging of sub-diffraction-limited objects is strongly influenced by the relationship between the far-field propagating wave and the near-field evanescent waves. The diffraction limit free evanescent waves are the key to achieving super-resolution imaging. This work shows that the MONS and SMON techniques can generate super-resolution through converting evanescent waves into propagating wave. The optical interactions with the microspheres were simulated using special software (DSIMie) and finite different in time domain numerical analysis software (CST Microwave Studio). The optical field structures are observed in the near-field of a microsphere. The photonic nanojets waist and the distance between single dielectric microsphere and maximum intensity position were calculated. The theoretical modelling was calculated for comparisons with experimental measurements in order to develop and discover super-resolution potential.

Nanoscopic spatial reorganization of selectin ligands, CD44 and PSGL-1, during the initial step of hematopoietic stem/progenitor cell (HSPC) homing, tethering and rolling of migrating cells over E-selectins, has been recently reported. However, the exact spatial distribution of these ligands and their spatial reorganization during the cell rolling on E-selectins are still an open question. The spatiotemporal characterization at the nanoscale level requires high resolution imaging methods. In this study, I quantitatively characterize nanoscopic spatiotemporal behavior of the selectin ligands on the migrating cells to understanding the molecular mechanism of the cell rolling at the nanoscale level by means of a microfluidics-based 3D super-resolution fluorescence microscopy technique. The obtained results suggest that PSGL-1 on the cell shows significant change in the axial distribution on the cell during the cell rolling on E-selectin whereas the spatial distribution of CD44 along the axial direction is not affected significantly by the cell rolling. These findings indicate that each selectin ligand has a distinct contribution to the initial step of the HSPC homing because of their distinct spatial localizations on the cells that regulate at least partly the accessibility of these ligands to the surface E-selectin.

Spatial and temporal dynamics of polymer chains play critical roles in their rheological properties, which have a significant influence on polymer processing and fabrication of polymer-based (nano) materials. Many theoretical and experimental studies have aimed at understanding polymer dynamics at the molecular level that give rise to its bulk phase properties. While much progress has been made in the field over the past ~60 years, many aspects of polymers are still not understood, especially in complicated systems such as entangled fluids and polymers of different topologies. In addition, the physical properties of biological macromolecules, i.e. DNA, are expected to affect the spatial organization of chromosome in a cell, which has the potential impact on a broad epigenetics research. Here, we propose new methods for simultaneous visualization of diffusive motion and conformational dynamics of individual polymer chains, two most important factors that characterize polymer dynamics, based on a new single-molecule tracking technique, cumulative-area (CA) tracking method. We demonstrate the applicability of the CA tracking to the quantitative characterization of the motion and relaxation of individual topological polymer molecules under entangled conditions, which is possible only by using the newly-developed CA tracking, using fluorescently-labeled linear and cyclic dsDNA as model systems. We further extend the technique to multi-color CA tracking that allows for the direct visualization and characterization of motion and conformation of interacting molecules. We also develop a new imaging method based on recently developed 3D super-resolution fluorescence microscopy technique, which allows direct visualization of nanoscale motion and conformation of the single molecules that is not possible by any other methods. Using these techniques, we investigate spatial and temporal dynamics of polymers at the single-molecule level, with special emphasis on the effect of topological forms of the molecules and the confined geometry on their spatiotemporal dynamics. Our results demonstrate that the new methods developed in this thesis provide an experimental platform to address key questions in the entangled topological polymer dynamics. The research will provide a platform for developing new polymer-based materials and open the possibility of studying spatial organization of DNA in a confined geometry from physics point of view.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23115号 / 医科博第126号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 伊佐 正, 教授 髙橋 良輔, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chez les bactéries, les pôles cellulaires servent de domaines subcellulaires impliqués dans plusieurs processus cellulaires. Chez l’agent pathogène du choléra, Vibrio cholerae, en forme de bâtonnet incurvé, le pole contenant l’unique flagelle est impliqué dans la virulence. La protéine d’ancrage polaire HubP interagit avec plusieurs ATPases telles que ParA1 (ségrégation des chromosomes), ParC (localisation polaire du système de chimiotaxie) et FlhG (biosynthèse des flagelles), organisant ainsi l'identité polaire de V. cholerae. Cependant, les mécanismes moléculaires exacts de cet ancrage polaire doivent encore être élucidés. L’objectif de cette thèse est d’établir une vue d'ensemble de l'organisation de pôle cellulaire ce qui implique le mécanisme d’orchestration des différentes fonctions cellulaires par l’identification de l’ensemble des partenaires d'interaction de HubP ainsi que la cartographie fine du pôle cellulaire par microscopie à super résolution (PALM). Afin d’identifier de nouveaux partenaires d'interaction de HubP, j'ai étudié la différence de composition en protéines polaires entre les contextes HubP+ et HubP-. La composition en protéines polaires a été quantifiée de manière relative et absolue en ajoutant des Tag isobares aux protéines extraites de mini-cellules. Ces mini-cellules correspondent des petits compartiments cellulaires issus d’un évènement de division anormal proche du pole et sont enrichies en protéines polaires. Parmi ~800 protéines identifiées, ~ 80 protéines ont été considérées comme enrichies en contexte HubP+ incluant de nombreuses protéines attendues (FlhG, ParC et en aval des protéines de chimiotaxie). J'ai étudié la localisation de 14 protéines par microscopie à fluorescence et pu révéler 4 nouvelles protéines présentant une localisation polaire dépendant de HubP : VbrX, VbrY, et 2 protéines hypothétiques MotV et MotW. La délétion de motV et motW provoque un défaut significatif de propagation dans une gélose molle suggérant une implication dans la chimiotaxie et/ou la motilité. Alors que la microscopie électronique a montré que les deux mutants ont bien un flagelle polaire unique, le suivi-vidéo de leur déplacement a révélé que les deux mutants présentaient des défauts de nage assez distincts: ∆motV est plutôt affecté dans le changement de direction et ∆motW dans la vitesse de déplacement. Des expériences de microscopie fluorescente ont montré que MotV, MotW et HubP présentaient des dynamiques de localisation polaire distinctes au cours du cycle cellulaire. Pour une observation fine du pôle cellulaire par PALM, de nouveaux outils d’analyse d’image à haut débit étaient exigés. La précision des contours des petites cellules bactériennes faiblement contrastées n’est pas suffisante par l’observation en fond clair, j'ai développé une nouvelle technique de marquage avec des protéines fluorescentes photo-activables pour un tracé précis de la membrane interne ou du périplasme. En outre, nous avons créé un logiciel utilisant Matlab appelé Vibio qui intègre le contour de cellule et la liste des molécules obtenues par microscopie à super résolution. La capacité d’analyse à haut débit du logiciel permet d’étudier la distribution des molécules de l’échelle de la cellule unique à une population en orientant les cellules par leur courbure longitudinale. J’ai pu révéler que HubP est principalement localisé du côté convexe du pôle de la cellule, tandis que ses partenaires se situaient principalement au milieu du pôle. Mon travail de thèse a révélé avec succès de nouveaux partenaires d'interaction de HubP et la fonction de certaines protéines dans la motilité cellulaire. J'ai développé une nouvelle technique de microscopie pour une localisation subpolaire précise qui fonctionne bien pour l'analyse d'images PALM dans Vibio. J’ai ainsi pu faire progresser les connaissances de l’orchestration des fonctions polaires chez V. cholerae. / In rod shaped bacteria, cell poles serve as important subcellular domains involved in several cellular processes including motility, chemotaxis, protein secretion, antibiotic resistance, and chromosome segregation. In the cholera pathogen Vibrio cholerae, vibrioid rod shape and single polarized flagellum involve in the virulence. Polar landmark protein HubP was shown to interact with multiple ATPases, such as ParA1 (chromosome segregation), ParC (polar localization of chemotaxis apparatus), and FlhG (flagella biosynthesis), thus organizing the polar identity of V. cholerae by tethering proteins to cell pole. However, the exact molecular mechanisms are yet to be elucidated. In this thesis, I tackled to unveil comprehensive view of the cell pole organization which implies the orchestration of different cellular functions, by identifying further interaction partners of HubP as well as drawing conceivable picture of the cell pole by super-resolution photoactivated localization microscopy. To identify new interaction partners of HubP, I used minicells in which cell poles were enriched as they derived from cell division near the cell pole. Difference in protein composition between HubP+ and HubP- minicells were examined by isobaric tags for relative and absolute quantitation. Among ~800 proteins identified, ~80 proteins were considered to be enriched in HubP+ minicells including many expected proteins (FlhG, ParC and downstream chemotaxis proteins). I chose 14 proteins to investigate their subcellular localization with fluorescent microscopy. In conclusion, I discovered 4 proteins that showed polar localization in a HubP-dependent manner. These proteins are VbrX, VbrY, and 2 hypothetical proteins MotV and MotW. ∆motV and ∆motW showed significant defect in a diameter of travel in soft agar plate that suggesting the possible involvement in chemotaxis and/or motility. Whereas electron microscopy showed that both mutants possess intact monotrichous flagellum, video-tracking revealed that the two mutants showed rather distinct defects during swimming: MotV is rather turning mutant while MotW is a speed mutant. Fluorescent microscopy experiments indicated that MotV, MotW and HubP showed distinct polar dynamics over cell cycle. For fine-scale observation of the cell pole by PALM, it was appreciated that novel tools for high-throughput analysis was demanded. Since brightfield images are not sufficient to have accurate contours of small and low contrast bacterial cells, I developed new labeling technique with photoactivatable fluorescent proteins for precise outlining at either inner membrane or periplasm. Furthermore, we created Matlab-based software called Vibio which integrates cell outline and the list of molecules obtained by super-resolution microscopy. High-throughput capability of the software enabled to analyze distribution of detected molecules from single cell to whole bunch of cells in a manner that cells are oriented by cell curvature. These allowed me to discover that HubP is mostly lopsided at the convex side of the cell pole, while its partners mostly located middle of the pole. Altogether, I successfully unveiled 4 novel interaction partners of HubP. I revealed of the function of hypothetical proteins that are involved in cell motility. I developed new labeling technique for precise polar localization that works well for PALM image analysis in Vibio. Therefore, I observed precise polar localization of HubP and other polar proteins.

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The kidney is one of the most important and complex organs in the human body, filtering hundreds of litres of blood daily. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understanding the function of the kidney in health and disease. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (on the sub-200 nm scale), and thus electron microscopy has been the only method of choice to visualize these minute structures. In one study, we show for the first time that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney, a structure around 75 nanometers in width, can now be resolved using light microscopy. In a second study, a novel sample preparation method, expansion microscopy, is utilized to physically expand kidney tissue samples. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragm using confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved further. In a third study, influences on the development of the kidney were studied. There is substantial knowledge regarding what genes (growth factors, receptors etc.) are important for the normal morphogenesis of the kidney. Less is known regarding the physiology behind how paracrine factors are secreted and delivered in the developing kidney. By depleting calcium transients in explanted rat kidneys, we show that calcium is important for the branching morphogenesis of the ureteric tree. Further, the study shows that the calcium-dependent initiator of exocytosis, synaptotagmin, is expressed in the metanephric mesenchyme of the developing kidney, indicating that it could have a role in the secretion of paracrine growth factors, such as GDNF, to drive the branching. / <p>QC 20170523</p>

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24304号 / 生博第487号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 渡邊 直樹, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM