Thèse de doctorat : Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 172-190.

Plusieurs types de bactéries peuvent se déplacer dans leur milieu à l’aide de flagelles rotatifs. Ces flagelles sont composés d’un moteur rotatif ainsi que d’un filament long qui peut atteindre plusieurs fois la longueur du corps (10-15 μm) à l’extérieur de la cellule. Ceux-ci sont composés d’un assemblage de plusieurs milliers de protéines identiques appelées flagelline (FliC). Chaque filament croît par auto-assemblage des unités de FliC à son extrémité extérieure. Chaque FliC doit donc être transportée, partiellement dépliées dans un mince canal à l’intérieur du filament. Au bout du filament se trouve une structure, nommée le cap, composée de 5 protéines FliD essentielles à la polymérisation de la flagelline. Le travail réalisé au cours de cette thèse porte sur deux aspects particuliers de l’assemblage d’un filament. Premièrement, un filament peutil continuer à s’assembler s’il est cassé ? Deuxièmement, quel est le taux de croissance d’un filament et est-ce que ce taux varie avec la longueur du filament ? En utilisant des impulsions laser ultrabrèves, nous avons coupé des filaments marqués d’un fluorophore pour observer s’ils continuaient à s’assembler. Suivant une période de croissance de 2 heures, les filaments étaient marqués de fluorophores différents et observées sous un microscope en épifluorescence. Une souche de Salmonella enterica génétiquement modifiée pour n’exprimer en moyenne qu’un seul filament par cellule a été utilisée. Cette modification assure que chaque filament revisité a bien été coupé auparavant. La même expérience fut aussi réalisée à l’aide d’une souche surexprimant la protéine FliD. En tout, 82 filaments bactériens furent ainsi coupés, puis observés après une période de croissance et aucune reprise de croissance ne fut observée. Ces résultats peuvent sembler surprenants à la lumière de récentes études qui ont rapporté que les filaments continuent de s’assembler lorsqu’ils sont cassés mécaniquement par des forces de cisaillement (”shearing”). En utilisant des marquages de couleurs différentes, nous avons aussi mesurer le taux de croissance du filament en fonction de sa longueur. Nos résultats démontrent que le taux de croissance diminue graduellement avec la longueur, allant de ∼ 4μm/h à 1μm jusqu’à ∼ 1μm/h à 10 μm. Ces résultats contrastent avec le taux constant de 2.3m/3h rapporté récemment dans la littérature. Les expériences décritent dans cette thèse ont donc permis d’élaborer un modèle novateur décrivant le mécanisme de croissance du filament bactérien combinant la simple diffusion avec un mécanisme actif qui introduit les flagellines à la base du filament. / Many types of bacteria swim using a rotary motor. These remarkable biological motors are composed of a stator, a rotor and a bacterial flagellar filament which extends many body lengths (10-15μm) outside of the cell. The filaments are constructed of as many as 20000- 30000 protein subunits (called flagellin). Each filament grows at its distal end by self-assembly of flagellin subunits that have to be exported in an unfolded conformation through the narrow channel inside the filament. At the very end of the filament, a “cap” structure made of the FliD protein is essential for flagellin self-assembly and polymerization. The work performed in this doctoral thesis focuses on two specific aspects of the filament assembly. First, we ask whether the filament can continue to grow after being cut? Secondly, the rate of growth is measured as a function of the filament’s length. Using femtosecond laser ablation, we cut individual bacterial filaments and observed whether they could regrow. Bacterial filaments were first labeled with an orange fluorophore, cut with the laser, and then re-labeled with a green fluorophore after a 2h regrowth period. The experiments were performed with a genetically modified Salmonella enterica strain that grows only one filament per cell. This modification allows us to make sure that we revisit (after the regrowth period) the exact individual filaments that were cut by the laser. The same experiment was also performed with a strain where the cap protein FliD could be overexpressed. Overall, 82 filaments were cut and we did not observe any regrowth. Interestingly, this conclusion differs from results reported recently using mechanically broken (sheared) Escherichia coli filaments. Using a similar approach (sequential labeling of filaments with different colors) we also investigated the rate at which bacterial filaments grow as a function of their initial length. Our results lead us to the conclusion that the growth rate decreases with length (from ∼ 4μm/h at 1μm down to ∼ 1μm/h at 10μm). These observations again contrast with the constant growth rate (2.3 μm/3h) reported in a recent study. Those two separate results helped in the developement of a new model for the mechanism behind the bacterial flagellar growth combining simple diffusion with an active mecanism feeding the flagellin proteins at the base of the filament.

El citoesqueleto de actina es imprescindible para el mantenimiento de la morfología celular, así como para realizar diversas funciones como la movilidad/migración y división celular. En términos generales, este componente del citoesqueleto está constituido por los filamentos de actina o microfilamentos (MFs) y por una serie de proteínas de unión y/o relacionadas con la actina, implicadas en la organización estructural de los MFs y en la regulación de la dinámica de polimerización/despolimerización de actina. Una forma sencilla y eficaz de interferir en la dinámica de actina es mediante el uso de toxinas de actina. En este trabajo se ha realizado un estudio exhaustivo de los efectos provocados por estas toxinas a nivel de la morfología celular, arquitectura y de salida de proteínas del aparato de Golgi, bien por depolimerización de los MFs (citocalasina D, latrunculina B, micalolide B o toxina botulínica C2) o bien por su polimerización aberrante o estabilización (jasplakinolide). El análisis de los efectos provocados por las distintas toxinas revela como éstos son dependientes del tipo celular, modo de acción de la toxina así como de la concentración y tiempo de exposición a esta. Hemos observado como los cambios de la morfología de las cisternas producto de la despolimerización de los MFs se correlacionan con variaciones en la homeostasis el pH de este orgánulo. Según lo cual, pensamos que los MFs participan en el diseño y mantenimiento de la forma aplanada de las cisternas del aparato de Golgi, probablemente regulando, en parte, la maquinaria molecular implicada en el mantenimiento de la homeostasis iónica de este orgánulo. Por otro lado, hemos observado como los MFs participan de forma variable en la salida del aparato de Golgi de cargo no asociado a balsas lipídicas con destino basolateral y apical. Sin embargo, son prescindibles en la salida de cargo asociado a balsas lipídicas con destino apical. Paralelamente a estos resultados que implican a los MFs en la morfo-funcionalidad del aparato de Golgi, hemos desarrollado un modelo celular para generación de agresomas de actina filamentosa, los cuales han sido descritos en distintas enfermedades como el Alzheimer o el acoholismo crónico bajo el nombre de cuerpos de Hirano. En este sentido, el jasplakinolide es capaz de inducir la formación reversible de un agresoma de actina filamentosa similar al cuerpo de Hirano cuya degradación está mediada por los sistemas proteasomal y lisosomal/autofagia. Por último, describimos por primera vez in vitro la generación y coexistencia de dos agresomas originados por mecanismos diferentes sin que tenga lugar en ningún momento la mezcla de sus componentes moleculares.

Le cytosquelette se compose de microfilaments (polymères d’actine), de microtubules (polymères de tubuline) et de filaments intermédiaires (polymères de desmine, de lamines …). Le sepsis défini par une infection associée à une réaction inflammatoire systémique est responsable de dysfonctions myocardiques de mauvais pronostique. Cette cardiomyopathie apparait dans les premières heures du sepsis et guérit en moins de deux semaines chez les survivants. Même si certaines études démontrent l’implication d’éléments du cytosquelette dans la cardiomyopathie septique, les rôles des microfilaments et des microtubules ne sont pas clairement établis.Macrophage migration inhibitory factor (MIF) est une cytokine pro-inflammatoire sécrétée en excès dans le sepsis qui serait responsable d’un ralentissement de la récupération myocardique. Dans un premier temps, notre travail a consisté à caractériser l’implication des microtubules dans la dysfonction musculaire cardiaque induite par MIF. Dans un modèle de trabécules auriculaires droites humaines nous avons démontré que MIF induit une hyperpolymérisation des microtubules responsable d’une hyperviscosité intracellulaire, d’une dysfonction mitochondriale et d’une dysfonction contractile. Nos résultats suggèrent qu’une hyperpolymérisation des microtubules induite par MIF pourrait être responsable d’un ralentissement de la récupération myocardique à la phase tardive de la myocardiopathie septique. Dans un second temps, nous avons évalué l’implication des microfilaments dans un modèle murin de dysfonction myocardique inflammatoire induite par l’injection d’une endotoxine bactérienne, le lipopolysaccharide. Nos résultats suggèrent qu’à la phase précoce de la cardiomyopathie inflammatoire il existe une hyperpolymérisation des microfilaments responsable de dysfonctions contractile et mitochondriale.Les connaissances fondamentales acquises au cours de ce travail de thèse suggèrent une implication directe des microtubules et des microfilaments dans la physiopathologie des cardiomyopathies inflammatoires. / The cytoskeleton is composed of intracellular microfilaments (actin polymers), microtubules (tubulin polymers) and intermediate filaments (desmin, lamin … polymers).Sepsis, the association of an infection and a systemic inflammatory response, induces myocardial dysfunction. Septic cardiomyopathy appears in the early phase of sepsis and is associated with fatal outcome. Complete recovery of myocardial function occurs within two weeks following the onset of myocardial dysfunction in surviving patients. Although several studies demonstrate a role of cytoskeleton in septic cardiomyopathy, the involvement of microfilaments and microtubules is not clear.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine secreted during sepsis that is suggested to postpone myocardium recovery. The first aim of the study was to characterize microtubule implications in MIF-induced cardiac muscle dysfunction. In a model of human right atrial trabecule we demonstrated that MIF induces microtubule stabilizations which are responsible for high intracellular viscosity, contractile and mitochondria dysfunctions. Our results suggest that MIF-induced microtubule stabilizations might be responsible for a delay of myocardial recovery during septic cardiomyopathy. The second aim of the study was to characterize microfilament involvements in a murine inflammatory cardiomyopathy induced by a lipopolysaccharid (LPS) injection. Our results suggest that microfilament stabilizations might be responsible for LPS-induced contractile and mitochondria dysfunctions in the early phase of inflammatory cardiomyopathy. Thus, these new fundamental mechanisms suggest a direct involvement of microtubules and microfilaments in the development and evolution of inflammatory cardiomyopathies.

<p> Amoeboid Motion is thought to be due to the action of an actomyosin- like protein present in the cytoplasm of amoeba. A co-ordinated net- 0 work of microfilaments of the actomyosin-like protein, 70 A in diameter, may be the mechanical means of accomplishing amoeboid motion. The microfilaments formed of the actomyosin-like protein, may be capable of rapid association and dissociation in vivo. In this thesis, the cytoplasm of Naegleria gruberi amoeba has been shown to possess a protein similar to actomyosin. Characterization of the ATPase activity, superprecipitating ability, electrophoretic behaviour and microfilament producing ability reveal that the actomyosin-like protein of Naegleria gruberi amoeba is quite similar to the analogous protein in Physarum polycephalum. Naeqleria gruberi may be an ideal organism in which to study the interconversion of one form of a biologically active macromolecule to another., In different stages of the life cycle, amoeboid motion, flagellar beating and mitotic spindles are present. It is possible that the same contractile molecules in different forms may perform different functions. </P> / Thesis / Master of Science (MSc)

A previous study in chickens revealed that myosin light chain kinase (MLCK), f actin, and myosin are found on the crystalline lens. Their polygonal arrangement at the posterior surface resembles a muscle tissue, which suggests that these proteins may have a contractile role in accommodation. The ciliary muscle in chickens is skeletal in nature and, therefore, chickens were used to test the hypothesis that contractile microfilaments play a role in accommodation. Ciliary nerve-induced accommodation was measured in the presence of an MLCK inhibitor 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7). Eyes of 6-day old white Leghorn chickens (gallus gallus domesticus) were enucleated in Tyrode’s saline solution while keeping the ciliary nerve intact. One eye was treated with ML-7 and the other eye was treated with vehicle only. Three concentrations of ML-7 were used: 1 µM, 10 µM, and 100 µM. Two experiments were carried out, one including a (3×10 min) wash and one without. Focal lengths of the vehicle- and ML-7-treated eyes were measured before, during and after accommodation. Immunoblots were used to detect the amount of phosphorylated myosin with and without the inhibitor. Focal lengths for accommodation were shorter than those at rest (p<0.001). In the wash experiment, vehicle-treated eyes had higher accommodative amplitudes compared to ML-7-treated eyes for all three dosage groups. In the no-wash experiment, only the 1 µM group demonstrated the same trend as the wash experiment. For the 10 µM and 100 µM groups, ML-7-treated eyes had higher accommodative amplitudes compared to vehicle-treated eyes. Immunoblots revealed varying amounts of inhibition within pairs of eyes as well as between birds for both experiments. Results from this experiment indicate that ML-7 was not effective at determining whether contractile microfilaments found on the lens contribute to accommodation.

A previous study in chickens revealed that myosin light chain kinase (MLCK), f actin, and myosin are found on the crystalline lens. Their polygonal arrangement at the posterior surface resembles a muscle tissue, which suggests that these proteins may have a contractile role in accommodation. The ciliary muscle in chickens is skeletal in nature and, therefore, chickens were used to test the hypothesis that contractile microfilaments play a role in accommodation. Ciliary nerve-induced accommodation was measured in the presence of an MLCK inhibitor 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7). Eyes of 6-day old white Leghorn chickens (gallus gallus domesticus) were enucleated in Tyrode’s saline solution while keeping the ciliary nerve intact. One eye was treated with ML-7 and the other eye was treated with vehicle only. Three concentrations of ML-7 were used: 1 µM, 10 µM, and 100 µM. Two experiments were carried out, one including a (3×10 min) wash and one without. Focal lengths of the vehicle- and ML-7-treated eyes were measured before, during and after accommodation. Immunoblots were used to detect the amount of phosphorylated myosin with and without the inhibitor. Focal lengths for accommodation were shorter than those at rest (p<0.001). In the wash experiment, vehicle-treated eyes had higher accommodative amplitudes compared to ML-7-treated eyes for all three dosage groups. In the no-wash experiment, only the 1 µM group demonstrated the same trend as the wash experiment. For the 10 µM and 100 µM groups, ML-7-treated eyes had higher accommodative amplitudes compared to vehicle-treated eyes. Immunoblots revealed varying amounts of inhibition within pairs of eyes as well as between birds for both experiments. Results from this experiment indicate that ML-7 was not effective at determining whether contractile microfilaments found on the lens contribute to accommodation.

xiii, 114 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Novel experiments that probe the dynamics of intracellular species, including the center-of-mass displacements and internal conformational transitions of biological macromolecules, have the potential to reveal the complex biochemical mechanisms operating within the cell. This work presents the implementation and development of Fourier imaging correlation spectroscopy (FICS), a phase-selective approach to fluorescence spectroscopy that measures the collective coordinate fluctuations of fluorescently labeled microscopic particles. In FICS experiments, a spatially modulated optical grating excites a fluorescently labeled sample. Phase-synchronous detection of the fluorescence, with respect to the phase of the exciting optical grating, can be used to monitor the fluctuations of partially averaged spatial coordinates. These data are then analyzed by two-point and four-point time correlation functions to provide a statistically meaningful understanding of the dynamics under observation. FICS represents a unique route to elevate signal levels, while acquiring detailed information about molecular coordinate trajectories. Mitochondria of mammalian cells are known to associate with cytoskeletal proteins, and their motions are affected by the stability of microtubules and microfilaments. Within the cell it is possible to fluorescently label the mitochondria and study its dynamic behavior with FICS. The dynamics of S. cerevisiae yeast mitochondria are characterized at four discrete length scales (ranging from 0.6 - 1.19 μm) and provide detailed information about the influence of specific cytoskeletal elements. Using the microtubule and microfilament destabilizing agents, Nocodazole and Latrunculin A, it is determined that microfilaments are required for normal yeast mitochondrial motion while microtubules have no effect. Experiments with specific actin mutants revealed that actin is responsible for enhanced mobility on length scales greater than 0.6 μm. The versatility of FICS expands when individual molecules are labeled with fluorescent chromophores. In recent experiments on the tetrameric fluorescent protein DsRed, polarization-modulated FICS (PM-FICS) is demonstrated to separate conformational dynamics from molecular translational dynamics. The optical switching pathways of DsRed, a tetrameric complex of fluorescent protein subunits, are examined. An analysis of PM-FICS coordinate trajectories, in terms of 2D spectra and joint probability distributions, provides detailed information about the transition pathways between distinct dipole-coupled DsRed conformations. This dissertation includes co-authored and previously published material. / Committee in charge: Tom Stevens, Chairperson, Chemistry; Andrew Marcus, Advisor, Chemistry; Peter von Hippel, Member, Chemistry; Marina Guenza, Member, Chemistry; John Toner, Outside Member, Physics

On the basis of detailed phenotypic examination of fh1 and fh2 mutants we observed that the main housekeeping Arabidopsis thaliana formin AtFH1 (At3g25500) and its closest relative, AtFH2 (At2g43800) are involved in both actin filaments and microtubule dynamics. fh1 mutants showed increased sensitivity to the actin polymerization inhibitor Latrunculin B (LatB). Formin mutants had cotyledon pavement cells which exhibited more pronounced lobes compared to the wild type, and alterations in vascular tissue patterning were found. The double fh1 fh2 homozygote was not obtained, suggesting that at least one functional formin gene is required for proper gametophyte development. Methods used to observe and quantify both architecture and dynamics of the cortical cytoskeleton from confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM) were standarized and allowed to find that mutants exhibited more abundant but less dynamic F- actin bundles and more dynamic microtubules than wild type seedlings, fh1 mutant phenotype observed in roots was further aggravated by a (heterozygous) fh2 mutation. The formin inhibitor SMIFH2 mimicked the alterations observed in fh1 mutants in plants, it has been the first report of this inhibitor in plants. Defects in membrane trafficking were...

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