Les protéines de la membrane plasmique jouent un rôle fondamental dans la détection des informations véhiculées par le milieu extracellulaire et l’adaptation des cellules aux variations de l’environnement. Elles font l’objet d’une régulation fine qui permet de moduler leur présence à la membrane et de contrôler les voies de signalisation en aval. Dans ce contexte, les arrestines qui constituent une superfamille de protéines adaptatrices, se sont imposées comme des régulateurs clés depuis la découverte des β-arrestines et arrestines visuelles, spécifiques des eucaryotes supérieurs, et de leur rôle dans la régulation des récepteurs couplés aux protéines G hétéro-trimériques, jusqu’à l’identification plus récente de nouveaux membres apparentés, présents des mammifères jusqu’aux protistes, et partageant un rôle commun de régulation de cargos membranaires. Ce travail de thèse porte sur la caractérisation fonctionnelle de deux représentants du clan arrestine de l’amibe Dictyostelium discoideum, les protéines AdcB et AdcC. Ces deux protéines partagent une même organisation multimodulaire, spécifique aux Dictyostélides, qui associe au cœur arrestine, un domaine putatif C2 de type calcium-binding et deux modules SAMs, respectivement aux extrémités N- et C-terminales des protéines. Nous avons établi que ces domaines apportent des fonctions spécifiques à ces arrestines en leur conférant la capacité de lier des lipides anioniques in vitro en réponse au calcium à travers leur module C2, et de former des structures homo- et hétéro-oligomériques via leurs domaines SAMs. En dépit de ces similarités, AdcB et AdcC présentent un comportement différent in cellulo dans la mesure où seul AdcC transloque à la membrane plasmique en réponse à une élévation du calcium cytosolique, provoquée par la stimulation des cellules par les chimioattractants AMPc et acide folique ou le calcium lui-même. Ces résultats ont été complétés par une étude phénotypique des mutants invalidés pour ces arrestines et la recherche de partenaires qui ouvrent des pistes pour des études futures. / Integral proteins of the plasma membrane play a major role in the detection of environmental cues and in the adaptation of cells to variations of their environment. Regulatory mechanisms modulate their presence at the cell surface and control the signaling cascades activated in response to their stimulation. In this context, members of the arrestin revealed to be key regulators, since the discovery of β- and visual arrestins and their well-described role in the regulation of G-protein coupled receptors in complex organisms, and the more recent identification of arrestin-related proteins, present from mammals to protists and sharing functions in membrane cargo trafficking. This work aims at the functional characterization of two arrestin-related proteins of the social amoeba Dictyostelium discoideum, the AdcB and AdcC proteins. These two members of the arrestin clan share a similar multimodular organization, specific to Dictyostelids, with a putative N-terminal calcium-binding type C2 domain and two C-terminal SAM domains surrounding the arrestin module. We showed that the C2 domain confers calcium-dependent binding properties to anionic lipids in vitro and that the SAM domains allow the self-association and hetero-interaction of the two proteins in complexes of high molecular weight. Despite these similarities, AdcB and AdcC harbor a distinct behavior in vivo as only AdcC translocates to the plasma membrane in response to an intracellular calcium rise triggered by the chemoattractants acid folic and cAMP or extracellular calcium. In parallel, a phenotypic characterization of adcB and adcC single or double null mutants and a search for partners were conducted, that open new avenues for future research on these adaptor proteins.

The major evolutionary transition from single-celled to multicellular life is believed to have occurred independently of the main metazoan lineages in the cellular slime moulds, of which Dictyostelium discoideum is the best-studied species. Unusually, in this case multicellular development is a facultative trait and part of an asexual life cycle. It is triggered by starvation and involves aggregation of hitherto independent and possibly unrelated free-living cells. The consequences of multicellularity in D.discoideum are strongly influenced by the environment and meaningful external perturbations are easily carried out. This makes the organism ideally suited to a study of epigenetic factors that regulate development. In an attempt to understand how conserved epigenetic pathways are integrated within the developmental framework, two likely players were chosen for investigation - heat shock protein 90 (Hsp90) and histone deacetylases (HDACs). Hsp90 has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, and morphological evolution. The role of Hsp90 in D.discoideum life cycle was studied using a specific inhibitor, geldanamycin. Inhibition of Hsp90 function in D.discoideum caused a delay in aggregation and an arrest of development at the ‘mound’ stage. A reduction in Hsp90activity in starving cells of D.discoideum resulted in the generation of a range of phenotypes. The study suggests that Hsp90 is required for a specific developmental transition of the social amoeba and is important in generating a reliable outcome of the developmental process. Histone acetylation regulates gene expression and leads to the establishment and maintenance of cellular phenotypes during development of plants and animals. To study the roles of HDACs in D.discoideum, biochemical, pharmacological and genetic approaches were employed. The inhibition of HDAC activity by trichostatin A resulted in histone hyperacetylation and a delay in cell aggregation and differentiation. Cyclic AMP oscillations were normal in starved amoebae treated with trichostatin A but the expression of a subset of cAMP-regulated genes was delayed. Bioinformatic analysis indicated that there are four genes encoding putative HDACs in D.discoideum. One of these four genes, hdaB, was found to be dispensable for growth and development under laboratory conditions; but formed spores with lower efficiency than the wild type in chimeras. The work shows that HDAC activity is important for regulating two aspects of multicellular development: (a) heterochrony, namely the relative timing of developmental events, and (b) modulating the behaviour of single cells in a manner that is sensitive to their social environment.

Cette thèse présente nouveaux organismes trouvés dans d'échantillons d'eaux usées proviennent la zone sud de Jeddah, en Arabie Saoudite. Legionella saoudiensis, Kaumoebavirus, moumouvirus saoudien (SDMV), Yasminevirus et Bunga messiliensis qui sont isolés par une méthode de co-culture amibienne d'infection par Dictyostelium discoideum ATCC 44841, Vermamoeba vermiformis CDC-19, Acanthamoeba polyphaga Linc AP-1 , et Acanthamoeba griffinii, respectivement. Legionella saoudiensis, une souche bactérienne Gram-négative, en forme de bacille, LS-1T appartient au genre Legionella de la famille des Legionellaceae, basée sur des séquences de gène 16S rRNA et d'autres 4 gènes (mip, rpoB, rnpB et 23S-5S). D'une part, le KAUmoebavirus a des capsides icosaédriques de ~ 250 nm-large, un génome d'ADN de 350 731 pb, et une densité de codage de 86%, correspondant à 465 gènes. La plupart de ces gènes (59%) sont étroitement liés aux gènes de Faustoviruses (43%) et Asfarviruses (23%). D'autre part, le moumouvirus saoudien est un nouveau virus géant appartenant à la lignée Mimivirus B, de l'hôpital universitaire King Abdulaziz à Djeddah, et a présenté des particules icosaédriques de 500 nm avec un génome de 1 046 087 pb, plus grand que les génomes moumouvirus qui ont été décrites dans le passé. Il a été prédit que son génome code pour 868 ORF, dont la taille varie de 54 à 2 914 acides aminés. En outre, il code pour 40 nouveaux gènes (ORFans) sans similitude avec d'autres séquences. Ces résultats montrent que la dispositiond’une carte élargie des protistes conduit à découvrir de nouveaux virus géants. / This thesis displays novel organisms that are found in sewage water samples from southern area of Jeddah, Saudi Arabia. These organisms are Legionella saoudiensis, Kaumoebavirus, Saudi moumouvirus (SDMV), Yasminevirus, and Bung messiliensis that are isolated by amoebal co-culture method of infection with Dictyostelium discoideum ATCC 44841, Vermamoeba vermiformis CDC-19, Acanthamoeba polyphaga Linc AP-1, and Acanthamoeba griffinii, respectively. Legionella saoudiensis, a Gram-negative, bacilli shaped bacterial strain, LS-1T belongs to the genus Legionella in the family Legionellaceae based on 16S rRNA gene sequences and other 4 genes (mip, rpoB, rnpB, and 23S-5S). On one hand, KAUmoebavirus has ~250-nm-large icosahedral capsids, a 350,731 bp DNA genome, and a coding density of 86%, corresponding to 465 genes. Most of these genes (59%) are closely related to genes from Faustoviruses (43%) and Asfarviruses (23%). On the other hand, Saudi moumouvirus is a new giant virus belonging to Mimivirus lineage B, from the King Abdulaziz University hospital in Jeddah, and presented 500 nm icosahedral particles with a 1,046,087 bp genome, which is larger than moumouvirus-like genomes which have been described in the past. Its genome was predicted to encode 868 ORFs, ranging in size from 54 to 2,914 amino acids. Furthermore, this genome was predicted to encode 40 new genes (ORFans) without similarity with other sequences. These findings show that the widen chart of protists apply lead to discover new giant viruses.

A key prerequisite for cellular and organismal health is a functional proteome. A variety of human protein misfolding diseases are associated with the occurrence of amyloid protein aggregates, such as amyotrophic lateral sclerosis (ALS) or Huntington’s disease. The proteins involved in disease manifestation all contain aggregation-prone sequences of low compositional complexity. Such sequences are also known as prion-like, because of their sequence similarity to yeast prions. Yeast prion proteins are a specific subset of amyloid forming proteins with distinct physio-chemical and functional features, which give them transmissible properties. The aggregation properties of yeast prions and disease-related prion-like proteins reside in structurally independent, prion-forming domains (PrDs). These domains are highly enriched for uncharged polar amino acids, such as glutamine (Q) and asparagine (N). These compositional features can be used to predict prion-like proteins bioinformatically. To investigate the prevalence of prion-like proteins across different organisms, we analyzed a range of eukaryotic proteomes. Our analysis revealed that the slime mold D. discoideum contains the highest number of prion-like N/Q-rich proteins of all organisms. Based on this finding, we hypothesized that D. discoideum could be a valuable model system to study protein homeostasis (proteostasis) and the molecular basis of protein misfolding diseases. To explore how D. discoideum manages its highly aggregation-prone proteome, we analyzed the behavior of several well-characterized misfolding-prone marker proteins (variants of the disease-causing exon 1 of the huntingtin protein as well as wildtype and variant versions of the Q/N-rich yeast prion Sup35NM). Intriguingly, these proteins did not form cytosolic aggregates in D. discoideum, as they do in other organisms. Aggregates, however, formed as a result of heat stress, which indicates that the tested proteins have the capacity to aggregate, but are kept under tight control under normal conditions. Furthermore, when the stress level was reduced, the stress-induced aggregates dissolved, suggesting that D. discoideum has evolved mechanisms to reverse aggregation after a period of acute stress. Together, these findings reveal an unusual resilience of D. discoideum to aggregation-prone proteins, which very likely results from specific adaptations in its proteostasis network. By studying these specific adaptations, we could get important insight into the strategies that nature employs to control and maintain a highly aggregation-prone proteome. So far, our experimental investigations have revealed evidence for three specific adaptations. First, we identified the disaggregase Hsp101 as a key player in the acute stress response of D. discoideum. A functional analysis of Hsp101 in yeast and D. discoideum revealed that it supports thermotolerance. Second, we found evidence for an important role of the nucleus and nucleolus in proteostasis. We discovered that a small fraction of highly aggregation-prone proteins accumulated in the nucleus or nucleolus of D. discoideum cells. The magnitude of this nuclear accumulation could be increased by proteasome impairment, which suggests that the ubiquitin-proteasome system (UPS) is involved. This finding is consistent with previous studies in other organisms and hints at the possibility that D. discoideum disposes of aggregation-prone proteins by degrading them in the nucleus/nucleolus. Third and finally, we found that cells containing nuclear accumulations are asymmetrically distributed in the multicellular developmental stage (slug), suggesting that D. discoideum employs cell-sorting mechanisms to dispose of cells with accumulated protein damage. Although our current understanding of proteostasis in D. discoideum is preliminary, we have gained important insight into the molecular mechanisms and cellular pathways that D. discoideum uses to counteract protein aggregation. Findings from this work will inform similar comparative studies in other organisms and will impact our molecular understanding of protein misfolding diseases and aging. / Eine wesentliche Voraussetzung für die Gesundheit von Zellen und Organismen ist ein funktionales Proteom. Eine Reihe von humanen Protein- Missfaltungs-Erkrankungen, wie Chorea Huntington und Amyotrophe Lateralsklerose (ALS) werden mit dem Auftreten von amyloiden Protein- Aggregaten in Verbindung gebracht. Sämtliche Proteine, die in der Pathogenese dieser Krankheiten eine Rolle spielen, enthalten aggregations-anfällige Sequenzen mit geringer Sequenzkomplexität. Solche Sequenzen werden als Prion-ähnlich bezeichnet, da sie in ihrer Zusammensetzung den Prionen aus der Hefe S. cerevisiae gleichen. Die Prion-Proteine der Hefe gehören zu einer Unterart von amyloid-aggregierenden Proteinen, die durch bestimmte physikochemische und funktionelle Eigenschaften einen infektiösen Charakter erhalten. Die Aggregations-Eigenschaften von Hefeprionen und aggregationsanfällige Proteinen, die mit Erkrankungen in Verbindung gebracht werden, basieren auf strukturell unabhängigen, Prion-bildenden Domänen (prion domain, PrD). Diese Domänen sind angereichert mit polaren Aminosäuren wie Glutamin und Asparagin. Diese Zusammensetzung kann dazu verwendet werden prion-ähnliche Proteine bioinformatisch vorherzusagen. Um die Verbreitung von Prion-ähnlichen Proteinen in verschiedenen Organismen zu untersuchen, analysierten wir eine Reihe von eukaryotischen Proteomen. Unsere Analyse zeigte, dass der Schleimpilz D. discoideum die höchste Anzahl von Prion-ähnlichen N/Q-reichen Proteinen aufzeigt. Aufgrund dieser Erkenntnisse erstellten wir die Hypothese, dass D. discoideum ein nützlicher Modellorganismus sein könnte, um Protein Homöostase (Proteostase) sowie die molekulare Basis von Proteins-Missfaltungs-Erkrankungen zu ergründen. Um zu analysieren, wie D. discoideum mit seinem höchst aggregations-anfälligen Proteom umgehen kann, untersuchten wir das Verhalten mehrerer bereits charakterisierter aggregations-anfälliger Marker-Proteine in D. discoideum. Hierbei verwendeten wir Varianten des krankheits-erzeugenden Exon 1 des humanen Huntingtin Protein sowie den wild-typ und Varianten des N/Q-reichen Hefe Prions Sup35. Interessanterweise bildeten diese Proteine, anders als in anderen Organismen, keine zytosolischen Aggregate in D. discoideum aus. Aggregate wurden jedoch unter Hitzestress-Bedingungen gebildet. Dies deutet darauf hin, dass die getesteten Proteine durchaus das Vermögen zu aggregieren besitzen, jedoch unter normalen Wachstumsbedingungen streng kontrolliert werden. Wenn, darüberhinaus das Stress- Level gesenkt wurde, kam es zur Auflösung der stress-induzierten Aggregate. Dies deutet darauf hin, dass D. discoideum Mechanismen entwickelt hat, um Aggregate nach Perioden von akutem Stress wieder aufzulösen. Zusammengenommen enthüllen diese Erkenntnisse eine ungewöhnliche Widerstandsfähigkeit gegenüber aggregations-anfälligen Proteinen. Diese beruht höchstwahrscheinlich auf spezifischen Modifikationen im Proteostase Netzwerk. Durch die Analyse dieser spezifischen Anpassungen könnten wichtige Einblicke in die Strategien gewährt werden, welche die Natur benutzt, um ein höchst aggregations-anfälliges Proteom zu erhalten und zu kontrollieren. Bisher erbrachten unsere Experimente Anhaltspunkte für drei spezifische Anpassungen. Erstens zeigten wir, dass die Disaggregase Hsp101 eine Schlüsselrolle in der akuten Stressantwort in D. discoideum einnimmt. Eine funktionale Analyse von Hsp101 in D. discoideum und Hefe zeigte, dass die Disaggregase Thermotoleranz fördert. Zweitens haben wir Anhaltspunkte, dass der Nukleus und der Nukleolus eine wichtige Rolle in der Proteostase einnehmen. Eine geringe Fraktion der überaus aggregations-anfälligen Proteine akkumuliert im Nukleus oder Nukleolus von D. discoideum. Das Ausmaß der nuklearen Akkumulation konnte erhöht werden, wenn das Proteasom beeinträchtigt wird. Dies deutet darauf hin, dass das Ubiquitin-Proteasom-System involviert sein könnte. Diese Beobachtung ist im Einklang mit jüngsten Berichten aus anderen Organismen und daraus folgt, dass D. discoideum möglicherweise aggregations-anfällige Proteine durch Abbau im Nukleus entsorgt. Drittens konnten wir feststellen, dass Zellen, die nukleare Akkumulationen enthalten, asymmetrisch in der multizellulären Entwicklungs-Struktur des Pseudoplasmodiums verteilt sind. Dies deutet darauf hin, dass D. discoideum möglicherweise den Zellsortierungsmechanismus während der Entwicklung nutzen kann, um Zellen mit angereicherten Protein-Schäden zu beseitigen. Auch wenn das gegenwärtige Verständnis der Proteostase in D. discoideum nur vorläufig ist, haben wir wichtige Einblicke in die molekularen Mechanismen und zellulären Prozesse erhalten, die D. discoideum verwendet, um Protein-Aggregation zu verhindern. Die Ergebnisse dieser Arbeit werden ähnliche vergleichende Studien in anderen Organismen beeinflussen und Auswirkungen auf unser molekulares Verständnis über Protein-Missfaltungs-Erkrankungen und das Altern haben.

Macropinocytosis and phagocytosis, two actin-dependent and clathrin independent events of endocytosis, enable the cells such as macrophages and neutrophils to either internalize pathogens and initiates the human innate immune response or serve as a direct entry route for productive infection of pathogen. Dictyostelium discoideum, soil-living amoeba, a unicellular eukaryote that could professionally internalize fluid phase or particles several folds more than that of macrophages and neutrophils. Additionally, multiple key signaling pathways are conserved between Dictyostelium and mammalian cells, including pathways affecting small GTPases Ras and Rac and their downstream effectors, and F-Actin remodeling. All these traits makes Dictyostelium an excellent model organism to study the process pf macropinocytosis and phagocytosis. Upon internalization of the prey, these macropinocytes and phagocytes are often in an environment of increased production of superoxide radicals in the prey-containing vesicles, which helps stimulates the downstream signaling pathways to digest the prey inside. However, the mechanism of how superoxide regulates the process of macropinocytosis and phagocytosis is not fully understood. We had previously reported that Dictyostelium cells lacking Superoxide dismutase C (SodC) exhibited aberrantly high level of active RasG, high basal level of Phosphatidylinositol-3,4,5-triphosphate (PIP3), and severe chemotaxis defects. Now we report that sodC- cells displayed aberrant endosomal vesicle trafficking, significantly compromised particle uptake and defective cell to substratum matrix adhesion compared to that of wild type cells. By using high resolution live imaging microscope we also show that sodC- cells have defects in F-Actin remodeling at the phagocytic rim extension and F-Actin depolymerization of the nascent phagosome. Interestingly, the introduction of overexpressing of cytoplasmic superoxide dismutase (SodA), redox insensitive RasG (C118A) or treatment of PI3K inhibitor LY294002 in sodC- cells significantly rescued the defects of endosomal vesicle trafficking, particle uptake and adhesion. This project suggests that superoxide dismutase C regulates the endosomal vesicle trafficking, phagocytosis and cell to substratum matrix adhesion through the RasG/PI3K signaling axis in Dictyostelium cells.

CE TRAVAIL A PORTE SUR L'ETUDE DE LA PROTEINE MULTIMODULAIRE ALIX CHEZ DICTYOSTELIUM DISCOIDEUM, UN ORGANISME EUCARYOTE Où DEVELOPPEMENT MULTICELLULAIRE ET PROGRAMME DE MORT CELLULAIRE SONT ETROITEMENT LIES. L'INVALIDATION D'ALX MONTRE QUE CETTE PROTEINE EST ESSENTIELLE A LA DIFFERENCIATION CELLULAIRE ET A LA MORPHOGENESE AU COURS DU DEVELOPPEMENT MULTICELLULAIRE MAIS PAS AU PROGRAMME DE MORT. UNE APPROCHE STRUCTURE/FONCTION A REVELE QUE LE DOMAINE SUSCEPTIBLE DE PARTICIPER A DES TORSADES D'HELICES DE CETTE PROTEINE EST INDISPENSABLE A SA FONCTION DEVELOPPEMENTALE. DE PLUS, SA DISTRIBUTION CELLULAIRE INDIQUE QU'ALIX EST LOCALISE DANS LA VOIE ENDOCYTAIRE EN ASSOCIATION AVEC LA MACHINERIE ESCRT (ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT), IMPLIQUEE DANS LE TRI ET LA FORMATION DU CORPS MULTIVESICULAIRE (MVB). CEPENDANT, L'INVALIDATION DES GENES CODANTS POUR DIFFERENTS COMPOSANTS DES COMPLEXES ESCRT INDUIT DES PHENOTYPES DISSEMBLABLES. CECI SUGGERE QUE SOIT CES PROTEINES ONT DES ROLES DANS D'AUTRES PROCESSUS CELLULAIRES SOIT QUE CETTE VOIE N'EST PAS PERTURBEE DE LA MEME FAÇON DANS LES DIFFERENTS MUTANTS.

DICTYOSTELIUM DISCOIDEUM EST UN ORGANISME UNICELLULAIRE SE NOURRISSANT PAR PHAGOCYTOSE DE MICROORGANISMES ET ENDOCYTOSE DE PHASE FLUIDE. ELLE EST CAPABLE DE MIGRATION SUR UN SUBSTRAT, PAR EMISSION DE PROTRUSIONS SUR LE FRONT CELLULAIRE AVANT, ET PAR RETRACTION DU FRONT ARRIERE. LA MOTILITE EST LIEE A L'ADHERENCE DES CELLULES SUR LE SUBSTRAT, SIEGE DE LA TRANSMISSION DES FORCES EXERCEES PAR LA CELLULE. SOUMISE A UNE FORCE DE CISAILLEMENT HYDRODYNAMIQUE, DICTYOSTELIUM SE DEPLACE DANS LE SENS DU FLUX. NOUS AVONS ETUDIE LES MECANISMES BIOCHIMIQUES MIS EN JEU EN REPONSE A LA FORCE OU A L'ETALEMENT. NOUS AVONS ANALYSER LE COMPORTEMENT DE CELLULES SAUVAGES ET DE MUTANTS D'INVALIDATION, A L'ECHELLE DE LA CELLULE ET DU BORD CELLULAIRE. NOUS MONTRONS PLUSIEURS RESULTATS. LE CALCIUM LIBRE EXTRACELLULAIRE AUGMENTE LA VITESSE DE MIGARTION DES CELLULES ET LEUR SENSIBILITE AUX FORCES. CECI EST DU A UNE AUGMENTATION DE LA DYNAMIQUE DES BORDS CELLULAIRES, LES PROTRUSIONS ETANT PLUS GRANDES ET LES RETRACTIONS PLUS EFFICACES. IL Y A DES OSCILLATIONS DES BORDS CELLULAIRES, AVEC DES PERIODES PROPRES DIFFERENTES A L'AVANT ET A L'ARRIERE DE LA CELLULE. CES PERIODES NE DEPENDENT NI DE LA CONCENTRATION NI DU TYPE CELLULAIRE. LE CALCIUM AUGMENTE LA CINETIQUE ET LA REGULARITE DE L'ETALEMENT, EN AGISSANT SUR LA POLYMERISATION D'ACTINE. L'ETALEMENT NE DEPEND QUE DE LA POLYMERISATION D'ACTINE ET DE L'EMISSION DE PROTRUSIONS, TANDIS QUE LES RETRACTIONS NE PEUVENT EXISTER SANS MYOSINE 2. ENFIN, LES PROTEINES G ET LES RECETEURS A L'IP3 SONT IMPLIQUES DANS LA SIGNALISATION CALCIQUE.

Recent work has shown that microorganisms are surprisingly like animals in having sophisticated behaviours such as cooperation, communication, and recognition, as well as many kinds of symbioses. Here we show first that the social amoeba Dictyostelium discoideum has a primitive farming symbiosis that includes dispersal and prudent harvesting of the crop. About one-third of wild-collected clones engage in husbandry of bacteria. Instead of consuming all bacteria in their patch, they stop feeding early and incorporate bacteria into their fruiting bodies. They then carry bacteria during spore dispersal and can seed a new food crop, which is a major advantage if edible bacteria are lacking at the new site. However, if they arrive at sites already containing appropriate bacteria, the costs of early feeding cessation are not compensated, which may account for the dichotomous nature of this farming symbiosis. We also observed farmer Dictyostelium discoideum clones carry bacteria that they do not use as food. We hypothesized that these bacteria may play a defensive role against other D. discoideum clones. In our second study, we investigated the impact of these bacteria-carrying farmers on non-farming D. discoideum clones. We found that the presence of farming clones reduces spore production in non-farmers. Furthermore, this effect increases with frequency of farming clones, demonstrating the vulnerability of non-farming clones to farmers though in this experiment we had not separated the effects of the farmer clone and the bacteria they carry. In our third study we exposed non-farmers to a filtered supernatant from the most common non-food carried bacterium, Burkholderia xenovorans . This supernatant is likely to carry whatever the bacteria are producing. We treated Dictyostelium clones at the beginning of the social stage and found that the supernatant enhanced spore production of farming clones and hurt spore production of non-farming clones. This study shows that the effects of the bacteria can be restricted to a filtered supernatant alone. This discovery of symbiosis of D. discoideum with bacteria, and its impact on social interactions among D. discoideum clones will provide a fertile ground for further experiments on the evolution of sociality.

The process of eukaryotic transcription and its regulation has been an interesting area of research for decades. With more insights into the process of transcriptional regulation of genes, studies have revealed a transcriptional regulation at the level of RNA polymerase II in response to nutritional stress. Further studies in our laboratory and others’, using Saccharomyces cerevisiae as a model system, had shown that two subunits of core RNA polymerase II, RPB4 and RPB7 play a crucial role in response to nutritional starvation. Similarly, these proteins are also known to play important roles in stress response in higher eukaryotes. Additionally, altering levels of Rpb4 and Rpb7 can differentially affect starvation response in S. cerevisiae (Singh et al., 2007). Multiple tissue blot analyses had shown that both these subunits are differentially expressed in different human tissues more significantly in heart, kidney and brain (Khazak et al., 1995; Khazak et al., 1998; Schoen et al., 1997). These findings have led us to investigate in Dictyostelium discoideum, a cellular slime mold, the possible role of these subunits during starvation-induced development. D. discoideum cells exist as unicellular amoebae in soil. In this organism, growth and differentiation phases are distinctly separated, which is an advantage for investigating the functions of these subunits during growth and development. Cells respond to nutritional starvation by undergoing a series of morphological changes coordinated with transcriptional changes giving rise to a terminally differentiated structure referred to as fruiting body which has live spores suspended on top of stalk of dead cells. Though starvation-induced development is accompanied by differential expression of genes, few studies related to the transcription machinery, RNA polymerase II have been reported so far. Purification and presence of all three RNA polymerases from D. discoideum had been reported earlier but the details of their structures and regulation have not been explored in detail (Pong and Loomis, 1973; Renart et al., 1985). One interesting observation reported by Lam and colleagues (Lam et al., 1992) was that CTD of the largest subunit of RNA polymerase II, Rpb1, is highly conserved with 24 heptapeptide repeats and expression of RPB1 transcript was regulated during development. Thus, we carried out experiments to characterize Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II to understand any role of these subunits during development. Identification of Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II To identify the homologs of S. cerevisiae Rpb4 and Rpb7 in D. discoideum, we employed bioinformatics and genetic approaches. Firstly, we searched D. discoideum database for all protein sequences of S. cerevisiae RNA polymerase II subunits. We could obtain sequences homologous to all twelve subunits in D. discoideum. Among the 12 subunits of D. discoideum RNA polymerase II, we chose to characterize two subunits, DdRpb4 and DdRpb7. We cloned the open reading frames of these two genes from D. discoideum Ax2 cells and cloned them in yeast expression vectors for complementation studies. In S. cerevisiae, Rpb4 is a non-essential protein but rpb4∆ cells show abnormal phenotypes. Few phenotypes of rpb4∆ cells, such as temperature sensitivity, defective in response to nutritional starvation and defective in activated transcription, were employed to identify the D. discoideum homolog of ScRpb4 (Woychik and Young, 1989; Pillai et al., 2001: Pillai et al., 2003). We observed that DdRPB4 can rescue temperature sensitivity corroborated with its ability to activate transcription from HSE containing promoters and sporulation defects of Scrpb4Δ mutant to the wild type. However, DdRPB4 can rescue neither the defect in activated transcription of GAL10 and INO1 promoters nor the elongated morphology exhibited by Scrpb4Δ mutant. On the other hand, we observed that DdRPB7 can complement the lethality associated with ScRPB7 deletion and can partially rescue the phenotypes associated with Scrpb4∆ strain similar to ScRPB7 (Sharma and Sadhale, 1999; Singh et al., 2004). Taken together, we have identified D. discoideum Rpb4 and Rpb7 as bona fide homologs of S. cerevisiae Rpb4 and Rpb7, respectively. Analysis of Rpb4 and Rpb7 in D. discoideum Since yeast RNA polymerase II subunits, Rpb4 and Rpb7, play an important role in the regulation of genes responsive to starvation stress, we carried out experiments to characterize Rpb4 and Rpb7 during growth and starvation-induced development in D. discoideum. Temporal and spatial expression profiles show avaried but similar pattern of RPB4 and RPB7 transcripts during D. discoideum development. We observed similarity between ScRpb4 and DdRpb4 in its ability to interact with DdRpb7 and to localise in both nuclear and cytoplasmic compartments. Attempts to knock out or reduce the levels of DdRpb4 and DdRpb7 by homologous recombination and antisense approaches, respectively, failed. However, since altering levels of Rpb4 and Rpb7 in S. cerevisiae can affect different stress response pathways, we had used overexpression to alter the level of Rpb4 and analysed its effect on D. discoideum development. We overexpressed DdRpb4 as GFP fusion protein in Ax2 cells and observed that D. discoideum cells overexpressing DdRpb4 showed normal growth and development similar to the wild type protein. Interestingly, we observed that Ax2 cells overexpressing DdRpb4 have drastically reduced levels of the endogenous protein. Thus, we have identified a post-transcriptional control on the level of Rpb4 in D. discoideum. Role of S. cerevisiae Rpb4/Rpb7 subcomplex in stress In S. cerevisiae, Rpb4 and Rpb7 interact with each other and carry out important functions (Choder, 2003; Sampath and Sadhale, 2004). Employing the functional conservation of Rpb4 and Rpb7 across various model systems, we further investigated the role of the subcomplex in S. cerevisiae. Since Rpb7 is an essential gene, we have generated rpb7Δstrain in the presence of plasmids expressing Rpb7 or its homologs. We have generated a S. cerevisiae strain lacking both RPB4 and RPB7 and introduced Rpb4 and Rpb7 homologs from either D. discoideum or C. albicans. We analysed these strains under stresses such as high temperature and nutrient starvation. The results of these experiments have provided how the differences in Rpb4 and Rpb7 proteins and their ability to form a subcomplex could be reflected in differential stress responses. Besides the high functional conservation of these proteins, their interaction with other regulatory proteins might also be critical for a proper response to nutritional stress.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 201-225). Also available on the Internet.