FAM49 - A novel regulator of the protrusive behaviour and motility of cells

Most eukaryotic cell motility relies on plasma membrane protrusions, which depend on the actin cytoskeleton and its tight regulation. The SCAR/WAVE complex, a pentameric assembly comprising SCAR/WAVE, Nap1, CYFIP/Pir121, Abi and HSPC300, is a key driver of actin-based protrusions such as pseudopods. SCAR/WAVE is thought to activate the Arp2/3 complex, a crucial actin nucleator, after being itself activated by upstream signals such as active Rac1. Despite recent progress on the study of the SCAR/WAVE complex, its regulation is still incompletely understood, with Nap1’s role being particularly enigmatic. Upon screening for potential Nap1 binding partners in the social amoeba Dictyostelium discoideum – a well established model organism in the study of the actin cytoskeleton and cell motility – we found FAM49, a ~36 kDa protein of unknown function which is highly conserved in Metazoa (animals) and evolutionarily closer species such as D. discoideum. Interestingly, D. discoideum’s FAM49 and its homologs contain a DUF1394 domain, which is also predicted in CYFIP/Pir121 proteins and most likely involved in their direct binding to active Rac1, which in turn contributes to SCAR/WAVE’s activation. FAM49’s unknown role, apparent high degree of conservation and potential connections to SCAR/WAVE and Rac1 persuaded us to start investigating its function and biological relevance in D. discoideum, leading to the work presented in this thesis. Several pieces of our data collectively support a function for FAM49 in modulating the protrusive behaviour, and ultimately motility, of D. discoideum cells, as well as a regulatory link between FAM49 and Rac1. FAM49’s involvement in protrusion regulation was first hinted at by our observation that GFP-tagged FAM49 is enriched in pseudopods. The possibility of a link with Rac1 was then strengthened by two additional observations: first, pseudopodial GFP-FAM49 is substantially co-enriched with active Rac, both showing fairly comparable spatio-temporal accumulation dynamics; second, when dominant-active (G12V) Rac1 is expressed in cells, it triggers the recruitment and persistent accumulation of GFP-FAM49 at the plasma membrane, where both become highly co-enriched. We subsequently determined that fam49 KO cells differ from wild-type cells in the way they protrude and move, as assessed in under-agarose chemotaxis assays. In particular, our data indicate that fam49 KO cells tend to display a lower degree of global protrusive activity, their protrusions extend more slowly and are less discrete, and the cells end up moving at lower speeds and with higher directional persistence. This phenotype was substantially rescued by FAM49 re-expression. While re-expressing FAM49 in fam49 KO cells we generated putative FAM49 overexpressor cells; compared to wild-type cells, they displayed atypically thin pseudopods and what seemed to be an excessively dynamic, and perhaps less coordinated, protrusive behaviour. Additional data in our study suggest that pseudopods made by fam49 KO cells are still driven by SCAR/WAVE, which is clearly not being replaced by WASP (as is now known to be the case in D. discoideum cells lacking a functional SCAR/WAVE complex). Nonetheless, the peculiar dynamics of those pseudopods imply that SCAR/WAVE’s activity is regulated differently when FAM49 is lost, though it remains to be determined how. This thesis is the first report of a dedicated study on FAM49 and lays the foundation for future research on it.

Shakespeare at the Festival d'Avignon : the Poetics of Adaptation of L. Lagarde (Richard III, 2007), T. Ostermeier (Hamlet, 2008) and I. van Hove (The Roman Tragedies, 2008)

International audience

Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies

International audience

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.

Forms of the Postmodern Historical Novel : Christoph Ransmayr, Daniel Kehlmann, Wolfgang Hildesheimer

L’émergence du postmodernisme aux Etats-unis, mouvement esthétique rejetant les dogmes modernistes, date des années 1960. En s’imposant, durant les dernières décennies du 20ème siècle, comme le paradigme esthétique de l’architecture, des arts et de la littérature, le postmodernisme a également créé les conditions propres à une renaissance du roman historique. Cependant, la fiction historique postmoderne constitue maintenant une nouvelle forme du genre basée sur la parodie, l’ironie et le scepticisme envers les discours dominants. Cette nouvelle forme ne se limite plus à la présentation des récits dans un cadre historique réaliste. Elle remet plutôt en question la validité et par conséquent la nature même du discours historique, problématisant et mettant ainsi à l’avant le processus d’interprétation et de reconstruction du passé. Dans cette optique, la fiction historique contemporaine reflète les débats actuels sur les formes de l’historiographie, débats lancés par Hayden White. Dans les années 1980, la fiction historique a de nouveau fleuri dans l'espace culturel allemand tout comme ailleurs. Le présent mémoire analyse des formes postmodernes de la fiction historique en se basant sur trois romans historiques de langue allemande parus entre 1981 et 2005: Die Schrecken des Eises und der Finsternis de Christoph Ransmayr, Die Vermessung der Welt de Daniel Kehlmann et Marbot : Eine Biographie de Wolfgang Hildesheimer. L’analyse s'appuie sur divers modèles de la fiction postmoderne, en particulier sur le schéma de catégorisation du roman historique élaboré par Ansgar Nünning. Le mémoire montre dans quelle mesure ces romans appliquent des moyens stylistiques typiques pour le postmodernisme et portent un regard critique ou comique sur l’histoire et la culture allemande et autrichienne.

Forms of the Postmodern Historical Novel : Christoph Ransmayr, Daniel Kehlmann, Wolfgang Hildesheimer

L’émergence du postmodernisme aux Etats-unis, mouvement esthétique rejetant les dogmes modernistes, date des années 1960. En s’imposant, durant les dernières décennies du 20ème siècle, comme le paradigme esthétique de l’architecture, des arts et de la littérature, le postmodernisme a également créé les conditions propres à une renaissance du roman historique. Cependant, la fiction historique postmoderne constitue maintenant une nouvelle forme du genre basée sur la parodie, l’ironie et le scepticisme envers les discours dominants. Cette nouvelle forme ne se limite plus à la présentation des récits dans un cadre historique réaliste. Elle remet plutôt en question la validité et par conséquent la nature même du discours historique, problématisant et mettant ainsi à l’avant le processus d’interprétation et de reconstruction du passé. Dans cette optique, la fiction historique contemporaine reflète les débats actuels sur les formes de l’historiographie, débats lancés par Hayden White. Dans les années 1980, la fiction historique a de nouveau fleuri dans l'espace culturel allemand tout comme ailleurs. Le présent mémoire analyse des formes postmodernes de la fiction historique en se basant sur trois romans historiques de langue allemande parus entre 1981 et 2005: Die Schrecken des Eises und der Finsternis de Christoph Ransmayr, Die Vermessung der Welt de Daniel Kehlmann et Marbot : Eine Biographie de Wolfgang Hildesheimer. L’analyse s'appuie sur divers modèles de la fiction postmoderne, en particulier sur le schéma de catégorisation du roman historique élaboré par Ansgar Nünning. Le mémoire montre dans quelle mesure ces romans appliquent des moyens stylistiques typiques pour le postmodernisme et portent un regard critique ou comique sur l’histoire et la culture allemande et autrichienne.

A novel non-peptidic agonist of the ghrelin receptor with orexigenic activity in vivo

Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand.

Soluble fish : picture words in a limitless sea — surrealist poetics and a history of the unconscious

This article discusses the Surrealist text 'Soluble Fish' by Andre Breton against prevailing and dominant paradigms of language and the unconscious. Drawing on the theories of Freud, Jacques Lacan, Gilles Deleuze and Felix Guattari, it considers desire as ‘lack’ versus desire as a productive force entirely necessary for life and ‘becoming’. In addition, using an extract of my own creative practice, I propose the taxonomy of ‘new Surrealism’—a contemporary interpretation of the Surrealist’s productive force, also known as the ‘Marvellous.’

The poetics of collaboration

The question of collaboration is one that arguably can't be ignored in contemporary academia, creative fields, or current philosophical and critical landscapes. The word ‘collaboration’ at once brings to mind the conspiratorial nature of crime as well as the cooperative nature of teamwork and the harmonious meeting of minds and practices. It is, then, a slippery word, and for this reason serves as a fertile provocation for the inquiries unpacked and developed in this special issue of Axon.

The development of 10 novel polymorphic microsatellite markers through next generation sequencing and a preliminary population genetic analysis for the endangered Glenelg spiny crayfish, Euastacus bispinosus

The Glenelg spiny crayfish, Euastacus bispinosus, is an iconic freshwater invertebrate of south eastern Australia and listed as 'endangered' under the Environment Protection and Biodiversity Conservation Act 1999, and 'vulnerable' under the International Union for Conservation of Nature's Red List. The species has suffered major population declines as a result of over-fishing, low environmental flows, the introduction of invasive fish species and habitat degradation. In order to develop an effective conservation strategy, patterns of gene flow, genetic structure and genetic diversity across the species distribution need to be clearly understood. In this study we develop a suite of polymorphic microsatellite markers by next generation sequencing. A total of 15 polymorphic loci were identified and 10 characterized using 22 individuals from the lower Glenelg River. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.80; mean expected heterozygosity = 0.36) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. Individuals from two additional sites (Crawford River, Victoria; Ewens Ponds Conservation Park, South Australia) were genotyped at all 10 loci and a preliminary investigation of genetic diversity and population structure was undertaken. Analyses indicate high levels of genetic differentiation among sample locations (F ST = 0.49), while the Ewens Ponds population is genetically homogeneous, indicating a likely small founder group and ongoing inbreeding. Management actions will be needed to restore genetic diversity in this and possibly other at risk populations. These markers will provide a valuable resource for future population genetic assessments so that an effective framework can be developed for implementing conservation strategies for E. bispinosus.

Isolation and polyphasic characterization of a novel hyper catalase producing thermophilic bacterium for the degradation of hydrogen peroxide.

A newly isolated microbial strain of thermophilic genus Geobacillus has been described with emphasis on polyphasic characterization and its application for degradation of hydrogen peroxide. The validation of this thermophilic strain of genus Geobacillus designated as BSS-7 has been demonstrated by polyphasic taxonomy approaches through its morphological, biochemical, fatty acid methyl ester profile and 16S rDNA sequencing. This thermophilic species of Geobacillus exhibited growth at broad pH and temperature ranges coupled with production of extraordinarily high quantities of intracellular catalase, the latter of which as yet not been reported in any member of this genus. The isolated thermophilic bacterial culture BSS-7 exhibited resistance against a variety of organic solvents. The immobilized whole cells of the bacterium successfully demonstrated the degradation of hydrogen peroxide (H2O2) in a packed bed reactor. This strain has potential application in various analytical and diagnostic methods in the form of biosensors and biomarkers in addition to applications in the textile, paper, food and pharmaceutical industries.

Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.