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Bovine Parvovirus (BPV) belongs to the genus Bocavirus, family Parvoviridae. BPV is the leading etiologic agent among the pathogens that cause primary gastroenteritis of cattle. Many of the intracellular events associated with virus replication are unknown. In this research project, we investigated BPV internalization into the host cell and trafficking in the cytosol. Preliminarily, EBTr cells had abundant clathrin, virus attached to purified clathrin, and EM micrographs revealed virus in endocytic vacuoles. Assays detecting virus infectivity (i.e. viral protein synthesis), virus production (completion of the replication cycle), and quantitative PCR (qPCR) to detect viral transcripts were used to evaluate virus uptake and subsequent trafficking events in the presence of selective inhibitors. Cell toxicity mediated by the drugs was evaluated by the MTT test. Virucidal effects of the drugs were assessed. A control virus was used to verify the inhibitor technology. Immunofluoresceinated virus particles were found in clathrin-rich early endosomes. Clathrin-mediated endocytosis (CME) was examined by clathrin polymerization inhibiting agent (chloropromazine), lysosomotropic agents (ammonium chloride and chloroquine), a vacuolar ATPase inhibitor (bafilomycin A1), and a blocker of transition between endosomes (brefeldin A). Caveosome pathway inhibitors included phorbol 12-myristate 13-acetate (a suppressor of caveolae formation), nystatin and methyl-beta-cyclodextrin (lipid raft blockers), and genistein (a tyrosine kinase phosphorylation inhibitor). Trafficking of BPV was investigated using specific inhibitors of proteasomal activity, actin-myosin function, and microtubule-dynein function. The proteasomal protease suppressor (lactacystin), and a proteasomal chymotrypsin inhibitor (epoxomicin) were used. The role of actin was probed by cytochlasin D, latrunculin A, and ML-7. The microtubule inhibitors nocodazole, vanadate, and EHNA were used to probe microtubule function. The inhibitors of CME reduced virus production and reduced infectivity, a result confirmed by qPCR. The blockers of caveolin-mediated entry did not interfere with virus production nor virus infectivity. Proteasome activity blockage did not affect the virus replication. But the virus cycle was affected by actin blockage and by microtubule blockage detected by qPCR. Taken together these data indicate that BPV uptake is mediated by clathrin coated pits and is acid-dependent. Further processing of BPV in the cytosol does not require proteasomal enzymes. Actin-associated vesicular transport appears to be essential to virus replication and trafficking to the nucleus appears to be mediated by microtubules.

yes / The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6. / British Heart Foundation (grant number PG/10/68/28528; http://www.bhf.org.uk)

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Endocytosis is both an ancient and a diverse feature of the eukaryotic cell. Studying how it evolved can provide insight into the nature of the last common eukaryotic ancestor, and the diversification of eukaryotes into the known extant lineages. In this thesis, I present two studies on the evolution of endocytosis. In the first part of the thesis I report results from a large-scale, phylogenetic and comparative genomic study of clathrin-mediated endocytosis (CME). The CME pathway has been studied to a great level of detail in yeast to mammal model organisms. Several protein families have now been identified as part of the complex set of protein-protein and protein-lipid interactions which mediate endocytosis. To investigate how such complexity evolved, first, I defined the modular nature of the CME interactome (CME-I) by literature review, and then I carried out a systematic phylogenetic and protein domain architecture analysis of the proteins involved. These data were used to construct a model of the evolution of the CME-I network, and to map the expansion of the network's complexity to the eukaryotic tree of life. In the second part of the thesis, I present results from evolutionary and functional studies of the eisosome, a protein complex which has been proposed to regulate the spatial distribution of endocytosis in S. cerevisiae. The phylogeny of eisosomes components Pil1 and Lsp1 reported here, suggests that eisosomes are likely to have originated at the base of the fungi, and then diversified significantly via multiple gene duplications. I thus studied the localisation and function of Pil1 and Lsp1 homologues in Magnaporthe oryzae to investigate the role of eisosomes in filamentous fungi. Results suggests that eisosomes are linked with septal formation and integrity in M. oryzae, and that the septal specific Pil2 paralogue was lost in budding yeasts. Together, the data presented in this thesis describe the evolutionary history of a complex biological system, but also highlights the problem of asymmetry in the understanding of endocytic diversity in the eukaryotes.

Dans les cellules eucaryotes, le trafic intracellulaire de nombreuses protéines est assuré par des vésicules de transport tapissées de clathrine. Les complexes adaptateurs de clathrine (AP) sont responsables de l’assemblage de ces vésicules et de la sélection des protéines qui seront transportées. Nous avons étudié cinq familles atteintes du syndrome neurocutané MEDNIK qui est caractérisé par un retard mental, une entéropathie, une surdité, une neuropathie périphérique, de l’icthyose et de la kératodermie. Tous les cas connus de cette maladie à transmission autosomique récessive sont originaires de la région de Kamouraska, dans la province de Québec. Par séquençage direct des gènes candidats, nous avons identifié une mutation impliquant le site accepteur de l’épissage de l’intron 2 du gène codant pour la sous-unité σ1 du complexe AP1 (AP1S1). Cette mutation fondatrice a été retrouvée chez tous les individus atteints du syndrome MEDNIK et altère l’épissage normal du gène, menant à un codon stop prématuré. Afin de valider l’effet pathogène de la mutation, nous avons bloqué la traduction de cette protéine chez le poisson zébré en injectant une séquence d’oligonucléotides antisenses spécifique à AP1S1. À 48 heures après la fertilisation, les larves knock down pour AP1S1 montrent une réduction de la pigmentation, une désorganisation de la structure de l’épiderme et une perturbation du développement moteur. Alors que la surexpression de l’AP1S1 humain dans ce modèle a permis la récupération du phénotype normal, l’expression de l’AP1S1 mutant fut sans effet sur les phénotypes moteurs et cutanés des larves knock down. Les résultats obtenus montrent que la mutation du AP1S1 responsable du syndrome de MEDNIK est associée à une perte de fonction et que la sous-unité σ1 du complexe AP1 joue un rôle crucial dans l’organisation de l’épiderme et le développement de la moelle épinière. / Intracellular protein transport between organelles is mainly mediated by clathrin coated vesicles. Clathrin adaptor protein (AP) complexes participate in clathrin coated vesicle formation and in sorting protein cargo. We studied 5 families with MEDNIK syndrome, which is characterized by mental retardation, enteropathy, deafness, neuropathy, ichtyosis and keratoderma. All families affected with this autosomal recessive syndrome originate from an isolated population in the Kamouraska region of Quebec. The candidate genes identified in the positive region were sequenced and a founder mutation was identified in the acceptor splice slice of intron 2 of the AP1S1 gene. This gene encodes for the small subunit σ1 of the complex adaptor 1 (AP1). This splicing mutation leads to a premature stop codon, which is predicted to alter the normal function of this protein. To validate the pathogenic effect of this mutation we blocked the AP1S1 protein translation in zebrafish by injecting an anti-sense oligonucleotide designed against AP1S1. At 48 hours post fertilisation, the knockdown larvae showed reduced pigmentation, perturbation of skin formation, and severe perturbation of motor development and function motor development. Over expression of the human AP1S1 rescued the normal phenotype whereas the expression of the mutant AP1S1 did not. These results show that this mutation is causative for MEDNIK syndrome and demonstrates a critical role of the small subunit σ1 in epidermal organisation and in the development of the spinal cord.