Tracking North Pacific Humpback Whales To Unravel Their Basin-Wide Movements

In 2014, Oregon State University (OSU) initiated a multi-year project to study humpback whale (Megaptera novaeangliae) migrations in the North Pacific Ocean using satellite tracking technology in combination with genetic and photo-identification (photo-ID) analyses. The study is highly relevant to management, given the need for new information arising from the recent separation of humpback whales into Distinct Population Segments (DPS) for listing under the US Endangered Species Act, including four DPSs in the North Pacific (“Western North Pacific”, “Hawaii”, “Mexico”, and “Central America”) with different conservation statuses. The project’s objective was to conduct a comprehensive characterization of humpback whale movements during breeding, migration, and feeding periods by tagging animals in both a feeding area (southeastern Alaska) and a breeding area (Hawaii). In order to obtain representative results, the sampling plan called for two field efforts at each site, with Pacific Life Foundation funding the southeastern Alaska portion of the project (2014 and 2015 seasons), and the Hawaii portion being cost-shared through a combination of sources including the Makana Aloha Foundation (2015 season) and the US Department of the Navy (2018 season). This final report provides the combined results and accomplishments from these efforts. Argos-based, fully implantable tags were deployed on 37 humpback whales in Seymour Canal and Frederick Sound, southeastern Alaska, in 2014 and 2015. Tracking periods ranged from 3.3 to 78.3 d (mean = 28.2 d, sd = 16.2 d), with distances traveled ranging from 73 to 6,503 km (mean = 2,010 km, sd = 1,649 km). The tracked locations for these animals ranged over 40 degrees of latitude, from Lynn Canal and Icy Strait (59°N) in southeastern Alaska to the southern tip of Hawaii Island (19°N) in the Hawaiian Archipelago. Genetic and photo-ID analyses revealed that two of the whales tagged in 2014 were re-tagged in 2015, providing a unique opportunity to compare movements between years for the same individuals. For one of these animals the movements and their timing were similar between years, as it moved from Seymour Canal into Frederick Sound with a difference of 4 d between years. However, early failure of the tag in 2014 (after 6.2 d) prevented a longer comparison. In contrast, the movements of the second animal within southeastern Alaska were similar but the timing was very different between the two years, despite a similar tracking period (21.9 d in 2014 versus 19.1 d in 2015). In 2014 this animal spent a substantial amount of time in Seymour Canal (17 d) before moving into Stephens Passage for the remainder of its tracking period, while in 2015 the animal moved into Stephens Passage soon after tagging and only for a brief period before it moved into Frederick Sound, from where it initiated the migration toward Hawaii. Differences in timing notwithstanding, the similarities in the tracks between years for both animals provided some evidence of route fidelity, as has been recently shown for several species of migratory marine animals. Twenty of the whales tagged in southeastern Alaska began their winter migration to a low-latitude breeding area, with start dates ranging from 19 November to 6 January. Three of these whales were tracked to breeding areas, two to Hawaii and one to the Mexican mainland. Another 16 whales were headed in the direction of Hawaii and one in the direction of Mexico when their tags quit. The duration and distance spent on migration for the three animals that reached a breeding area ranged from 29 to 46 d and from 4,200 to 4,700 km, respectively. The two animals that arrived in Hawaii entered the archipelago at Hawaii Island. Forty-five tags were deployed on humpback whales off Maui, Hawaii, in 2015 and 2018. Two of these tags provided no locations. Tracking periods for the remaining 43 whales ranged from 0.1 to 147.2 d (mean = 20.8 d, sd = 29.0 d), with distances ranging from 13 to 11,302 km (mean = 1,217 km, sd = 2,348 km). The tracked locations for these animals ranged over 43 degrees of latitude, from the south coast of Maui (21°N) to the Bering Sea (64°N). While in Hawaiian waters, the majority of locations were in the Maui Nui region (the waters between the islands of Maui, Lanai, Molokai and Kahoolawe), during both in 2015 and 2018. Penguin Bank was another area heavily frequented by the tagged whales. Most tagged whales moved in a predominant northwesterly direction after tagging, with animals leaving Maui headed for Lanai, Molokai, and/or Penguin Bank. Several whales were also tracked to Oahu, and one whale was further tracked to both Kauai and Niihau. Only one whale was tracked southeast to Hawaii Island in 2015, but other tagging studies have documented eastward movements to Oahu, Penguin Bank, and Maui Nui, so it is apparent that whales may move extensively between islands, both in westerly and easterly directions. Nine of the whales tagged in Hawaii began their migration to a high-latitude feeding area, with departure dates ranging from 29 January to 11 April. Four of these whales were tracked to feeding areas, three to northern British Columbia and one to the eastern Aleutian Islands. Another four whales were headed on a northeasterly trajectory toward northern British Columbia and three more on a northerly or northwesterly trajectory toward destinations in the Aleutian Island chain when their tags quit. The three whales that were tracked to northern British Columbia arrived in the Haida Gwaii Archipelago after having spent 30-44 d and 4,300-5,000 km on migration. The animal that migrated north to the eastern Aleutians arrived at an area approximately 200 km south of Unimak Pass, 28 d and 3,775 km after departing Hawaii. These results, together with those obtained from animals tagged in southeastern Alaska that migrated to a breeding area (Hawaii or Mexico), provide evidence that the travel time and distance covered by humpback whales while on migration across the North Pacific Basin can vary widely, with overall ranges of 28-46 d and 3,775-5,000 km, respectively. A 50-km buffer zone around southeastern Alaska and Hawaii was used for purposes of characterizing whale movement speeds and residence times in the feeding and breeding areas (inside the buffer zones), as well as during migration (outside the buffer zones). Residence time was computed as the time period from tag deployment to when a whale crossed the buffer zone boundary as it departed on migration. Residence time in southeastern Alaska in late fall was estimated for 20 whales, ranging from 4.4 to 49.1 d (mean = 17.3 d), although additional information from earlier tagging studies indicated that individual humpback whales may use this feeding area for periods of up to four to five months. In contrast, residence time in Hawaii was estimated for nine whales, ranging from 3.3 to 23.2 d (mean = 14.8 d), consistent with earlier photo-ID and telemetry studies and lending support to the notion that that there is a rapid turnover of individuals in this breeding area during the winter season. In any case, overall true residence time in these areas is likely longer than the minimum values we report based on satellite telemetry, as we cannot know the time a whale had spent in an area prior to tagging. Movement speeds during the different phases of the migration (feeding, breeding, migrating) were calculated based on the portions of the tracks that occurred inside or outside the 50-km buffer zones. Whales tagged in southeastern Alaska moved at a mean speed of 1.01 km/h (median = 0.47 km/h, sd = 1.28 km/h) while in the southeastern Alaska feeding area; 5.51 km/h (median = 5.63 km/h, sd = 1.98 km/h) while migrating; and 1.49 km/h (median = 1.01 km/h, sd = 1.36 km/h) once they arrived in the Hawaii breeding area. Whales tagged in Hawaii moved at a mean speed of 1.36 km/h (median = 1.00 km/h, sd = 1.21 km/h) while in the Hawaii breeding area; 4.44 km/h (median = 4.32 km/h, sd = 2.18 km/h) while migrating; and 2.00 km/h (median = 1.53 km/h, sd = 1.53 km/h) once they arrived in the southeastern Alaska feeding area. These results showed that whales moved much slower while in the feeding and breeding areas than while migrating, and that travel speed from the feeding to the breeding areas was somewhat faster than from the breeding to the feeding areas. Biopsy samples were collected from 27 of the whales tagged in southeastern Alaska in 2014 and 2015, and from 39 of the whales tagged in Hawaii in 2015 and 2018. These 66 samples were identified by a unique multi-locus genotype of at least 14 microsatellite loci, which indicated they represented 64 unique individuals (after accounting for the two animals that were re-tagged). The 25 individuals tagged in southeastern Alaska represented 14 females and 11 males. The 39 individuals tagged in Hawaii represented four females and 35 males. The DNA profiles of the 64 individuals were compared to a reference database of 1,805 individuals sampled from 2004 to 2006 in the North Pacific by the program SPLASH, which revealed nine matches (i.e., genotype recaptures). Of these, six matches were recaptures within an area (four within southeastern Alaska and two within Hawaii) and three were recaptures between whales tagged in Hawaii and sampled previously on feeding areas in either northern British Columbia (n = 2) or southeastern Alaska (n = 1). Mitochondrial deoxyribonucleic acid (mtDNA) sequences of the 64 individuals resolved seven haplotypes for the consensus region of 500 base-pairs. All seven haplotypes had been previously described for North Pacific humpback whales by SPLASH, but only two occurred in the southeastern Alaska samples while all seven occurred in the Hawaii samples, supporting earlier results indicating a greater haplotypic diversity in the Hawaii breeding area than in the southeastern Alaska feeding area. Further, pairwise tests of differentiation between the tagging areas and the 18 SPLASH regional strata were consistent with those reported in that study, supporting our current understanding of humpback whale population structure, migratory destinations, and site fidelity in the North Pacific. Photo-IDs (fluke photographs) were obtained from 30 whales tagged in southeastern Alaska and from 24 whales tagged in Hawaii. Comparisons with the online Happywhale photo-ID database as well as with OSU’s own ID catalog revealed matches for 25 of the tagged whales (18 from southeastern Alaska and seven from Hawaii). Thirty-five percent of the tagged whales with an ID were found in Happywhale and 13 percent in OSU’s catalog. Most matches (19 of 25) were made within the same area in which the whale was tagged, with time spans between sightings of up to 14 years. Two whales tagged in southeastern Alaska in 2014 each had only one photo-ID match in a different area than the one in which they were tagged. Both had been previously photographed in Hawaii, one in 1997 (17 years apart) and in 2004 (10 years apart). The remaining four resighted tagged whales had both within- and between-area matches. Three of these latter whales were tagged in southeastern Alaska, with two of them matching sightings in Hawaii (1987 and 2019, respectively), and the third one being resighted in central California on two consecutive years (2017 and 2018). The fourth whale was tagged in Hawaii and matched sightings over six consecutive years (2013-2018) in southern British Columbia/northern Washington. An additional 26 matches were found in Happywhale from among 149 fluke photographs of untagged whales collected by OSU in Hawaii. Of these, 13 matches were made within Hawaii (with a maximum time span between sightings of 21 years); nine matches were made between Hawaii and different parts of Alaska, including southeastern Alaska, Kodiak Island, Cook Inlet, and the Shumagin Islands; four matches were made between Hawaii and Washington State and Vancouver Island, British Columbia; and one match was made between Hawaii and the Chukchi Sea, near Kolyuchin Island, northeastern Russia. Through the combined use of satellite tagging, genetics, and photo-ID, we characterized the patterns of humpback whale occupation in both a breeding and a feeding area in the North Pacific Ocean, as well as the long-distance migratory movements that these animals undertake seasonally between these areas. The results of this study revealed the complex migratory linkages between Hawaii and the high-latitude feeding areas with unprecedented detail. Genotype and photo-ID recaptures of multiple individuals between migratory destinations supported previously known strong connections between breeding and feeding areas (e.g., Hawaii and southeastern Alaska/northern British Columbia, and Hawaii and Washington/southern British Columbia). Satellite tracking also revealed the movements and migratory connections between Hawaii and feeding areas in the Aleutian Islands and the Bering Sea, while photo-ID recaptures demonstrated additional connections between Hawaii and feeding areas in the northern Gulf of Alaska (Shumagin Islands, Kodiak Island, Cook Inlet) and the Chukchi Sea. Additional years of sampling during different parts of the reproductive season and in other parts of the main Hawaiian islands (e.g., Kauai and Hawaii), as well as in the northwestern Hawaiian Islands, would provide valuable information to address outstanding questions about the humpback whale DPS using this extensive breeding area, as well as its broader connections to remote feeding areas throughout the North Pacific Basin, most of which are poorly known. Also, while the majority of whales tracked from southeastern Alaska showed a strong connection to the Hawaii breeding area, a small proportion of these animals demonstrated a connection to the Mexican mainland breeding area, indicating some mixing of the Hawaii and Mexico DPSs in the southeastern Alaska feeding area. These animals are of particular interest, as in their transit along the western coast of North America they overlap with animals from the Central America DPS, which forages off California and Oregon. Further tagging work to better understand the patterns of habitat use and the extent of the overlap between the Mexico and Central America DPSs in this region would greatly help current needs to improve how animals are assigned to DPS for management purposes in the context of relative exposure to anthropogenic activities, given their different conservation statuses

Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

A genome-scale specificity and interaction map for yeast SH3 domain-containing proteins reveal how family members show selective binding to target proteins and predicts the dynamic localization of new candidate endocytosis proteins

ETUDE FONCTIONNELLE DE LA PROTEINE APPARENTEE A L'ACTINE ARP5 ET D'UN DE SES PARTENAIRES CELLULAIRES AFILP, DANS LA LEVURE SACCHAROMYCES CEREVISIAE

CE TRAVAIL DE THESE PORTE SUR LA CARACTERISATION FONCTIONNELLE DE LA PROTEINE ARP5. LE GENE ARP5 (YNL059C) A ETE IDENTIFIE AU COURS DU SEQUENCAGE SYSTEMATIQUE DU GENOME DE LA LEVURE S. CEREVISIAE EN 1995. IL CODE POUR UNE PROTEINE HYPOTHETIQUE DE 755 AA QUI PRESENTE 26% D'IDENTITE ET 51% DE SIMILARITE A L'ACTINE. DES SOUCHES DE LEVURE DELETEES DU GENE ARP5 MONTRENT QU'IL EST TRES IMPORTANT VOIRE ESSENTIELLE POUR LEUR CROISSANCE CELLULAIRE (SELON LEUR CONTEXTE GENETIQUE). LES MUTANT ARP5-D, AINSI QUE LES MUTANTS PONCTUELS ARP5-TS, PRESENTENT DES PHENOTYPES PLEIOTROPES (INCAPACITE A METABOLISER DIFFERENTES SOURCES DE CARBONE, FRAGILITE DE PAROI CELLULAIRE, ETC). L'INTEGRATION D'UNE FUSION ARP5-GFP (GREEN FLUORESCENT PROTEIN) AU LOCUS CHROMOSOMIQUE ARP5 A PERMIS DE MONTRER QUE LA PROTEINE ARP5 EST NUCLEAIRE TOUT AU LONG DU CYCLE CELLULAIRE. AFIN DE MIEUX APPREHENDER SES FONCTIONS NUCLEAIRES, NOUS AVONS RECHERCHE DES PARTENAIRES FONCTIONNELS OU PHYSIQUES D'ARP5P PAR L'INTERMEDIAIRE DE CRIBLES DE SUPPRESSEURS-MULTICOPIES ET DE DOUBLE-HYBRIDES RESPECTIVEMENT. LA PROTEINE JUSQU'ALORS INCONNUE AFILP (ARP-FIVE-INTERACTING) EST LA SEULE A AVOIR ETE SELECTIONNEE DANS LES DEUX CRIBLES. LA PROTEINE DE FUSION AFIL-GFP EST LOCALISEE DANS LE NOYAU TOUT AU LONG DU CYCLE. LA DIVERSITE DES PHENOTYPES OBSERVES POUR LES MUTANTS ARP5 ET AFIL NOUS A SUGGERE L'IMPLICATION D'ARP5P ET AFILP DANS UNE FONCTION NUCLEAIRE GENERALE TELLE QUE LA TRANSCRIPTION OU LA MATURATION DES ARNM. DES MARQUAGES IMMUNOLOGIQUES SUR CHROMOSOMES ONT MONTRE QU'ARP5P ET AFILP SE FIXENT A LA CHROMATINE SOUS FORME DE POINTS BIEN PRECIS (ENV 15 A 60 PAR NOYAU). PAR AILLEURS, LA CARACTERISATION D'UN PHENOTYPE SPT (SUPPRESSOR OF TY) DANS LES MUTANTS ARP5-D SUGGERE QU'ARP5P EST IMPLIQUEE PLUS PRECISEMENT DANS LA REGULATION DE LA STRUCTURE CHROMATINIENNE. DE PLUS, LA SENSIBILITE DES MUTANTS ARP5 ET AFIL A DES AGENTS DOMMAGEABLES POUR L'ADN INDIQUE QU'ARP5P ET AFILP POURRAIENT ETRE IMPLIQUEES DANS LA REPARATION DE L'ADN. LES RESULTATS DECRITS DANS CE TRAVAIL MONTRE QU'ARP5P ET AFILP INTERAGISSENT PHYSIQUEMENT POUR REMPLIR UNE FONCTION COMMUNE. ILS CONVERGENT VERS L'IMPLICATION D'ARP5P DANS LE CONTROLE TRANSCRIPTIONNEL OU LA REPARATION DE L'ADN VIA LE REMODELAGE DE LA CHROMATINE.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Evolution des réseaux d'interaction des domaines protéiques SH3 dans la levure (Fonctions et interactions d'une famille de protéines composée de Lsb1 et Lsb2)

Chez S. cerevisiae, les nutriments, les lipides et les protéines membranaires sont internalisés par endocytose. Ce processus requiert des filaments d actine dynamiques. Un facteur clé pour la polymérisation de l actine est le nucléateur Arp2/3, activé par des facteurs de promotion de la nucléation (NPF). Pendant l endocytose, la polymérisation de l actine est initiée, entre autre, par les NPFs Las17 et les myosines de type I. Nous avons caractérisé deux protéines, Lsb1 et Lsb2 qui lient Las17 par leurs domaines SH3 et qui inhibent in vitro la polymérisation de l actine dépendante du complexe Arp2/3. Lsb1 et Lsb2 colocalisent avec des protéines du cytosquelette, Las17, Abp1 et Sla1 et influencent la stabilité de Las17 in vivo. Nous avons ainsi identifié deux nouveaux régulateurs négatifs de l activité NPF de Las17 ce qui permet de mieux comprendre son rôle dans la polymérisation de l actine et l endocytose. Nous avons également caractérisé chez des levures S. cerevisiae, A. gossypii, C. albicans et S. pombe des réseaux d interactions protéine-protéine à travers des spécificités de liaison des domaines SH3 par la technique SPOT. Une conservation de la spécificité des myosines de type I, connu par ailleurs, a été montrée par analyse bioinformatique et valide notre méthode. De plus, nous montrons que la fonction de la myosine I de recruter la machinerie de polymérisation dans un extrait de S. cerevisiae est conservée de S. cerevisiae à A. gossypii. Nous faisons des analyses similaires pour de nombreuses autres protéines à domaine SH3 ce qui nous permettra de prédire des interactions protéines-protéines ainsi de mieux comprendre l évolution des réseaux d interaction protéique.In S. cerevisiae, nutrients, lipids and membrane proteins are internalized by endocytosis. These processes require dynamic actin filament assembly. A key factor for actin polymerization is the nucleating complex Arp2/3 that is activated by nucleation promoting factors (NPF). During the process of endocytosis, a strong NPF activity is exhibited by Las17, the unique S. cerevisiae homolog of WASP and the type I myosin, Myo5, among others. Here, we characterize two SH3 domain containing proteins Lsb1 and Lsb2. We could show that both proteins bind to the proline rich sequence of the NPF Las17 via their SH3 domains and efficiently inhibit Las17 induced Arp2/3-dependent actin polymerization in vitro. We could also show that Lsb1 and Lsb2 partially colocalize with Las17 and that they influence the stability of Las17 in vivo. However, we did not detect any defect in endocytosis for the single or double deletion mutants of LSB1 and LSB2. In conclusion, we identified two new negative regulators of the NPF activity of Las17 that will help us to further understand actin nucleation and endocytosis. The characterization of the SH3 domain binding specificity in four yeast species S. cerevisiae, A. gossypii, C. albicans and S. pombe showed high conservation of the type I myosin specificity during evolution, validating our method. The ability of type I myosin to recruit the actin polymerization machinery in S. cerevisiae is conserved from S. cerevisiae to A. gossypii. Similar analysis of numerous other SH3 domains, including Lsb1 and Lsb2, is in progress, which will allow us to predict new protein-protein interactions and to gain insights into the evolution of protein interaction networks.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Testing the effectiveness of an acoustic deterrent for gray whales along the Oregon coast

This study was conducted to determine whether a low-powered sound source could be effective at deterring gray whales from areas that may prove harmful to them. With increased interest in the development of marine renewal energy along the Oregon coast the concern that such development may pose a collision or entanglement risk for gray whales. A successful acoustic deterrent could act as a mitigation tool to prevent harm to whales from such risks. In this study, an acoustic device was moored on the seafloor in the pathway of migrating gray whales off Yaquina Head on the central Oregon coast. Shore-based observers tracked whales with a theodolite (surveyor’s tool) to accurately locate whales as they passed the headland. Individual locations of different whales/whale groups as well as tracklines of the same whale/whale groups were obtained and compared between times with the acoustic device was transmitting and when it was off. Observations were conducted on 51 d between January 1 and April 15, 2012. A total of 143 individual whale locations were collected for a total of 243 whales, as well as 57 tracklines for a total of 142 whales. Inclement weather and equipment problems resulted in very small sample sizes, especially during experimental periods, when the device was transmitting. Because of this, the results of this study were inconclusive. We feel that another season of field testing is warranted to successfully test the effectiveness of the deterrent, but recommend increasing the zone of influence to 3 km to ensure the collection of adequate sample sizes. Steps have been taken to acquire the necessary federal research permit modification to authorize the increased zone of influence and to modify the acoustic device for the increased power. With these changes we are confident we will be able to determine whether the deterrent is effective at deflecting gray whales. A successful deterrent device may serve as a valuable mitigation tool to protect gray whales, and other baleen whales, in the event that marine energy development poses a collision or entanglement risk

Interactions entre la protéine Hof1 (de la famille PCH) et la protéine Vrp1p/WIP (Wiskott-Aldrich Syndrome Protein interacting protein) dans la régulation de la division cellulaire et du transport membranaire

Chez la levure (Saccharomyces cerevisiae), le phénomène d'endocytose comprend l'endocytose aspécifique (de fluides et de membranes) et l'endocytose dépendant de récepteurs spécifiques (protéines membranaires). Les deux processus nécessitent l'assemblage de filaments d'actine. Les facteurs clefs permettant cette polymérisation sont le complexe Arp2/3 ainsi qu'un certain nombre de facteur de nucléation de la polymérisation de l'actine que l'on nomme les NPFs (pour Nucleation Promoting Factors); les NPFs régulent de façon spatiotemporelle l'activité du complexe Arp2/3. Chez la levure, en plus de Las17p, qui est l'orthologue de la protéine humaine WASP (Wiskott-Aldrich syndrome protein), une myosine non conventionnelle de type I (Myo5p) présente une forte activité NPF lorsqu'elle l'interagit avec le domaine WH2 (WASP Homology 2) de la protéine Vrp1p, l'orthologue levure de la protéine WIP (WASP Interacting Protein). Au laboratoire, nous avons trouvé chez la protéine Vrp1 un autre domaine clef, le domaine HOT (Hof One Trap) qui est important pour la fonction de Vrp1p in vivo. Il fixe directement le domaine SH3 de la protéine de la cytokinese, Hof1p. La fonction du domaine HOT est de contrecarrer l'effet inhibiteur du domaine SH3 de Hof1p dans les processus d'assemblage de l'actine et d'endocytose stimulés par la protéine Myo5p. Un nouveau domaine de liaison aux monomères d'actine a été révélé chez Vrp1p (VH2) qui présente une redondance fonctionnelle avec le domaine WH2. L'endocytose récepteurs spécifiques nécessite une interaction stable entre Vrp1p et Las17p. Pourtant, il s'avère que l'import aspécifique de fluide et de membranes peut se faire sans l'association Vrp1p-Las17p et avec seulement un domaine WH2 et le domaine HOT de Vrp1p. Finalement, en utilisant une approche de purification par affinité nous avons identifié d'autres protéines pouvant interagir avec le domaine SH3 de Hof1p. Ces profils d'interaction ont été comparés avec ceux d'autres domaines SH3 que l'on trouve chez les protéines de la levure. L'interaction unique entre Vrp1p-Hof1p nous permet de mieux appréhender la pathologie du syndrome Wiskott-Aldrich.In the budding yeast Saccharomyces cerevisiae, endocytosis comprises bulk uptake (fluids and membranes) and receptor-mediated internalisation (membrane proteins). Both processes require efficient actin filament assembly. Key factors that nucleate the assembly of actin filaments are the Arp2/3 complex and a number of NPFs (Nucleation Promoting Factors), which are responsible for temporal and spatial regulation of Arp2/3 activity. In yeast, in addition to Las17p, the orthologue of WASP (Wiskott-Aldrich syndrome protein), one type I unconventional myosin (Myo5p) exhibits strong NPF activity through coordination with the WH2 (WASP Homology 2) domain containing protein Vrp1p, the yeast orthologue of WIP (WASP Interacting Protein). Here, we identified another key Vrp1p domain (Hof One Trap/HOT), which binds directly to the SH3 domain of the cytokinesis protein Hof1p, is important for Vrp1p function in vivo. The key function of the Vrp1p HOT domain is to counteract the inhibitory effect of the Hof1p SH3 domain in Myo5p-stimulated actin assembly and endocytosis. We have also revealed a novel actin monomer binding domain (VH2) in Vrp1p, which is functionally redundant with the WH2 domain. Receptormediated endocytosis requires stable interaction of Vrp1p with Las17p. However, we find that bulk uptake of fluid and membrane takes place without Vrp1p-Las17p association and requires only functional WH2 and HOT domains of Vrp1p. Finally, we identified a number of other potential Hof1p SH3 domain interactors using an affinity isolation approach and compared this interaction profile with those of several other yeast SH3 domains. The unique Vrp1p-Hof1p interaction pattern allows us to gain insight into the pathology of Wiskott-Aldrich syndrome.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceAustraliaFRA

Etude du lien entre le cytosquelette d'actine et les peroxysomes dans la levure Saccharomyces cerevisiae (et détermination du rôle intracellulaire de la myotubularine humaine dans le système modèle de levure Saccharomyces cerevisiae)

Chez la levure Saccharomyces cerevisiae Las17p est l homologue de WASp (Wiskott-Aldrich syndrome protein des cellules mammifères), responsable de l activation de la polymérisation d actine dépendante d Arp2/3 qui forme des tâches corticales. Les résultats préliminaires du laboratoire avaient démontré que la peroxine Pex13p est capable d interagir via son domaine Src homology 3 (SH3) avec Las17p, in vitro dans un essai GST pull-down. Cette étude est alors basée sur la recherche d un lien éventuel entre la machinerie de polymérisation d actine et les peroxysomes. La peroxine 13 (Pex13p) comme Pex14p et Pex5p fait partie intégrante du complexe d amarrage à la membrane peroxysomale et est essentielle pour l import dans les peroxysomes. Mes résultats favorisent l hypothèse que le cytosquelette d actine pourrait affecter les peroxysomes non pas directement sur l'import mais à travers un effet sur leur biogénèse, non encore précisé ayant un impact sur leur taille et leur nombre.Le gène hMTM1 sous une forme mutée est responsable de la myopathie myotubulaire liée au chromosome X (XLMTM). Il code pour une phosphatase à phosphoinositides, membre de la famille des myotubularines comprenant un unique homologue levure, Ymr1p. L objet de la deuxième partie de ma thèse était de mieux comprendre la fonction intracellulaire et éventuellement la préférence de substrat de cette enzyme en utilisant le modèle levure S. cerevisiae. Suivant les résultats obtenus, l expression hétérologue de la myotubularine humaine chez la levure S. cerevisiae produit une enzyme active, provoquant l élargissement des vacuoles. Le phénotype observé est dû à l activité phosphatase et donc à la diminution du taux de PtdIns(3,5)P2. Les résultats suggèrent un rôle de hMTM1 dans le trafic membranaire et la maturation des endosomes précoces et tardifs et dans l homéostasie vacuolaire.In yeast Saccharomyces cerevisiae Las17p is the yeast homologue of WASP (Wiskott-Aldrich syndrome protein) in mammalian cells and a principal activator of actin polymerization Arp2/3 dependant that forms actin patches. Preliminary results from a GST pull-down assay in the laboratory had shown that Pex13p was able to interact, via its domain Src homology domain 3 (SH3), with Las17p. This study is based on the search for an eventual link between components of the actin polymerization machinery and peroxisomes. The peroxine Pex13p, together with Pex14p and Pex5p, is a part of the import complex at the peroxysomal membrane. My results favour the hypothesis that the actin cytoskeleton might regulate not the import into peroxisomes directly, but through an effect on peroxysomal biogenesis having an impact on peroxysomal size and number.Mutated forms of the hMTM1 gene are responsible for the X-linked myotubular myopathy (XLMTM) and it encodes a phosphoinositides phosphatase, a member of the myotubularine family, having an unique homologue in yeast S. cerevisiae, Ymr1p. According to the results, heterologous expression of hMTM1 in yeast S. cerevisiae leads to the production of an active enzyme, causing an enlarged vacuole phenotype. This phenotype is due to the phosphatase activity and therefore to the decrease in the level of PtdIns(3,5)P2 . The results suggest a role for hMTM1 in membrane trafficking, in early and late endosome maturation and in maintaining vacuolar homeostasy.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Un fluorophore photoactivable pour des études spatio-temporelles de la dynamique du cytosquelette d'actine (les interactions des protéines à domaine SH3, le cas de Bzz1p)

Le cytosquelette cellulaire est constitué d'un réseau de protéines comme les filaments d'actine. Mon objectif de thèse consistait à développer une sonde fluorescente photoactivable afin d étudier la dynamique des protéines affectant le complexe d activation de la polymérisation de l actine. Une coumarine photoactivable avec un motif Ni-NTA capable de se lier à une étiquette protéique 6xHis a été synthétisée. Les propriétés photochimiques ont été déterminées. Pour une entrée efficace de cette molécule dans des cellules, un acide du motif NTA doit être acétylé. Mes études ont aussi porté sur la dynamique et les interactions fonctionnelles d une protéine, Bzz1p. Cette dernière est une protéine avec deux domaines SH3 et un domaine FCH. Des interactions génétiques et physiques (protéines) ont été recherchées. Des partenaires lipidiques du domaine FCH de Bzz1p ont été identifiés. De nouveaux partenaires suggèrent que Bzz1p pourrait agir au niveau de différentes membranes de la cellule.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF