Identification of candidate regions for a novel Usher syndrome type II locus

PURPOSE: Chronic diseases affecting the inner ear and the retina cause severe impairments to our communication systems. In more than half of the cases, Usher syndrome (USH) is the origin of these double defects. Patients with USH type II (USH2) have retinitis pigmentosa (RP) that develops during puberty, moderate to severe hearing impairment with downsloping pure-tone audiogram, and normal vestibular function. Four loci and three genes are known for USH2. In this study, we proposed to localize the gene responsible for USH2 in a consanguineous family of Tunisian origin. METHODS: Affected members underwent detailed ocular and audiologic characterization. One Tunisian family with USH2 and 45 healthy controls unrelated to the family were recruited. Two affected and six unaffected family members attended our study. DNA samples of eight family members were genotyped with polymorphic markers. Two-point and multipoint LOD scores were calculated using Genehunter software v2.1. Sequencing was used to investigate candidate genes. RESULTS: Haplotype analysis showed no significant linkage to any known USH gene or locus. A genome-wide screen, using microsatellite markers, was performed, allowing the identification of three homozygous regions in chromosomes 2, 4, and 15. We further confirmed and refined these three regions using microsatellite and single-nucleotide polymorphisms. With recessive mode of inheritance, the highest multipoint LOD score of 1.765 was identified for the candidate regions on chromosomes 4 and 15. The chromosome 15 locus is large (55 Mb), underscoring the limited number of meioses in the consanguineous pedigree. Moreover, the linked, homozygous chromosome 15q alleles, unlike those of the chromosome 2 and 4 loci, are infrequent in the local population. Thus, the data strongly suggest that the novel locus for USH2 is likely to reside on 15q. CONCLUSIONS: Our data provide a basis for the localization and the identification of a novel gene implicated in USH2, most likely localized on 15q

Régulation de la macropinocytose constitutive dans les fibroblastes transformés par les oncogènes

La macropinocytose désigne la formation de grandes vésicules endocytaires primaires de taille et de forme irrégulière, produites par évagination de la membrane péricellulaire, qui permet l'accumulation accélérée de solutés extracellulaires. Il existe une forte similitude entre les macropinosomes et les "phagosomes spacieux" induits par des bactéries entéropathogènes telles que Salmonella et Shigella. Les macropinosomes de forment préférentiellement au front de migration et peuvent fusionner avec les lysosomes ou recycler leur contenu vers le milieu extracellulaire (régurgitation). La transformation de fibroblastes Rat-1 par les oncogènes v-Src ou K-Ras induit la formation constitutive de macropinosomes au niveau des zones du "ruffling". Notre travail s'est intéressé à la dynamique de cette macropinocytose constitutive et à ses régulateurs potentiels. La première partie de ma thèse concerne le sort des macropinosomes dans les fibroblastes transformés par v-Src. Ceux-ci ne fusionnent pas avec les endosomes contenant la transferrine. Pour tester l'effet de l'AMP-cyclique sur cette macropinocytose, nous avons utilisé deux analogues perméants de l'AMPc, le dibutyryl AMP-cylique et le 8-bromo-AMP-cyclique, ainsi qu'un activateur de l'adénylate cyclase, la forskoline. Ces trois agents ralentissent d'environ 35% la vitesse de l'accumulation nette d'un traceur de l'endocytose fluide, la peroxydase de raifort, dans les cellules transformées par v-Src, mais n'ont pas d'effet dans la lignée parentale. Cependant, au contraire d'inhibiteurs de la phospho-inositide 3-kinase (PI3K) ou d'inhibiteurs de la phospholipase C spécifique du phosphatidylinositol (PI-PLC), le dibutyryl AMP-cyclique ne ramène pas le taux d'accumulation de la peroxydase des fibroblastes transformés par v-Src au niveau des fibroblastes parentaux et n'empêche pas la formation de macropinosomes, visualisée en microscopie confocale. L'analyse détaillée de la cinétique d'accumulation de la peroxydase dans les cellules transformées a révélé que le dibutyryl AMP-cyclique ralentit l'accumulation de la peroxydase seulement après un temps supérieur à 5 minutes, en accélérant sa régurgitation, sans affecter le recyclage de la transferrine. Ces résultats montrent clairement que, dans les fibroblastes transformés par v-Src, la macropinocytose et la micropinocytose sont deux voies distinctes et que l'AMP-cyclique n'affecte ni la micropinocytose, ni la formation de macropinosomes, mais active sélectivement la régurgitation à partir de ceux-ci. La seconde partie de ma thèse s'intéresse à la régulation de la formation des macropinosomes. La transformation des fibroblastes Rat-1 par les oncogènes v-Src ou K-Ras ainsi que la transfection stable par un vecteur d'expression de la sous-unité régulatrice sauvage p85a, créant un "dominant-positif" de la PI3K, provoquent la destruction des câbles de tension, le recrutement de l'actine corticale et la formation du "ruffling" qui génère les macropinosomes, ce qui est reflété par l'accélération sélective de l'endocytose en phase fluide. Ces altérations corrèlent étroitement avec l'activation de la PI3K et de la PI-PLC, mesurées par la production des phospho-inositides D3 in situ et in vitro et par le niveau de l'IP3 respectivement, et sont abolies après transfection stable par un vecteur d'expression pour la sous-unité p85a tronquée, agissant comme "dominant-négatif" de la PI3K, ou après traitement des cellules avec les inhibiteurs pharmacologiques de la PI3K et de la PI-PLC. Ceci démontre que ces deux enzymes sont nécessaires dans la signalisation de la macropinocytose constitutive. Ces inhibiteurs diminuent fortement le pourcentage des cellules montrant un "ruffling" membranaire mais n'affectent pas l'endocytose de la transferrine.[...]Macropinocytosis refers to the formation of large primary endocytic vesicles of irregular size and shape, generated by actin-driven evaginations of the plasma membrane, whereby cells avidly incorporate extracellular solutes. Macropinosomes resemble "empty" phagosomes and show no difference with the "spacious phagosomes" triggered by the enteropathogenic bacteria Salmonella and Shigella. Macropinosomes are formed at the leading edge and appear tightly regulated. They may fuse with lysosomes or regurgitate their content back to the extracellular space. Transformation of fibroblasts by Src or Ras results into the constitutive formation of macropinosomes at "ruffling" zones. My thesis adresses the dynamic of the constitutive macropinocytosis and its potential regulators. The first part of the thesis deals with the fate of macropinosomes. We found that, in v-Src transformed fibroblasts, macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase, forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer, horseradish peroxidase, in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phosphatidylinositol 3-kinase (PI3K) inhibitors and phosphatidylinositol-specific phospholipase C (PI-PLC), dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of peroxidase entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited its accumulation only after intervals >5 minutes, due to accelerated regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom. The second part of my thesis deals with the regulation of macropinocytosis. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection with an expression vector for wild-type PI3K regulatory subunit p85a, acting as dominant-positive PI3K, constitutively lead to stress fibers disruption, cortical actin recruitment, extensive ruffling and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and PI-PLC, as assayed by 3-phosphoinositides synthesis in situ and in vitro and IP3 steady state levels, respectively, and were abolished by stable transfection of Src-transformed cells with an expression vector for truncated p85a, acting as dominant-negative PI3K, as well as by pharmacological inhibitors of PI3K and PI-PLC, indicating requirement for both enzymes. These inhibitors strongly decreased the population of cells showing active membrane ruffling and did not detectably affect receptor-mediated endocytosis of transferrin. Interestingly, the effect of Src and dominant-positive p85a transfection on the actin cytoskeleton could be reversed within 30-60 min by the pharmacological inhibitor of PI3K, wortmannin. [...](MED 3)--UCL, 200

Genetics of Arteriovenous Malformations

Arteriovenous malformations (AVMs) and arteriovenous fistulas (AVFs) are abnormal fast-flow connections between arterial and venous circulation, without a normal intervening capillary bed. They are congenital developmental lesions seen in various sites of the body. Pathogenesis is still largely unknown, and no specific biomarkers have been identified to study e.g. evolution of lesions

v-Src accelerates spontaneous motility via phosphoinositide 3-kinase, phospholipase C and phospholipase D, but abrogates chemotaxis in Rat-1 and MDCK cells.

In Rat-1 fibroblasts, v-Src causes a profound remodelling of cortical actin cytoskeleton. This transformation includes membrane ruffling, a hallmark of the leading edge in migrating cells, and results from activation of phosphoinositide 3-kinase (PI 3-kinase), phospholipase C (PLC) and phospholipase D (PLD). We therefore reexamined whether motility is constitutively triggered by v-Src and studied whether this response is controlled by the same signalling pathway. The study was performed using Rat-1/tsLA29 and MDCK/tsLA31 cells, each harbouring a different thermosensitive v-Src kinase, active at 34 degrees C but inactivated at 40 degrees C. In both cell lines, overnight v-Src activation induced transformation and accelerated spontaneous motility by approximately twofold, as evidenced by wound-healing assay and by single-cell track, time-lapse recording in Dunn chambers. Inhibitors of PI 3-kinase, PLC and PLD selectively abrogated acceleration of motility by v-Src. Since mechanisms that co-ordinate spontaneous, as distinct from oriented, cell migration are separable, we further analysed in Dunn chambers chemotactic response of Rat-1/tsLA29 cells to PDGF and of MDCK/tsLA31 cells to EGF. In both cases, v-Src decreased the steady-state level of growth factor receptors at the cell surface twofold, and abrogated movement directionality at comparable level of occupancy as in non-transformed cells. The burst of pinocytosis in response to growth factors was also abolished by v-Src. Altogether, these results indicate that v-Src triggers motility in a PI 3-kinase-, PLC- and PLD-dependent manner, but abrogates directionality by suppressing polarised signalling downstream of growth factor receptors.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Regulation of macropinocytosis in v-Src-transformed fibroblasts: cyclic AMP selectively promotes regurgitation of macropinosomes.

Stable transformation of Rat-1 fibroblasts by the v-Src oncoprotein results into the constitutive formation of macropinosomes. In the present report, we found that macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer horseradish peroxidase at intervals >5 minutes in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate or the phosphatidylinositol 3-kinase inhibitor wortmannin, dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of tracer entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited peroxidase accumulation only after intervals >5 minutes, due to accelerated peroxidase regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom