Positive-strand RNAviruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear endoplasmic reticulum (ER) membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that ER-vesicle protein of 14 kD (Erv14), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, the perinuclear ER localization of BMV 1a is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coat components (Sec13, Sec24 or Sec31). The requirement of Erv14 for the localization of BMV 1a is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by both Erv14 and Sec24 for the proper localization of BMV 1a. The COPII pathway is well known for being involved in protein secretion; our data suggest that a subset of COPII coat proteins have an unrecognized role in targeting proteins to the perinuclear ER membrane.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-20T12:06:06Z No. of bitstreams: 1 Renata Triane da Silva Felix.pdf: 2513974 bytes, checksum: be007467b0e1d516329887a18d273370 (MD5) / Made available in DSpace on 2017-02-20T12:06:06Z (GMT). No. of bitstreams: 1 Renata Triane da Silva Felix.pdf: 2513974 bytes, checksum: be007467b0e1d516329887a18d273370 (MD5) Previous issue date: 2008-04-01 / We evaluated the reproductive aspects of the organization and characterization of oocytes and the maturational stages of Plagioscion squamosissimus in the reservoir of stone, river of Auditors, Bahia. The collections were bimonthly, conducted between november/04 and september/06. It was recorded during reproductive activity every year, with peaks in march/05 and from july/06. The average total fertility rate was 22,610 oocytes and fertility on the length was higher than the fertility on the weight when the standard length was more than 212mm. The proportion sexual presented significant difference (p <0.05) for the months of January, May, July and september/05 and, for the second year, a significant difference occurred in January and march/06. There was a concentration of lengths in modal class of 120 to 240mm for males and females in Year 1 and 2 males occurred in the larger class size. The weight-length relationship had reversed values for males and females in two years, registering for females increase in year 1 allometric negative and positive in year 2. The split was considered spawning and size of the first maturity was 150 for males and females. The GSR and the condition factor (allometric) when compared showed reverse in almost every period. The microscopic analyses were made through cuts in histology stained with hematoxylin-eosin-phloxine and mixture of Gomori’s tricromic. The gonads showed differentiation of color under the cages, suggesting a classification of cages. Through the macro and microscopic analysis identified five maturational stages. For the development oocitário, were defined six phases: oogonia and perinucleolus; vitelogenic and initial lipid; vitelogenic and lipid intermediate and advanced lipid vitelogenic and pre-ovulation. It was observed structures similar to that of marine fish, as droplets of oil in the final stages and a tendency to hydration pre-ovulatory. This is explained by the family seafood Sciaenidae, suggesting more detailedstudies to define these structures and stages of development ovocity. Despite the species to be well adapted to the environment, spawning the central body of the tank, it is necessary to enlarge the studies in this place, to establish parameters that will influence in its mechanism reprodutivo.Observou up structures similar to that of marine fish . As droplets of oil in the final stages and a tendency to hydration pre-ovulatory. This is explained by the family seafood Sciaenidae, suggesting more detailed studies to define these structures and stages of development ovocity. / Foram avaliados os aspectos reprodutivos e caracterização da organização dos oócitos e os estádios maturacionais de Plagioscion squamosissimus no reservatório de Pedra, rio de Contas, Bahia. As coletas foram bimestrais, realizadas entre novembro/04 e setembro/06. Foi registrada atividade reprodutiva durante todo ano, com picos em março/05 e a partir de julho/06. A fecundidade absoluta média foi de 22.610 ovócitos e a fecundidade relativa ao comprimento foi superior à fecundidade relativa ao peso quando o comprimento padrão foram acima de 212mm. A proporção sexual apresentou diferença significativa ( p< 0,05) para os meses de janeiro, maio, julho e setembro/05, e, para o segundo ano, ocorreu diferença significativa em janeiro e março/06. Houve concentração de comprimentos na classe modal de 120 a 240mm para machos fêmeas e no ano 1 e 2 os machos ocorreram nas maiores classes de tamanho. A relação peso-comprimento apresentou valores invertidos para machos e fêmeas nos dois anos, registrando para as fêmeas incremento alométrico negativo no ano 1 e positivo no ano 2. A desova foi considerada parcelada e o tamanho de primeira maturação foi de 150mm para machos e fêmeas. O RGS e o fator de condição (alométrico) quando comparados, apresentaram valores inversos em quase todo período. As análises microscópicas foram feitas através de histologia em cortes corados com hematoxilina – eosina – floxina e mistura tricrômica de Gomori. As gônadas apresentaram diferenciação de cor de acordo com o desenvolvimento gonadal, sugerindo uma classificação de desenvolvimento gonadal. Através da análise macro e microscópica, foram identificados cinco estádios maturacionais. Para o desenvolvimento oocitário, foram definidas seis fases: oogônia e perinucleolar; vitelogênica e lipídica iniciais; vitelogênica e lipídica intermediária; vitelogênica e lipídica avançada e pré-ovulação. Observou-se estruturas semelhantes à de peixes marinhos, comogotículas de óleo nas fases finais e uma tendência à hidratação pré-ovulatória. Esta característica é explicada pela origem marinha da família Sciaenidae, sugerindo estudos mais detalhados para definição dessas estruturas e fases de desenvolvimento ovocitário. Apesar da espécie encontrar-se bem adaptada ao ambiente, desovando no corpo central do reservatório, faz-se necessário ampliar os estudos neste local, afim de determinar parâmetros que venham a influenciar em seu mecanismo reprodutivo.Observou-se estruturas semelhantes à de peixes marinhos, como gotículas de óleo nas fases finais e uma tendência à hidratação pré-ovulatória. Esta característica é explicada pela origem marinha da família Sciaenidae, sugerindo estudos mais detalhados para definição dessas estruturas e fases de desenvolvimento ovocitário.

Le cytosquelette d'actine est une structure dynamique capitale pour la cellule, qui intervient dans les processus de signalisation et génère des forces mécaniques pour compléter des fonctions aussi diverses que l'adhésion, la migration, la division ou la différenciation. Les protéines qui régulent cette structure sont capables de moduler ces fonctions. J'ai identifié une nouvelle famille de protéines régulatrices de l'actine, les protéines Refilines (RefilineA et RefilineB), dont l'expression est corrélée avec l'engagement des cellules dans des programmes de différenciation. La RefilineA est induite lors de la différenciation des cellules précurseurs neurales multipotentes en cellules progénitrices gliales. La RefilineB est stabilisée dans les cellules épithéliales lors de la transition épithélio-mésenchymateuse (TEM) induite par le TGF-β. Dans ces cellules, les Refilines agissent en se complexant à la FilamineA, une protéine qui se lie aux filaments d'actine et forme le maillage. Des syndromes génétiques de mutations sur le gène de la FilamineA entrainent d'importants défauts développementaux, cependant la fonction précise de la protéine reste à ce jour obscure. Le complexe Refiline/FilamineA induit la formation de câbles d'actine et génère également une nouvelle structure d'actine périnucléaire appelée coiffe d'actine (« actin cap ») ou « ligne TAN» qui s'ancre à l'enveloppe nucléaire pour réguler les mouvements et la morphologie du noyau. Les Refilines sont les seules protéines identifiées à ce jour capables de catalyser la formation de structures périnucléaires d'actine. Ces résultats ouvrent donc de nouvelles perspectives pour appréhender les fonctions de la FilamineA ainsi que la biologie et les fonctions des structures périnucléaires d'actine. / The actin cytoskeleton is a highly dynamic structure involved in cell signaling and that creates mechanical force for the completion of diverse functions such as adhesion, migration, division or differentiation. Proteins that regulate this structure can modulate its function. We identified a new protein family that regulates the actin cytoskeleton, Refilin proteins (RefilinA and RefilinB), and whose expression correlates with differentiation switches. RefilinA is induced during differentiation of neural multipotent precursors into glial progenitors, while RefilinB is stabilized in epithelial cells during epithelial-mesenchymal transition (EMT) induced by TGF-β. In cells, Refilins interact with FilaminA, a protein that binds actin filaments to organize them into a network. Genetic syndromes where the FilaminA gene is mutated lead to important developmental defects, The Refilin/FilaminA complex generates actin cables as well as a new perinuclear structure called « actin cap » or «TAN line» that interacts with the nuclear envelope to regulate nuclear movement and shape. Refilin proteins are the only proteins identified so far that induce the formation of perinuclear actin structures. These results open up new perspective for the understanding of FilaminA's function as well as for the biology and functions of perinuclear actin structures.

Nucleus is the largest cellular organelle in animal cells. Due to its bulky nature and the stiffness of nuclear lamina the nucleus constitutes the substantial problem for migrating cells where nucleus has to move. The actomyosin generated forces and LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, that is composed of SUN and nesprin proteins, play key role in nuclear movement. LINC complex mechanically couples nuclear lamina to the cytoskeleton and allows the forces exerted by the cytoskeleton to move the nucleus. Perinuclear actin fibers, also termed actin cap, mechanically link focal adhesions with nucleus and they may generate forces that position the nucleus in a way that is optimal for cellular movement. However, molecular mechanism of how perinuclear actin fibers and LINC complex orchestrate the nuclear movement and functional significance of this process remain poorly understood. The specific aim was to determine the mechanisms by which perinuclear actin fibers are formed and how are these mechanisms employed to facilitate cell migration. The role of LPA-RhoA signaling axis and LINC complex in the formation of perinuclear actin fibers was also examined. It was confirmed that LPA is essencial stimulus during actin cap formation. On the other hand, FAK kinase was found necessary for...