Localization of Ras signaling complex in budding yeast

In Saccharomyces cerevisiae, cAMP/pKA pathway plays a major role in metabolism, stress resistance and proliferation control. cAMP is produced by adenylate cyclase, which is activated both by Gpr1/Gpa2 system and Ras proteins, regulated by Cdc25/Sdc25 guanine exchange factors and Ira GTPase activator proteins. Recently, both Ras2 and Cdc25 RasGEF were reported to localize not only in plasma membrane but also in internal membranes. Here, the subcellular localization of Ras signaling complex proteins was investigated both by fluorescent tagging and by biochemical cell membrane fractionation on sucrose gradients. Although a consistent minor fraction of Ras signaling complex components was found in plasma membrane during exponential growth on glucose, Cdc25 appears to localize mainly on ER membranes, while Ira2 and Cyr1 are also significantly present on mitochondria. Moreover, PKA Tpk1 catalytic subunit overexpression induces Ira2 protein to move from mitochondria to ER membranes. These data confirm the hypothesis that different branches of Ras signaling pathways could involve different subcellular compartments, and that relocalization of Ras signaling complex components is subject to PKA control.     

Rhabdomyosarcomas: an overview on the experimental animal models

Rhabdomyosarcomas (RMS) are aggressive childhood soft-tissue malignancies deriving from mesenchymal progenitors that are committed to muscle-specific lineages. Despite the histopathological signatures associated with three main histological variants, termed embryonal, alveolar and pleomorphic, a plethora of genetic and molecular changes are recognized in RMS. Over the years, exposure to carcinogens or ionizing radiations and gene-targeting approaches in vivo have greatly contributed to disclose some of the mechanisms underlying RMS onset. In this review, we describe the principal distinct features associated with RMS variants and focus on the current available experimental animal models to point out the molecular determinants cooperating with RMS development and progression.     

Landscape genetic structure of chestnut (Castanea sativa Mill.) in Spain

The current need for forest conservation and management has driven a rapid expansion of landscape genetics approach. This discipline combines tools from molecular genetics, landscape ecology and spatial statistics and is decisive for improving not only ecological knowledge but also for properly managing population genetic resources. This approach could be appropriate to sweet chestnut (Castanea sativa Mill.), a multipurpose species of great economic importance in the Mediterranean basin and a species considered to be a good model of integration between natural and human-driven distribution of diversity. Sixteen chestnut populations, covering the distribution range of the species in Spain, were analysed using seven microsatellite markers. Results revealed a high level of genetic diversity in Spanish chestnut populations, which in part followed a geographical pattern, although distribution was not homogeneous. Likewise, areas particularly rich in diversity were detected, facilitating the development of a hypothesis about the history of chestnut in Spain. In conclusion, these results provide valuable baseline data for more in-depth studies on chestnut landscape genetics that can contribute to its conservation.     

The RhoA-associated protein Citron-N controls dendritic spine maintenance by interacting with spine-associated Golgi compartments

Dendritic spines are highly dynamic protuberances that are thought to be crucial for learning and memory. Although it is well known that actin filaments and membrane dynamics regulate spine plasticity, how these two events are linked locally is less clear. Here, we provide evidence that Citron-N (CIT-N), a binding partner of the small GTPase RhoA, is associated with the actin filaments and Golgi compartments of dendritic spines. We also show that CIT-N is required for recruiting F-actin and Golgi membranes at spines of in vitro-grown neurons. Studies in knockout mice show that this protein is essential for the maturation of dendritic spines. We suggest that CIT-N might function as a scaffold protein in spine organization through its ability to bind to Golgi membranes and by affecting actin remodelling.     

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.