ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.     

Tsunami impact on the population development of a critically endangered hummingbird species of a Pacific island

It is a long-standing question how tsunamis can influence wild populations of animals and plants. Here, we assessed short-term changes in the population of the critically endangered Juan Fernández Firecrown (Sephanoides fernandensis) by using abundance data recorded 1 year before and 1 year after the 2010 Chilean tsunami. We tested that the abundance of Firecrowns declined in the areas where the tsunami caused the massive loss of Cabbage Trees, an important seasonal nectar source for Firecrowns. The abundance of Juan Fernández Firecrowns decreased after the tsunami, but also was affected by the habitat type, altitude, and the abundance of Cabbage Trees. Firecrowns tended to be more abundant in settlement areas than in native forest whereas the reduction in Firecrown abundance after the tsunami was more intense in settlement areas than in native forest. As expected, this habitat effect was dependent on the massive loss of Cabbage Trees in settlement areas following the tsunami. In spite of the short-term nature of our data, our results are conclusive in showing that the loss of an important food source causes short-term changes in the distribution and abundance of Firecrowns, which, in turn, could contribute to population decline.     

Wnt directs the endosomal flux of LDL-derived cholesterol and lipid droplet homeostasis

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.     

American bullfrog invasion in Argentina: where should we take urgent measures?

Argentina is the country with the most geographically extended biological invasion of the American bullfrog (Lithobates catesbeianus) in South America after Brazil. Here, we used a maximum entropy ecological niche modeling algorithm (using records of the native range of American bullfrog) to project the model onto the whole of Argentina. We determined the most suitable habitats for this invasive alien species and where we consider urgent measures should be taken. Our projections showed good agreement with known feral populations of American bullfrog in Argentina. By implementing the “Multivariate Environmental Similarity Surface” analysis, we be able to determine that factors such as low precipitations or highest altitudes could be limiting the species’ ability to invade the west and south of the country. We suggest that strategies should focus on detecting established feral populations of the American bullfrog and preventing further introductions or range expansion of feral populations in the northeast portion of the country. Lastly, we report a new feral population of bullfrogs in Argentina.     

Expression of a highly differentiated phenotype and hepatic functionality markers in gilthead seabream (Sparus aurata L.) long-cultured hepatocytes: first morphological and functional in vitro characterization

The development of primary cultures and cell lines from aquatic organisms is a valuable tool for a wide range of research activities applied to aquaculture. Despite several efforts, derivation and long-term culturing of primary hepatocytes from marine vertebrates are still rare and unsuccessful. This is the first report to fully characterize long-term cultures of primary hepatocytes from the European seabream, Sparus aurata L. (Osteichthyes, Sparidae) (SaHePs). In this new model, hepatocyte cells were long-term viable, active proliferating, and fully retained liver function up to 3 weeks. SaHePs expressed a differentiated phenotype, owing to the reacquisition of the peculiar cytoarchitecture with the complete assembly of cytoskeletal and junctional network, as shown by the production and immunolocalization of several polarity markers and cytoskeletal proteins (MDR1, ZO-2, C-CAM1, Vimentin, Cadherin, ??-Tubulin, ??-Catenin, ??-Actin). Cytostructural analysis to identify polarized expression and bile canaliculi formation was performed by immunofluorescence and contrast phase microscopy. Long cultured SaHePs also demonstrated evidence of Albumin, ??1-Antitrypsin (AAT) and ??-Fetoprotein (AFP) synthesis, expression of the detoxifying metabolic enzyme cytochrome P-4501A (CYP 1A), and production of hepatocyte specific cytoskeleton proteins, such as Cytokeratin 8 (CK8) and Cytokeratin 18 (CK 18). The presence of specific markers for hepatic phenotype, detected by immunocytochemistry and Western blot analysis, is suggestive of the full maintenance of a highly differentiated phenotype and hepatic maturation. These data demonstrate that SaHePs can be long cultured without losing the hepatic functionality. This study provides a useful tool for innovative research applications in fish toxicological, pathological, and physiological studies, as one of the few hepatic, functionally active, in vitro model from marine fish.     

Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant-Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence.