A GENETIC MARKER TO SEPARATE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) MORPHOTYPES1

Emiliania huxleyi (Lohm.) Hay and Mohler is a ubiquitous unicellular marine alga surrounded by an elaborate covering of calcite platelets called coccoliths. It is an important primary producer involved in oceanic biogeochemistry and climate regulation. Currently, E. huxleyi is separated into five morphotypes based on morphometric, physiological, biochemical, and immunological differences. However, a genetic marker has yet to be found to characterize these morphotypes. With the use of sequence analysis and denaturing gradient gel electrophoresis, we discovered a genetic marker that correlates significantly with the separation of the most widely recognized A and B morphotypes. Furthermore, we reveal that the A morphotype is composed of a number of distinct genotypes. This marker lies within the 3′ untranslated region of a coccolith associated protein mRNA, which is implicated in regulating coccolith calcification. Consequently, we tentatively termed this marker the coccolith morphology motif.     

Rotating for elongation: Fat2 whips for the race

Dynamic rearrangements of the actin cytoskeleton are crucial for cell shape and migration. In this issue, Squarr et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508081) show that the cadherin superfamily protein Fat2 regulates actin-rich protrusions driving collective cell migration during Drosophila melanogaster egg morphogenesis through its interaction with the WAVE regulatory complex.Collective cell migration is a hallmark of tissue remodeling during embryonic development, as well as of tissue repair and cancer invasion (Rørth, 2012). A novel type of collective cell migration has emerged in recent years from studies of Drosophila follicles (Haigo and Bilder, 2011), highlighting what seems to be a potentially conserved, intrinsic property of epithelial cells growing in constricted environments. The Drosophila follicle or egg chamber is a spherical assembly of germ cells surrounded by an epithelium of somatic follicle cells. The egg chamber elongates to an ellipsoid during oogenesis, thereby conferring the egg its appropriate shape (Fig. 1). Egg chamber elongation is guaranteed by a molecular corset, formed by the follicle cells and the basal ECM, which directs the growth of the egg chamber along the anterior–posterior axis by constraining the central area of the egg (He et al., 2010). In particular, an ordered array of contractile actin filaments within the follicle cells, running perpendicular to the anterior–posterior axis of the egg chamber, contributes to this “corset” (He et al., 2010). Collective migration of follicle cells around the anterior–posterior axis of the egg chamber is required to promote the global polarization of these parallel actin bundles and of the follicular basement membrane (Haigo and Bilder, 2011; Cetera et al., 2014). This is a remarkable type of cell migration as it leads to a rotational movement of a group of cells within a constrained space and without an identified collective leading edge. Nonetheless, it turns out that actin-rich protrusions, which are typical of a leading edge, are present at the basal side of each individual follicle cell. These protrusions point toward the direction of rotation and are necessary to generate the collective rotational movement (Cetera et al., 2014).Open in a separate windowFigure 1.The actin-rich structures underlying egg chamber elongation. Maturing egg chambers contain germ cells (yellow), including the oocyte (D, brown), covered by a layer of follicle cells (light blue). During early maturation stages, the follicle cells drive the rotation of the egg chamber over the ECM (dark blue) and pull the germ cells along. This rotation promotes the elongation of egg chambers along the antero–posterior axis, observed from stage 7/8. (A) Schematic representation of an egg chamber at stage 5/6, during the collective rotation movement in the direction indicated by the arrow. The axis cross applies to A, C, and D. (B) A schematic of the follicle epithelium (transversal section) indicating its apical side, oriented toward the germ cells, and the basal membrane, laying over the ECM. Basal actin filaments are depicted in red. (C) Higher magnification of the follicle cells visualized from their basal side. Actin-rich structures are depicted in red. They include actin-rich protrusions at the leading edge of each cell and whip-like protrusions at tripartite junctions between cells. Within each cell, the bundles of actin filaments (basal actin) run parallel to the direction of follicle epithelium rotation (black arrow). (D) Illustration of the elongated egg chamber in the next maturation stages (7/8) in Drosophila development.Presence of these actin protrusions at the leading edge of follicle cells requires the WASP family verprolin homologous protein (WAVE) and the WAVE regulatory complex (WRC; Cetera et al., 2014), known activators of actin nucleation through the Arp2/3 complex, as depletion of WAVE or of the WRC component Abi eliminates all actin-based protrusions in follicle cells (Cetera et al., 2014).The details of the control of WAVE and WRC activation have been under scrutiny for many years (Stradal and Scita, 2006; Ismail et al., 2009; Chen et al., 2010; Mendoza, 2013). Nonetheless, a recent discovery opened the possibility of a novel and conserved mechanism for WRC activation (Chen et al., 2014). A combination of structural and biochemical approaches revealed that the WRC can be recruited to the membrane by an array of membrane proteins sharing a conserved peptide motif, the WRC interacting receptor sequence (WIRS). The binding surface for WIRS is provided by two WRC subunits, including Abi, and leads to activation of WAVE toward the Arp2/3 complex (Chen et al., 2014). Could a WIRS domain-containing molecule be involved in the localized activation of WAVE during the rotational movement of follicle cells? This attractive hypothesis is supported by the observation that disruption of the WIRS interface in the WRC leads to the generation of round eggs (Chen et al., 2014). The round-egg phenotype characteristically results from mutations in genes promoting the rotational movement of the follicle cells or organizing the underlying ECM (Gutzeit et al., 1991; Bateman et al., 2001; Frydman and Spradling, 2001; Deng et al., 2003; Schneider et al., 2006). One of these round-egg genes is the cadherin superfamily fat2/kugelei (Gutzeit et al., 1991). Individual follicle cells from fat2 mutants have parallel-arranged actin filaments. However, these actin filaments are no longer perpendicular to the anterior–posterior axis and their coordinated organization in the tissue is lost (Gutzeit et al., 1991; Viktorinová et al., 2009). Moreover, large clones of fat2 mutant follicle cells lead to uncoordinated arrangement of actin bundles in the wild-type neighboring cells (Viktorinová et al., 2009). Collectively, these previous results suggested that Fat2 coordinates actin organization in follicle cells.In this issue, Squarr et al. identified Fat2 as a novel WIRS domain–containing molecule that acts through the WRC to control collective cell migration during Drosophila oogenesis. Squarr et al. (2016) elegantly used live in vivo imaging and genetically encoded probes to characterize different types of actin-rich protrusions during egg chamber maturation. Their analysis showed that small filopodial protrusions extend at regular intervals in a polarized fashion at the basolateral cell border, whereas on the apical side filopodial protrusions are not polarized along the migration direction (Cetera and Horne-Badovinac, 2015; Squarr et al., 2016). Stunning time-lapse movies remarkably revealed an additional type of actin-rich whip-like protrusion at tricellular junctions (Fig. 1). All these types of actin protrusions depend on the WRC, as they are largely missing in follicle cells with impaired WRC function. At defined stages of egg chamber maturation, the WRC is prominently enriched at tricellular junctions, a localization that resembles that reported for Fat2 at the same stages (Viktorinová et al., 2009; Squarr et al., 2016). Motivated by this observation and in search of the molecular mechanisms driving the formation of the actin-rich structures, Squarr et al. (2016) asked whether Fat2 is functionally related to the WRC-dependent organization of actin. Indeed, they identified three conserved WIRS motifs in the cytoplasmic tail of Fat2 and demonstrated a direct interaction of Fat2’s cytoplasmic tail with the WRC through in vitro binding assays, establishing Fat2 as a novel WIRS ligand.They additionally proved that functional WIRS interactions are necessary for the formation of whip-like protrusions and polarized protrusions as well as egg chamber elongation by analyzing flies lacking the conserved WIRS binding surface in the WRC. To test the hypothesis that Fat2 is involved in recruiting the WRC, the researchers examined fat2 mutant cells, which displayed impaired WRC localization to the basal follicle side and to tricellular junctions as well as reduced actin-rich protrusions at the basal side. Thus, Fat2 contributes to the localization of the WRC in these egg chambers. Interestingly, analysis of additional mutant lines showed that neither loss of the WRC nor loss of the Fat2–WRC interaction affected the distribution of Fat2, suggesting it acts upstream of the WRC to control WRC localization and formation of polarized cell protrusions.Additional paths of WRC regulation might involve the ECM receptor phosphatase Dlar, as dlar mutants also exhibit a round-egg phenotype (Bateman et al., 2001). Indeed, Squarr et al. (2016) observed that Dlar and WRC subunit localization partially overlap during the early stages of egg chamber maturation and provide biochemical evidence for an indirect molecular interaction in vivo between Dlar and the WRC. Importantly, in dlar mutants, less WRC localized to the basal side and actin-rich protrusions along the membrane were also reduced. Nonetheless, the localization of the WRC at tricellular junctions is partially maintained and so is actin accumulation at these sites. Collectively, these data establish a model in which Fat2 and Dlar are linked in recruiting the WRC to induce the formation of polarized cell protrusions, contributing to different aspects of WRC regulation for collective follicle cell migration during Drosophila oogenesis.Squarr et al. (2016) combined genetic, biochemical, and live-imaging analyses in Drosophila to gain insight into molecular actin dynamics in vivo. Recent data from time-lapse imaging suggested that collective rotational movement is an intrinsic property of epithelial cells and a feature of developing glandular tissues in mammals (Ewald et al., 2008; Tanner et al., 2012). Squarr et al. (2016) confirmed that nonmalignant human breast epithelial cell lines form spheres and undergo multiple rotations (Tanner et al., 2012). To ask whether the actin-rich protrusions that they observed in fly follicle cells represent a conserved structure underlying epithelial rotational migration, they conducted live imaging of mammary epithelial cells expressing the actin marker LifeAct-EGFP to highlight actin organization. In this system, they observed actin accumulation at the basal side of the rotating spheres and actin-rich protrusions at basal intercellular junctions. The function of these actin whips and protrusions across species is not yet clear; Squarr et al. (2016) speculate that whip-like protrusions might interact with the ECM to synchronize directed cell migration and to drive the morphogenetic movement. The similar morphology of the protrusions in Drosophila and human epithelial cells additionally suggests that the molecular mechanisms driving tissue rotation might be similar, a hypothesis compatible with the known localization of the human homologue of Fat2 at intercellular epithelial junctions. It will be of great interest to address whether the Fat2-, Dlar-, and WRC-dependent molecular mechanisms described in this study are conserved in other rotational collective cell migration processes.     

An integrated pan‐tropical biomass map using multiple reference datasets

We combined two existing datasets of vegetation aboveground biomass (AGB) (Proceedings of the National Academy of Sciences of the United States of America, 108 , 2011, 9899; Nature Climate Change, 2 , 2012, 182) into a pan‐tropical AGB map at 1‐km resolution using an independent reference dataset of field observations and locally calibrated high‐resolution biomass maps, harmonized and upscaled to 14 477 1‐km AGB estimates. Our data fusion approach uses bias removal and weighted linear averaging that incorporates and spatializes the biomass patterns indicated by the reference data. The method was applied independently in areas (strata) with homogeneous error patterns of the input (Saatchi and Baccini) maps, which were estimated from the reference data and additional covariates. Based on the fused map, we estimated AGB stock for the tropics (23.4 N–23.4 S) of 375 Pg dry mass, 9–18% lower than the Saatchi and Baccini estimates. The fused map also showed differing spatial patterns of AGB over large areas, with higher AGB density in the dense forest areas in the Congo basin, Eastern Amazon and South‐East Asia, and lower values in Central America and in most dry vegetation areas of Africa than either of the input maps. The validation exercise, based on 2118 estimates from the reference dataset not used in the fusion process, showed that the fused map had a RMSE 15–21% lower than that of the input maps and, most importantly, nearly unbiased estimates (mean bias 5 Mg dry mass ha^?1 vs. 21 and 28 Mg ha^?1 for the input maps). The fusion method can be applied at any scale including the policy‐relevant national level, where it can provide improved biomass estimates by integrating existing regional biomass maps as input maps and additional, country‐specific reference datasets.     

GAS2 and GAS4, a pair of developmentally regulated genes required for spore wall assembly in Saccharomyces cerevisiae

The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral.     

Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens

Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/β-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.     

Modulation of MCP-1 and iNOS by 50-Hz sinusoidal electromagnetic field.

The purpose of this study was to investigate whether overnight exposure to 1 mT-50 Hz extremely low-frequency sinusoidal electromagnetic field (EMF) affects the expression and production of inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1) in human monocytes. RT-PCR and Western blot analysis demonstrate that EMF exposure affects the expression of iNOS and MCP-1 in cultured human mononuclear cells at the mRNA level and protein synthesis. Interestingly, the effects of EMF exposure clearly differed with respect to the potentiation and inhibition of iNOS and MCP-1 expression. Whereas iNOS was down-regulated both at the mRNA level and at the protein level, MCP-1 was up-regulated. These results provide helpful information regarding the EMF-mediated modulation of the inflammatory response in vivo. However, additional studies are necessary to demonstrate that EMF acts as a nonpharmacological inhibitor of NO and inducer of MCP-1 in some diseases where the balance of MCP-1 and NO may be important.     

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.     

Endothelial and vascular smooth muscle cell dysfunction mediated by cyclophylin A and the atheroprotective effects of melatonin

AimsThis study evaluated the role of cyclophilin A (CyPA) in early phase of atherosclerosis and also examined the atheroprotective effects of melatonin due to its antioxidant properties.Main methodsAPOE null mice at 6 and 15 weeks of age were treated with melatonin at a dose of 0.1 mg/kg/day or 10 mg/kg/day. We evaluated both histopathological alterations in endothelial and vascular smooth muscle cells by CyPA and rolling mononuclear cell expression during the early phase of atherosclerosis development.Key findingsOur study showed that CyPA expression increases and may modulate inflammatory cell adhesion and interleukin-6 expression inducing vascular smooth muscle cell migration and inflammatory cell extravasation in a time-dependent manner. Moreover, we observed an indirect atheroprotective effect of melatonin on vascular injury; it inhibited CyPA mediated inflammatory cell extravasation and oxidative stress.SignificanceThe melatonin treatment may represent a new atheroprotective approach that contributes to reducing the early phase of atherosclerosis involving the rolling of monocytes, their passage to subendothelial space and inhibition of CyPA expression.