GLUT4 in murine bone growth: from uptake and translocation to proliferation and differentiation

Skeletal growth, taking place in the cartilaginous growth plates of long bones, consumes high levels of glucose for both metabolic and anabolic purposes. We previously showed that Glut4 is present in growing bone and is decreased in diabetes. In the present study, we examined the hypothesis that in bone, GLUT4 gene expression and function are regulated via the IGF-I receptor (IGF-IR) and that Glut4 plays an important role in bone growth. Insulin and IGF-I actions on skeletal growth and glucose uptake were determined using mandibular condyle (MC) organ cultures and MC-derived primary cell cultures (MCDC). Chondrogenesis was determined by following proliferation and differentiation activities using immunohistochemical (IHC) analysis of proliferating cell nuclear antigen and type II collagen expression, respectively. Overall condylar growth was assessed morphometrically. GLUT4 mRNA and protein levels were determined using in situ hybridization and IHC, respectively. Glut4 translocation to the cell membrane was assessed using confocal microscopy analysis of GFP-Glut4 fusion-transfected cells and immunogold and electron microscopy on MC sections; glucose uptake was assayed by 2-deoxyglucose (2-DOG) uptake. Both IGF-I and insulin-stimulated glucose uptake in MCDC, with IGF-I being tenfold more potent than insulin. Blockage of IGF-IR abrogated both IGF-I- and insulin-induced chondrogenesis and glucose metabolism. IGF-I, but not insulin, induced Glut4 translocation to the plasma membrane. Additionally, insulin induced both GLUT4 and IGF-IR gene expression and improved condylar growth in insulin receptor knockout mice-derived MC. Moreover, silencing of GLUT4 gene in MCDC culture abolished both IGF-I-induced glucose uptake and chondrocytic proliferation and differentiation. In growing bone, the IGF-IR pathway stimulates Glut4 translocation and enhances glucose uptake. Moreover, intact Glut4 cellular levels and translocation machinery are essential for early skeletal growth.     

The DCR Protein TTC3 Affects Differentiation and Golgi Compactness in Neurons through Specific Actin-Regulating Pathways

In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR), prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.     

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.     

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.     

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.     

Hematopoietic progenitor cell liabilities and alarmins S100A8/A9‐related inflammaging associate with frailty and predict poor cardiovascular outcomes in older adults

Frailty affects the physical, cognitive, and social domains exposing older adults to an increased risk of cardiovascular disease and death. The mechanisms linking frailty and cardiovascular outcomes are mostly unknown. Here, we studied the association of abundance (flow cytometry) and gene expression profile (RNAseq) of stem/progenitor cells (HSPCs) and molecular markers of inflammaging (ELISA) with the cardiorespiratory phenotype and prospective adverse events of individuals classified according to levels of frailty. Two cohorts of older adults were enrolled in the study. In a cohort of pre‐frail 35 individuals (average age: 75 years), a physical frailty score above the median identified subjects with initial alterations in cardiorespiratory function. RNA sequencing revealed S100A8/A9 upregulation in HSPCs from the bone marrow (>10‐fold) and peripheral blood (>200‐fold) of individuals with greater physical frailty. Moreover higher frailty was associated with increased alarmins S100A8/A9 and inflammatory cytokines in peripheral blood. We then studied a cohort of 104 more frail individuals (average age: 81 years) with multidomain health deficits. Reduced levels of circulating HSPCs and increased S100A8/A9 concentrations were independently associated with the frailty index. Remarkably, low HSPCs and high S100A8/A9 simultaneously predicted major adverse cardiovascular events at 1‐year follow‐up after adjustment for age and frailty index. In conclusion, inflammaging characterized by alarmin and pro‐inflammatory cytokines in pre‐frail individuals is mirrored by the pauperization of HSPCs in frail older people with comorbidities. S100A8/A9 is upregulated within HSPCs, identifying a phenotype that associates with poor cardiovascular outcomes.     

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.