A Spectraplakin Is Enriched on the Fusome and Organizes Microtubules during Oocyte Specification in Drosophila

AbstractBackground: During Drosophila oogenesis a membranous organelle called the fusome has a key function in the establishment of oocyte fate and polarity, ultimately leading to the establishment of the major body axes of the animal. The fusome is necessary for the microtubule-driven restriction of markers of oocyte fate to the oocyte, but the mechanism by which the fusome organizes the microtubules is not known.Results: We have identified the spectraplakin Short stop (Shot) as a new component of the fusome. Spectraplakins are giant cytoskeletal linker proteins, with multiple isoforms produced from each gene. Shot is the sole spectraplakin in Drosophila. The phenotype caused by the absence of Shot is not similar to that of other components of the fusome but instead is similar to the absence of the downstream components that interact with microtubules: the dynein/dynactin-complex-associated proteins Egalitarian and BicaudalD. Shot is required for the association of microtubules with the fusome and the subsequent specification of the oocyte in 16-cell cysts. Shot is also required for the concentration of centrosomes into the oocyte, a process thought to be independent of microtubules because it still occurs in the presence of microtubule depolymerizing drugs. This suggests that Shot may protect some microtubules from depolymerization and that these microtubules are sufficient for this process.Conclusions: Shot provides the missing link between the fusome and microtubules within meiotic cysts, which is essential for the establishment of the oocyte. Shot associates with the fusome and is required for microtubule organization. We suggest that it does this directly, via its microtubule binding GAS2 domain

Drosophila p120-catenin is crucial for endocytosis of the dynamic E-cadherin-Bazooka complex.

The intracellular functions of classical cadherins are mediated through the direct binding of two catenins: β-catenin and p120-catenin (also known as CTNND1 in vertebrates, and p120ctn in Drosophila). Whereas β-catenin is crucial for cadherin function, the role of p120-catenin is less clear and appears to vary between organisms. We show here that p120-catenin has a conserved role in regulating the endocytosis of cadherins, but that its ancestral role might have been to promote endocytosis, followed by the acquisition of a new inhibitory role in vertebrates. In Drosophila, p120-catenin facilitates endocytosis of the dynamic E-cadherin-Bazooka subcomplex, which is followed by its recycling. The absence of p120-catenin stabilises this subcomplex at the membrane, reducing the ability of cells to exchange neighbours in embryos and expanding cell-cell contacts in imaginal discs.We thank John Overton, the Gurdon Institute Imaging Facility, Maria J. Gomez-Lamarca, and Benjamin Klapholz for their help. Supported by BBSRC grant BB/K00056X/1.This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/jcs.17752

'Calving laws', 'sliding laws' and the stability of tidewater glaciers

A new calving criterion is introduced, which predicts calving where the depth of surface crevasses equals ice height above sea level. Crevasse depth is calculated from strain rates, and terminus position and calving rate are therefore functions of ice velocity, strain rate, ice thickness and water depth. We couple the calving criterion with three 'sliding laws', in which velocity is controlled by (1) basal drag, (2) lateral drag and (3) a combination of the two. In model 1, velocities and strain rates are dependent on effective pressure, and hence ice thickness relative to water depth. Imposed thinning can lead to acceleration and terminus retreat, and ice shelves cannot form. In model 2, ice velocity is independent of changes in ice thickness unless accompanied by changes in surface gradient. Velocities are strongly dependent on channel width, and calving margins tend to stabilize at flow-unit widenings. Model 3 exhibits the combined characteristics of the other two models, and suggests that calving glaciers are sensitive to imposed thickness changes if basal drag provides most resistance to flow, but stable if most resistance is from lateral drag. ice shelves can form if reduction of basal drag occurs over a sufficiently long spatial scale. In combination, the new calving criterion and the basal-lateral drag sliding function (model 3) can be used to simulate much of the observed spectrum of behaviour of calving glaciers, and present new opportunities to model ice-sheet response to climate change.</p

Integrins in Development Moving on, Responding to, and Sticking to the Extracellular Matrix

AbstractIntegrins are cell surface receptors of the extracellular matrix present in all animals. Genetic analysis in worms, flies, and vertebrates has revealed integrin involvement in key developmental processes, and we focus here on examples of integrin functions that are comparable across these model organisms. Integrins contribute to cell movement by providing traction to migrating cells, through assembly of extracellular matrices that can serve as tracks for migration, and by transmitting guidance signals that direct cells or cell processes to their targets. Integrins also participate in signaling events that govern tissue differentiation and organogenesis. Finally, adhesion by integrin-mediated junctions allows tissues to withstand mechanical load and is essential for tissue integrity

Maintaining epithelial integrity: a function for gigantic spectraplakin isoforms in adherens junctions

The Short stop (Shot/Kakapo) spectraplakin is a giant cytoskeletal protein, which exists in multiple isoforms with characteristics of both spectrin and plakin superfamilies. Previously characterized Shot isoforms are similar to spectrin and dystrophin, with an actin-binding domain followed by spectrin repeats. We describe a new large exon within the shot locus, which encodes a series of plakin repeats similar to the COOH terminus of plakins such as plectin and BPAG1e. We find that the plakin repeats are inserted between the actin-binding domain and spectrin repeats, generating isoforms as large as 8,846 residues, which could span 400 nm. These novel isoforms localized to adherens junctions of embryonic and follicular epithelia. Loss of Shot within the follicle epithelium leads to double layering and accumulation of actin and ZO-1 in between, and a reduction of Armadillo and Discs lost within, mutant cells, indicative of a disruption of adherens junction integrity. Thus, we identify a new role for spectraplakins in mediating cell–cell adhesion

Drug Discovery Opportunities and Challenges at G Protein Coupled Receptors for Long Chain Free Fatty Acids

Discovery of G protein coupled receptors for long chain free fatty acids (FFAs), FFA1 (GPR40) and GPR120, has expanded our understanding of these nutrients as signaling molecules. These receptors have emerged as important sensors for FFA levels in the circulation or the gut lumen, based on evidence from in vitro and rodent models, and an increasing number of human studies. Here we consider their promise as therapeutic targets for metabolic disease, including type 2 diabetes and obesity. FFA1 directly mediates acute FFA-induced glucose-stimulated insulin secretion in pancreatic beta-cells, while GPR120 and FFA1 trigger release of incretins from intestinal endocrine cells, and so indirectly enhance insulin secretion and promote satiety. GPR120 signaling in adipocytes and macrophages also results in insulin sensitizing and beneficial anti-inflammatory effects. Drug discovery has focused on agonists to replicate acute benefits of FFA receptor signaling, with promising early results for FFA1 agonists in man. Controversy surrounding chronic effects of FFA1 on beta-cells illustrates that long term benefits of antagonists also need exploring. It has proved challenging to generate highly selective potent ligands for FFA1 or GPR120 subtypes, given that both receptors have hydrophobic orthosteric binding sites, which are not completely defined and have modest ligand affinity. Structure activity relationships are also reliant on functional read outs, in the absence of robust binding assays to provide direct affinity estimates. Nevertheless synthetic ligands have already helped dissect specific contributions of FFA1 and GPR120 signaling from the many possible cellular effects of FFAs. Approaches including use of fluorescent ligand binding assays, and targeting allosteric receptor sites, may improve further pre-clinical ligand development at these receptors, to exploit their unique potential to target multiple facets of diabetes

Microtubule organization is determined by the shape of epithelial cells.

Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia.This work was supported by grant BB/K00056X/1 from the UK Biotechnology, Biological Sciences Research Council. Gurdon Institute core funding was provided by the Wellcome Trust (092096) and Cancer Research UK (C6946/A14492). L.C. was supported by the Royal Society of Edinburgh/Scottish Government

Building a pipeline to solicit expert knowledge from the community to aid gene summary curation.

Brief summaries describing the function of each gene's product(s) are of great value to the research community, especially when interpreting genome-wide studies that reveal changes to hundreds of genes. However, manually writing such summaries, even for a single species, is a daunting task; for example, the Drosophila melanogaster genome contains almost 14 000 protein-coding genes. One solution is to use computational methods to generate summaries, but this often fails to capture the key functions or express them eloquently. Here, we describe how we solicited help from the research community to generate manually written summaries of D. melanogaster gene function. Based on the data within the FlyBase database, we developed a computational pipeline to identify researchers who have worked extensively on each gene. We e-mailed these researchers to ask them to draft a brief summary of the main function(s) of the gene's product, which we edited for consistency to produce a 'gene snapshot'. This approach yielded 1800 gene snapshot submissions within a 3-month period. We discuss the general utility of this strategy for other databases that capture data from the research literature. Database URL: https://flybase.org/

Alternative mechanisms for talin to mediate integrin function.

Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. We show that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. We confirmed these alternatives by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, we show that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.This work was supported by grants from the Wellcome Trust (069943 and 086451) and the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/L006669/1) to N.H.B., a BBSRC studentship to J.W. (BB/D526102/1), and a grant from the Royal Society and Medical Research Council (MR/K015664/1) to M.P.This is the final published version. It first appeared at http://www.cell.com/current-biology/fulltext/S0960-9822%2815%2900075-5

Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis

BackgroundAtopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear.ObjectiveWe sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin.MethodsWe applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set.ResultsTwo thousand four hundred thirty differentially expressed genes (false discovery rate, P < .05) were identified, of which 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for “extracellular space” and “defense response” were enriched, whereas “lipid metabolic processes” were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferon–mediated stress response.ConclusionThese analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development