Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton

AbstractBackground: Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Many aspects of connexin function, for example cellular transport, plaque assembly and stability, and channel conductivity, are finely tuned and likely involve proteins that bind to connexins' cytoplasmic domains. However, little is known about such regulatory proteins. To identify novel proteins that interact with the COOH-terminal domain of Connexin-43 (Cx43), the most widely expressed connexin family member, we applied a proteomics approach to screen fractions of mouse tissue homogenates for binding partners.Results: Drebrin was recovered as a binding partner of the Cx43 COOH-terminal domain from mouse brain homogenate. Drebrin had previously been described as an actin binding protein that diminishes in brains during Alzheimer's disease. The novel Drebrin-Cx43 interaction identified by proteomics was confirmed by colocalization of endogenous proteins in astrocytes and Vero cells, coimmunoprecipitation, electron microscopy, electrophysiology, coexpression of both proteins with fluorescent tags, and live-cell FRET analysis. Depletion of Drebrin in cells with siRNA results in impaired cell-cell coupling, internalization of gap junctions, and targeting of Cx43 to a degradative pathway.Conclusions: We conclude that Drebrin is required for maintaining Cx43-containing gap junctions in their functional state at the plasma membrane. It is thus possible that Drebrin may interact with gap junctions in zones of cell-cell contacts in a regulated fashion in response to extracellular signals. The rearrangement or disruption of interactions between connexins and the Drebrin-containing submembrane cytoskeleton directs connexins to degradative cellular pathways

English version of the food disgust scale: Optimization and other considerations

© 2021 Wiley Periodicals LLC. The disgust elicited by food plays an important role in food choice and consumption. Recently, Hartmann and Siegrist (Food Quality and Preference, 2018, 63, 38–50) developed and validated in German the food disgust scale (FDS), a 32-item instrument designed to measure visceral disgust elicited by food. In Study 1, we tested the English language translation of the FDS and its shortened version (FDS-SHORT) in England (n = 85) and Canada (n = 70). The internal reliability (Cronbach's alpha and mean interitem correlation [MCI]) was acceptable for both the FDS (α =.90, MIC =.22) and the FDS-SHORT (α =.73, MIC =.25). Exploratory factor analysis revealed that the English and German versions of the FDS had similar underlying structure and good discriminant validity. In Study 2, female participants (n = 159) who completed the FDS where the anchor term disgusted was used had higher FDS-SHORT scores than either their male counterparts or females for whom the anchor term grossed out was used (F[2, 266] = 11.1, p less than  .001). As grossed out captures only visceral rather than moral disgust, we recommend its adoption in English versions of these scales. These studies confirm that, as modified, the English FDS and FDS-SHORT are reliable and can be used with confidence in future research

Structural basis for the binding of tryptophan-based motifs by δ-COP.

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.We should like to thank the beamline scientists at the Diamond Light Source and Mike Lewis (MRC LMB), Gerry Johnston (Dalhousie University), and Mark Rose (Princeton University) for helpful discussions and technical advice. RJS and DJO are funded by a Wellcome Trust fellowship to DJO (090909). PPP was funded by Canadian Institute of Health Research. RD acknowledges support from the DFG Excellence Cluster “Inflammation and Interfaces” (ECX306) and the University of Lübeck. SMT and FMH acknowledge support from NIH (GM071574). PRE is funded by MRC grant U105178845This is the author accepted manuscript. The final version is available from PNAS via http://dx.doi.org/10.1073/pnas.150618611

Provisionally pregnant: uncertainty and interpretive work in accounts of home pregnancy testing

Upon their availability for purchase in the 1970s, home pregnancy testing devices were hailed as a ‘revolution’ for women’s reproductive rights. Some authors, however, have described these technologies as further enabling the medicalisation of pregnancy and as contributing to the devaluing of women’s embodied knowledge. The home pregnancy test is one of many technological devices encountered by women experiencing pregnancy in the United Kingdom today. Existing literature has described how engagement with medical technologies during pregnancy might address uncertainties experienced at this time, providing women with reassurance and alleviating anxieties. Drawing on interviews with women living in Scotland, this article explores accounts of testing for a first pregnancy, and women’s descriptions of the impacts of home pregnancy testing upon experiences of early gestation. Participants engaged with pregnancy tests in varying ways, with uses shaping and shaped by their experiences of early pregnancy more broadly. Particular technical characteristics of the home pregnancy test led many participants to question their interpretation of a positive result, as well as the accuracy of the test itself. Rather than addressing the unknowns of early gestation by confirming a suspected pregnancy, a positive result could thus exacerbate uncertainty. Through participants’ accounts, this article shows how uncertainty is lived out by users of mundane techno-medical artefacts and sheds new light on women’s experiences of the first trimester of pregnancy

Real-time investigation of dynamic protein crystallization in living cells

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitroL.R., M.K., D.R., and C.B. thank the German Federal Ministry for Education and Research (BMBF) for funding (Grant Nos. 01KX0806 and 01KX0807). L.R., M.D., and C.B. acknowledge support from the BMBF in the context of the Röntgen-Angström-Cluster (Grant No. 05K12GU3). J.M.-C. and A.B.-N. acknowledge support from the Spanish Ministerio Economía y Competitividad (MINECO, Grant No. BFU2013-43513-R). I.V.M., R.D., and L.R. are grateful for support from the DFG Cluster of Excellence “Inflammation at Interfaces” (EXC 306)S