The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events

Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome

First systematic analysis of the evolutionary conserved InR/TOR pathway interaction proteome in Drosophila.Quantitative mass spectrometry revealed that 22% of identified protein interactions are regulated by the growth hormone insulin affecting membrane proximal as well as intracellular signaling complexes.Systematic RNA interference linked a significant fraction of network components to the control of dTOR kinase activity.Combined biochemical and genetic data suggest dTTT, a dTOR-containing complex required for cell growth control by dTORC1 and dTORC2 in vivo

Lesson from the Stoichiometry Determination of the Cohesin Complex: A Short Protease Mediated Elution Increases the Recovery from Cross-Linked Antibody-Conjugated Beads

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analysesssuch as the stoichiometry determination of protein complexesshave become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periodssas performed in standard on-bead digestionsled to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with

An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations

Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein's molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.Peer reviewe

Caring in mind? Professionals’ awareness of young carers and young adult carers in Switzerland

Findings from international research emphasis the need of these young people to be identified and recognised. Therefore, a nationwide quantitative study of professionals' awareness was conducted in the Swiss context. Data were collected from professionals working in education, healthcare and social services. The study examined professionals’ familiarity with the terms used to describe young people with caring responsibilities; their ability to identify the population in their professional context; their perception of the relevance of the issue; their ability to support them; and their own training needs. Potential study participants were contacted via email. Data were collected using an online survey, which was open for 14 weeks during the period of September to December 2016. Survey data from 2,311 professionals, who reported being regularly in contact with young people with caring responsibilities in their occupational context, were included for analysis. Of all terms presented in the survey, young carer (YC) was the term most familiar to all professionals. Among healthcare and education sector, the percentage of professionals familiar with this term was higher than that among professionals from social services. Professionals from social services were the most likely to consider the issue relevant to their work, those from education were the least likely. Professionals who were familiar with the terms describing YC were more likely to consider the issue relevant to their work. Study results showed that more than half of the respondents had not identified young people with caring responsibilities, and that the ability of participants to identify YC was related to their occupational sector. The perceived ability to support YC was related to their reported ability to recognise them. In order to enable professionals to support YC, raising awareness, providing training for professionals and a national network of existing organisations should be well considered

Macromolecular 3D SEM reconstruction strategies: signal to noise ratio and resolution

Three-dimensional scanning electron microscopy generates quantitative volumetric structural data from SEM images of macromolecules. This technique provides a quick and easy way to define the quaternary structure and handedness of protein complexes. Here, we apply a variety of preparation and imaging methods to filamentous actin in order to explore the relationship between resolution, signal-to-noise ratio, structural preservation and dataset size. This information can be used to define successful imaging strategies for different applications