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Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission is, however, sparse. Here, we used microscopy-based tracking of individual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.

The Clinical Academic Careers Framework proposes an over-arching structure to develop the clinical academic workforce whose activities have patient benefit within a clear UK programme. Traditionally this has centred on professionals from medicine and dentistry, but in the last ten years has developed into a more inclusive career framework for non-medical health professions which includes Nurses, Midwives and Allied Health Professionals (NMAHPs) and Healthcare Scientists (HCSs) that provide NHS services. As such, it is reported that clinical academic NMAHPs and HCSs can contribute to the generation and translation of new knowledge to help improve outcomes and experiences for patients. In this article, we explore key issues relating to the impact of clinical academic NMAHPs and HCSs on clinical practice in a UK context, as well as some measurements of impact, including the value and limitations of currently used metrics (such as Key Performance Indicators, or KPIs). We report that measuring the learning in practice of this novel role will need to include smart metrics alongside a person-centred approach. We share four national case studies, all of which are drawn from clinical academic researchers from different UK settings to show the real variety and differences in roles. We argue that this is the key both to learning in practice about this role, and to witnessing the real differences clinical academics make.

Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions., eLife digest When cells become cancerous they often stop making certain proteins. This includes a protein known as cavin3 which resides in bulb-shaped pits of the membrane that surrounds the cell called caveolae. These structures work like stress detectors, picking up changes in the membrane and releasing proteins, such as cavin3, into the cell’s interior. Past studies suggest that cavin3 might interact with a protein called BRCA1 that suppresses the formation of tumors. Cells with mutations in the gene for BRCA1 struggle to fix damage in their DNA, and have to rely on other repair proteins, such as PARPs (short for poly (ADP-ribose) polymerases). Blocking PARP proteins with drugs can kill cancer cells with problems in their BRCA1 proteins. However, it was unclear what role cavin3 plays in this mechanism. To investigate this, McMahon et al. exposed cells grown in the laboratory to DNA-damaging UV light to stimulate the release of cavin3 from caveolae. This revealed that cavin3 interacts with BRCA1 when cells are under stress, and helps stabilize the protein so it can perform DNA repairs. Cells without cavin3 showed decreased levels of the BRCA1 protein, but compensated for the loss of BRCA1 by increasing the levels of their PARP proteins. These cells also had increased DNA damage following treatment with drugs that block PARPs, similar to cancer cells carrying mutations in the gene for BRCA1. These findings suggest that cavin3 helps BRCA1 to suppress the formation of tumors, and therefore should be considered when developing new anti-cancer treatments.

Identification of protein interaction networks is key for understanding intricate biological processes, but mapping such networks is challenging with conventional biochemical methods, especially for weak or transient interactions. Proximity-dependent biotin labelling (BioID) using promiscuous biotin ligases and mass spectrometry (MS)-based proteomics has emerged in the past decade as a powerful method for probing local proteomes and protein interactors. Here, we describe the application of an engineered biotin ligase, TurboID, for proteomic mapping and interactor screening in vivo in zebrafish. We generated novel transgenic zebrafish lines that express TurboID fused to a conditionally stabilised GFP-binding nanobody, dGBP, which targets TurboID to the GFP-tagged proteins of interest. The TurboID-dGBP zebrafish lines enable proximity-dependent biotin labelling in live zebrafish simply through outcrossing with existing GFP-tagged lines. Here, we outline a detailed protocol of the BLITZ method (Biotin Labelling In Tagged Zebrafish) for utilising TurboID-dGBP fish lines to map local proteomes and screen novel interactors. Graphic abstract: [Image: see text] Schematic overview of the BLITZ method. TurboID-dGBP fish are crossed with GFP-tagged lines to obtain embryos co-expressing TurboID-dGBP (indicated by mKate2) and the GFP-POI (protein of interest). Embryos expressing only TurboID are used as a negative control. Embryos (2 to 7 dpf) are incubated overnight with a 500 μM biotin-supplemented embryo medium. This biotin incubation step allows TurboID to catalyse proximity-dependent biotinylation in live zebrafish embryos. After biotin incubation, embryos are solubilised in lysis buffer, and free biotin is removed using a PD-10 desalting column. The biotinylated proteins are captured by streptavidin affinity purification, and captured proteins are analysed by MS sequencing.

Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in diverse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.

Caveolae-associated protein 3 (cavin3), a putative tumor suppressor protein, is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays, we show a direct interaction between BRCA1 and cavin3. Association of BRCA1 and cavin3 occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Supporting a role in DNA repair, cavin3-deficient cells were sensitized to the effects of PARP inhibition, which compromises DNA repair, and showed reduced recruitment of the BRCA1 A-complex to UV DNA damage foci. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. We conclude that cavin3 functions together with BRCA1 in multiple pathways that contribute to tumorigenesis.

This research paper centres on a pilot study where a/r/tography, an arts-based methodology, was implemented into a sport and exercise research context. A/r/tography is yet to be employed in this particular research context; therefore, an emphasis is placed on exploring the versatility and feasibility of this methodology when applied to the field of sport and exercise. In addition, we explore whether a/r/tography offers anything new and/or unique in comparison to other arts-based research that has already been conducted in this domain. In the paper that follows, we outline what a/r/tography is; why it could be considered as a methodological approach in sport and exercise research; and how it has been used in other research domains such as the social sciences. The remainder of the paper is dedicated to outlining the method that was undertaken in the a/r/tographic inquiry. A/r/tography was implemented with one swimmer participant and focused on the ‘normalised’ yet destructive ‘slim to win’ body prac...

Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P₂) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P₂, or disabling the interaction between FMNL2 and PtdIns(4,5)P₂. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton. Refereed/Peer-reviewed

In solid tumours, elevated interstitial fluid pressure (osmotic and hydrostatic pressure) is a barrier to drug delivery and correlates with poor prognosis. Glioblastoma (GBM) further experience compressive force when growing within a space limited by the skull. Caveolae are proposed to play mechanosensing roles, and caveola‐forming proteins are overexpressed in GBM. We asked whether caveolae mediate the GBM response to osmotic pressure. We evaluated in vitro the influence of spontaneous or experimental down‐regulation of caveola‐forming proteins (caveolin‐1, CAVIN1) on the proteolytic profile and invasiveness of GBM cells in response to osmotic pressure. In response to osmotic pressure, GBM cell lines expressing caveola‐forming proteins up‐regulated plasminogen activator (uPA) and/or matrix metalloproteinases (MMPs), some EMT markers and increased their in vitro invasion potential. Down‐regulation of caveola‐forming proteins impaired this response and prevented hyperosmolarity‐induced mRNA expression of the water channel aquaporin 1. CRISPR ablation of caveola‐forming proteins further lowered expression of matrix proteases and EMT markers in response to hydrostatic pressure, as a model of mechanical force. GBM respond to pressure by increasing matrix‐degrading enzyme production, mesenchymal phenotype and invasion. Caveola‐forming proteins mediate, at least in part, the pro‐invasive response of GBM to pressure. This may represent a novel target in GBM treatment.

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat., Caveolae are spherical nanodomains of the plasma membrane generated by assembly of caveolin and cavin proteins. Here, the authors show that fuzzy electrostatic interactions between caveolin-1 and Cavin1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

Mechanical tension governs epithelial morphogenesis and homeostasis, but its regulation remains poorly understood. Tension is commonly contractile, arising when the actomyosin cortices of cells are mechanically coupled together by cadherin adhesion. Here we report that caveolae control levels of epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) in the surrounding epithelium, but not in the oncogene-expressing cells, blocked extrusion leading to the retention and proliferation of transformed cells within the monolayer. Tensile stress was aberrantly elevated in CAV1-depleted monolayers due to elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) causing increased recruitment of the formin, FMNL2. Oncogenic extrusion was restored to CAV1-deficient monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, by controlling lipid signalling to the actin cytoskeleton, caveolae regulate mechanical tension for epithelial homeostasis.

SummaryReliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.

Cavin1 degradation is primarily mediated by the ubiquitin proteasome system. The phosphoinositide-binding region in cavin1 acts as a molecular switch for cavin1 degradation upon release of cavins in cytosol. This mechanism may help to maintain low levels of free cytosolic cavins at steady state., Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae.

Caveolae are plasma membrane invaginations involved in transport, signalling and mechanical membrane sensing in metazoans. Their formation depends upon multiple interactions between membrane-embedded caveolins, lipids and cytosolic cavin proteins. Of the four cavin family members, only cavin1 is strictly required for caveola formation. Here, we demonstrate that an eleven residue (undecad) repeat sequence (UC1) exclusive to cavin1 is essential for caveolar localization and promotes membrane remodelling through binding to phosphatidylserine. In the notochord of mechanically stimulated zebrafish embryos, the UC1 domain is required for caveolar stability and resistance to membrane stress. The number of undecad repeats in the cavin1 UC1 domain varies throughout evolution, and we find that an increased number also correlates with increased caveolar stability. Lastly, we show that the cavin1 UC1 domain induces dramatic remodelling of the plasma membrane when grafted into cavin2 suggesting an important role in membrane sculpting. Overall, our work defines a novel conserved cavin1 modular domain that controls caveolar assembly and stability.

I recently had the privilege of attending the 2016 Robert Burns Humanitarian Awards. At the awards ceremony, a Scottish government minister spoke of the very real potential of a collective sense of despair in the light of recent and current global events. The shortlisted candidates, he said, demonstrated that when humanity seems to be at its lowest point, the true potential of the human condition most often shines through. The humanitarian acts of the three men shortlisted for the 2016 award demonstrated just that. The message from the minister was, essentially, don’t despair, be inspired. It was indeed both humbling to learn of the personal sacrifice and commitment of each of the shortlisted candidates and it was inspiring to hear of the impact of their endeavours. The winner of the 2016 award was David Nott, a UK surgeon who for over 20 years has practised surgery in conflict and natural disaster zones including Bosnia, Afghanistan, Pakistan, Iraq, Syria, Yemen, Liberia, Chad, Ivory Coast, Libya, Sierra Leone, Central African Republic, Democratic Republic of the Congo and Haiti, very often at great personal risk. Clearly a man with exceptional skills and intellect, nevertheless his acceptance speech was tinged with humility. I could only imagine the Scottish poet Robert Burns, in whose name the awards are made, would have been impressed. The egalitarian Burns would expect no less, that each and every one of us use our skills and talents for our collective wellbeing ‘A man’s a man for a’ that’. In my experience as a nurse, I have been humbled and inspired almost every day of my career by colleagues who presume their extraordinary acts of human kindness and compassion to be nothing more than ordinary. They are not, and we should do much much more to acknowledge the contribution of nursing, not just in the face of conflict and natural disasters, but in the mundane messiness of everyday practice. The trouble is’ ‘though that good nursing care is often only visible to the untrained eye when it is set against poor care (or indeed no care). Arguably this can make it something of a challenge to defend the value of nursing if it is only recognised or valued when it is suboptimal. And it is ironic that it is the most expert of nurses, whose impact on the lives of individuals in their care is arguably the most profound, whose contribution is most poorly understood, or valued, by those who hold the purse strings.

This edition of JRN publishes six papers and their associated reviews that examine aspects of quality and safety in healthcare from a global nursing and healthcare perspective. There is increasing interest worldwide in the quality and safety of healthcare from governmental, organisational, professional and public perspectives and JRN has and will continue to contribute to these debates. The US Institute of Medicine (IOM) claims

The UK’s Research Excellence Framework (REF) 2014 rated the research from 154 universities, and the impact of research was evaluated in 6975 impact case studies. Nursing was returned within unit of assessment (UoA) 3, which also included Dentistry, Pharmacy and Allied Health Professions, although nursing research was also submitted within other UoAs. The study aim was to collate and categorise available REF impact case studies involving nursing researchers or on topics of relevance to nursing. Using nurs* as a search term, 469 case study entries were retrieved from the REF database and placed into three categories determined by the level of involvement of nurses. Some 80 impact case studies were submitted by nurses across 11 units of assessment, the majority being in UoA 3 ( n = 55). A further 50 revealed some relevant impact, although nurses did not have an obvious research role. A total of 248 case studies described actual or potential impact on health or social care but were not associated specifically with nursing. Nursing research has demonstrable impact; however, there is a significant body of research with relevance for nursing that has not been associated with the profession in the REF. More attention should be paid to the ‘hidden impact’ of nursing research to ensure the full impact of nursing is recognised.