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Caveolae are abundant plasma membrane pits formed by the coordinated action of peripheral and integral membrane proteins and membrane lipids. Here, we discuss recent studies that are starting to provide a glimpse of how filamentous cavin proteins, membrane-embedded caveolin proteins, and specific plasma membrane lipids are brought together to make the unique caveola surface domain. Protein assembly involves multiple low-affinity interactions that are dependent on 'fuzzy' charge-dependent interactions mediated in part by disordered cavin and caveolin domains. We propose that cavins help generate a lipid domain conducive to full insertion of caveolin into the bilayer to promote caveola formation. The synergistic assembly of these dynamic protein complexes supports the formation of a metastable membrane domain that can be readily disassembled both in response to cellular stress and during endocytic trafficking. We present a mechanistic model for generation of caveolae based on these new insights.

SH2 domain containing tyrosine phosphatase 2 (Shp2; PTPN11) regulates several intracellular pathways downstream of multiple growth factor receptors. Our studies implicate that Shp2 interacts with Caveolin-1 (Cav-1) protein in retinal ganglion cells (RGCs) and negatively regulates BDNF/TrkB signaling. This study aimed to investigate the mechanisms underlying the protective effects of shp2 silencing in the RGCs in glaucomatous conditions. Methods: Shp2 was silenced in the Cav-1 deficient mice and the age matched wildtype littermates using adeno-associated viral (AAV) constructs. Shp2 expression modulation was performed in an acute and a chronic mouse model of experimental glaucoma. AAV2 expressing Shp2 eGFP-shRNA under a strong synthetic CAG promoter was administered intravitreally in the animals' eyes. The contralateral eye received AAV-eGFP-scramble-shRNA as control. Animals with Shp2 downregulation were subjected to either microbead injections or acute ocular hypertension experimental paradigm. Changes in inner retinal function were evaluated by measuring positive scotopic threshold response (pSTR) while structural and biochemical alterations were evaluated through H&E staining, western blotting and immunohistochemical analysis of the retinal tissues. Results: A greater loss of pSTR amplitudes was observed in the WT mice compared to Cav-1-/- retinas in both the models. Silencing of Shp2 phosphatase imparted protection against inner retinal function loss in chronic glaucoma model in WT mice. The functional rescue also translated to structural preservation of ganglion cell layer in the chronic glaucoma condition in WT mice which was not evident in Cav-1-/- mice retinas. Conclusions: This study indicates that protective effects of Shp2 ablation under chronic experimental glaucoma conditions are dependent on Cav-1 in the retina, suggesting in vivo interactions between the two proteins.

Epithelial collective cell migration requires the intrinsic locomotor activity of cells to be coordinated across populations. This coordination is governed by the presence of cell-cell adhesions as well as the cooperative behaviour of cells within the monolayer. Here, we report a role for Caveolin-1 (CAV1) in epithelial collective cell migration. CAV1 depletion reduced the migratory behaviour of AML12 liver epithelial cells when grown as monolayers, but not as individual cells. This suggested that CAV1 is a component of the process by which multicellular collectivity regulates epithelial motility. The correlation length for migration velocity was increased by CAV1 RNAi, a possible sign of epithelial jamming. However, CAV1 RNAi reduced migration, even when monolayers were allowed to migrate into unconfined spaces. The migratory defect was ameliorated by simultaneous depletion of the FMNL2 formin, whose cortical recruitment is increased in CAV1 RNAi cells. We therefore suggest that CAV1 modulates intraepithelial motility by controlling the cortical availability of FMNL2. Although epithelial collective cell migration has been observed in multiple contexts both in vivo and in vitro, the inherent coupling and coordination of activity between cells within the monolayer remain incompletely understood. Our study highlights a role for CAV1 in regulating intraepithelial motility, an effect that involves the formin FMNL2.

None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β(2)-adrenoreceptor (β(2)-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β(2)-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β(2)-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.

Background and Aims Growth hormone (GH) is important for liver regeneration after partial hepatectomy (PHx). We investigated this process in C57BL/6 mice that express different forms of the GH receptor (GHR) with deletions in key signaling domains. Approach and Results PHx was performed on C57BL/6 mice lacking GHR (Ghr −/−), disabled for all GH‐dependent Janus kinase 2 signaling (Box1 −/−), or lacking only GH‐dependent signal transducer and activator of transcription 5 (STAT5) signaling (Ghr391 −/−), and wild‐type littermates. C57BL/6 Ghr −/−mice showed striking mortality within 48 hours after PHx, whereas Box1 −/− or Ghr391 −/− mice survived with normal liver regeneration. Ghr −/− mortality was associated with increased apoptosis and elevated natural killer/natural killer T cell and macrophage cell markers. We identified H2‐Bl, a key immunotolerance protein, which is up‐regulated by PHx through a GH‐mediated, Janus kinase 2–independent, SRC family kinase–dependent pathway. GH treatment was confirmed to up‐regulate expression of the human homolog of H2‐Bl (human leukocyte antigen G [HLA‐G]) in primary human hepatocytes and in the serum of GH‐deficient patients. We find that injury‐associated innate immune attack by natural killer/natural killer T cell and macrophage cells are instrumental in the failure of liver regeneration, and this can be overcome in Ghr −/− mice by adenoviral delivery of H2‐Bl or by infusion of HLA‐G protein. Further, H2‐Bl knockdown in wild‐type C57BL/6 mice showed elevated markers of inflammation after PHx, whereas Ghr −/− backcrossed on a strain with high endogenous H2‐Bl expression showed a high rate of survival following PHx. Conclusions GH induction of H2‐Bl expression is crucial for reducing innate immune‐mediated apoptosis and promoting survival after PHx in C57BL/6 mice. Treatment with HLA‐G may lead to improved clinical outcomes following liver surgery or transplantation.

The blood-brain barrier (BBB) is a dynamic diffusional barrier regulating the molecular and chemical flux between the blood and brain, thereby preserving cerebral homeostasis. Endothelial cells form the core anatomical component of the BBB based on properties such as specialized junctional complexes between cells, which restricts paracellular transport, and extremely low levels of vesicular transport, restricting transcytosis. In performing its protective function, the BBB also constrains the entry of therapeutics into the brain, hampering the treatment of various neurological disorders. Focused ultrasound is a novel therapeutic modality that has shown efficacy in transiently and non-invasively opening the BBB for the targeted delivery of therapeutics to the brain. Although the ability of ultrasound to disrupt the junctional assembly of endothelial cells has been partially investigated, its effect on the transcellular mode of transport has been largely neglected. In this study, we found that ultrasound induces a pronounced increase in the levels of the vesicle-forming protein caveolin-1. In order to investigate the role of vesicle-mediated transcytoplasmic transport, we compared the leakage of various cargo sizes between a mouse model that lacks caveolin-1 and wild-type mice following sonication of the hippocampus. The absence of caveolin-1 did not lead to overt abnormalities in the cerebral vasculature in the mice. We found that caveolin-1 has a critical role specifically in the transport of large (500 kDa), but not smaller (3 and 70 kDa) cargoes. Our findings indicate differential effects of therapeutic ultrasound on cellular transport mechanisms, with implications for therapeutic interventions.

Retinal ganglion cell degeneration is a characteristic feature of glaucoma, and accordingly, protection of these cells constitutes a major therapeutic objective in the disease. Here, we demonstrate the key influence of caveolin (Cav) in regulating the inner retinal homeostasis in two models of experimentally elevated intraocular pressure (IOP). Two groups of Cav-1-/- and wild-type mice were used in the study. Animals were subjected to experimentally induced chronic and acutely elevated IOP and any changes in their retinal function were assessed by positive scotopic threshold response recordings. TUNEL and cleaved caspase-3 assays were performed to evaluate apoptotic changes in the retina while Brn3a immunostaining was used as a marker to assess and quantify ganglion cell layer (GCL) changes. H&E staining was carried out on retinal sections to evaluate histological differences in retinal laminar structure. Cav-1 ablation partially protected the inner retinal function in both chronic and acute models of elevated IOP. The protective effects of Cav-1 loss were also evident histologically by reduced loss of GCL density in both models. The phenotypic protection in Cav-1-/- glaucoma mice paralleled with increased TrkB phosphorylation and reduced endoplasmic reticulum stress markers and apoptotic activation in the inner retinas. This study corroborated previous findings of enhanced Shp2 phosphorylation in a chronic glaucoma model and established a novel role of Cav-1 in mediating activation of this phosphatase in the inner retina in vivo. Collectively, these findings highlight the critical involvement of Cav-1 regulatory mechanisms in ganglion cells in response to increased IOP, implicating Cav-1 as a potential therapeutic target in glaucoma.

TLR‐inducible zinc toxicity is an antimicrobial mechanism utilized by macrophages, however knowledge of molecular mechanisms mediating this response is limited. Here, we show that E. coli exposed to zinc stress within primary human macrophages reside in membrane‐bound vesicular compartments. Since SLC30A zinc exporters can deliver zinc into the lumen of vesicles, we examined LPS‐regulated mRNA expression of Slc30a/SLC30A family members in primary mouse and human macrophages. A number of these transporters were dynamically regulated in both cell populations. In human monocyte‐derived macrophages, LPS strongly up‐regulated SLC30A1 mRNA and protein expression. In contrast, SLC30A1 was not LPS‐inducible in macrophage‐like PMA‐differentiated THP‐1 cells. We therefore ectopically expressed SLC30A1 in these cells, finding that this was sufficient to promote zinc‐containing vesicle formation. The response was similar to that observed following LPS stimulation. Ectopically expressed SLC30A1 localized to both the plasma membrane and intracellular zinc‐containing vesicles within LPS‐stimulated THP‐1 cells. Inducible overexpression of SLC30A1 in THP‐1 cells infected with the Escherichia coli K‐12 strain MG1655 augmented the zinc stress response of intracellular bacteria and promoted clearance. Furthermore, in THP‐1 cells infected with an MG1655 zinc stress reporter strain, all bacteria contained within SLC30A1‐positive compartments were subjected to zinc stress. Thus, SLC30A1 marks zinc‐containing compartments associated with TLR‐inducible zinc toxicity in human macrophages, and its ectopic over‐expression is sufficient to initiate this antimicrobial pathway in these cells. Finally, SLC30A1 silencing did not compromise E. coli clearance by primary human macrophages, suggesting that other zinc exporters may also contribute to the zinc toxicity response., Graphical Abstract The zinc transporter SLC30A1 is LPS‐inducible in human macrophages and can deliver a zinc toxicity response against intracellular Escherichia coli.

It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies., Levels of the transcription factor Myc are deregulated in a range of different cancers. Here the authors show that Myc induces rapid transcriptional responses in some mouse tissues (liver), but not others (heart), and that transcriptional and proliferative responses in cardiomyocytes depend on the positive transcription elongation factor (P-TEFb).

The rapid spread of arthropod-borne Zika virus poses a serious public health threat that calls for effective ways of controlling and treating viral infection. This in turn necessitates better understanding of the mechanisms of virus assembly and its interaction with the host cells. In order to facilitate such efforts, we developed a new multihost expression vector pmCellFree that allows rapid and multiplexed production of ZIKV proteins in any in vitro translation system as well as in mammalian cells. Using a combination of in vitro expression in Leishmania cell-free system and AlphaLISA interaction assay, pairwise protein interactions of all ZIKV proteins were systematically tested. We identified thirty-three intraviral binary protein interactions, of which 13 interactions are novel. These findings were further validated by expressing selected protein pairs in mammalian HEK293T cell line and assessing their interactions in the cellular lysate. The results of these interaction assays were identical to those obtained with in vitro expressed proteins. The observed novel protein-protein interactions were further validated using a pulldown assay. The unrevealed novel protein interactions may point to the previously unappreciated complexity of the ZIKV assembly process and may play an important role in the infection process. These interactions may represent new targets for antiviral drug development.

Cells remodel their structure in response to mechanical strain. However, how mechanical forces are translated into biochemical signals that coordinate the structural changes observed at the plasma membrane (PM) and the underlying cytoskeleton during mechanoadaptation is unclear. Here, we show that PM mechanoadaptation is controlled by a tension-sensing pathway composed of c-Abl tyrosine kinase and membrane curvature regulator FBP17. FBP17 is recruited to caveolae to induce the formation of caveolar rosettes. FBP17 deficient cells have reduced rosette density, lack PM tension buffering capacity under osmotic shock, and cannot adapt to mechanical strain. Mechanistically, tension is transduced to the FBP17 F-BAR domain by direct phosphorylation mediated by c-Abl, a mechanosensitive molecule. This modification inhibits FBP17 membrane bending activity and releases FBP17-controlled inhibition of mDia1-dependent stress fibers, favoring membrane adaptation to increased tension. This mechanoprotective mechanism adapts the cell to changes in mechanical tension by coupling PM and actin cytoskeleton remodeling., Mechanical forces are sensed by cells and can alter plasma membrane properties, but biochemical changes underlying this are not clear. Here the authors show tension is sensed by c-Abl and FBP17, which couples changes in mechanical tension to remodelling of the plasma membrane and actin cytoskeleton.

Cell morphology and tissue integrity are essential for embryogenesis. Caveolins are membrane proteins that induce the formation of surface pits called caveolae that serve as membrane reservoirs for cell and tissue protection during development. In vertebrates, caveolin 1 (Cav1) and caveolin 3 (Cav3) are required for caveola formation. However, the formation of caveola and the function of caveolins in invertebrates are largely unknown. In this study, three caveolins, Cav‐a, Cav‐b, and CavY, are identified in the genome of the invertebrate chordate Ciona spp. Based on phylogenetic analysis, Cav‐a is found to be closely related to the vertebrate Cav1 and Cav3. In situ hybridization shows that Cav‐a is expressed in Ciona embryonic notochord and muscle. Cell‐free experiments, model cell culture systems, and in vivo experiments demonstrate that Ciona Cav‐a has the ability to induce membrane curvature at the plasma membrane. Knockdown of Cav‐a in Ciona embryos causes loss of invaginations in the plasma membrane and results in the failure of notochord elongation and lumenogenesis. Expression of a dominant‐negative Cav‐a point mutation causes cells to change shape and become displaced from the muscle and notochord to disrupt tissue integrity. Furthermore, we demonstrate that Cav‐a vesicles show polarized trafficking and localize at the luminal membrane during notochord lumenogenesis. Taken together, these results show that the invertebrate chordate caveolin from Ciona plays crucial roles in tissue integrity and morphology by inducing membrane curvature and intracellular vesicle trafficking during embryogenesis.

Bosch et al. discuss the role of lipid droplets as a front-line defense in the immune system., In the ongoing conflict between eukaryotic cells and pathogens, lipid droplets (LDs) emerge as a choke point in the battle for nutrients. While many pathogens seek the lipids stored in LDs to fuel an expensive lifestyle, innate immunity rewires lipid metabolism and weaponizes LDs to defend cells and animals. Viruses, bacteria, and parasites directly and remotely manipulate LDs to obtain substrates for metabolic energy, replication compartments, assembly platforms, membrane blocks, and tools for host colonization and/or evasion such as anti-inflammatory mediators, lipoviroparticles, and even exosomes. Host LDs counterattack such advances by synthesizing bioactive lipids and toxic nucleotides, organizing immune signaling platforms, and recruiting a plethora of antimicrobial proteins to provide a front-line defense against the invader. Here, we review the current state of this conflict. We will discuss why, when, and how LDs efficiently coordinate and precisely execute a plethora of immune defenses. In the age of antimicrobial resistance and viral pandemics, understanding innate immune strategies developed by eukaryotic cells to fight and defeat dangerous microorganisms may inform future anti-infective strategies.

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.

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target., Author summary All life depends on a cell envelope to enclose the chemical reactions that make life possible. But how do cell envelopes grow? How each component of the cell envelope is incorporated into the envelope at the correct amount, in the correct place, and at the correct time, to prevent cell death, has been a long-standing question in bacteriology. Using a unique combination of high throughput chemical genetic screens we identified yhcB, a conserved gene of unknown function, required for the maintenance of cell envelope integrity in Escherichia coli. Loss of YhcB results in aberrant cell size driven by the production of excess membrane phospholipids. Subsequent molecular and biochemical analyses suggest YhcB influences the spatiotemporal biogenesis of LPS, peptidoglycan and membrane phospholipids. Our data indicate YhcB is a key regulator of cell envelope growth in Gram-negative bacteria playing a crucial role in coordinating cell width, elongation, and division to maintain cell envelope integrity.

Classical cadherin cell adhesion receptors are integral membrane proteins that mediate cell–cell interactions, tissue integrity and morphogenesis. Cadherins are best understood to function as membrane-spanning molecular composites that couple adhesion to the cytoskeleton. On the other hand, the membrane lipid environment of the cadherins is an under-investigated aspect of their cell biology. In this review, we discuss two lines of research that show how the membrane can directly or indirectly contribute to cadherin function. Firstly, we consider how modification of its local lipid environment can potentially influence cadherin signalling, adhesion and dynamics, focusing on a role for phosphoinositide-4,5-bisphosphate. Secondly, we discuss how caveolae may indirectly regulate cadherins by modifying either the lipid composition and/or mechanical tension of the plasma membrane. Thus, we suggest that the membrane is a frontier of cadherin biology that is ripe for re-exploration.

Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus., Caveolae are plasma membrane invaginations containing cavin proteins that are disrupted upon stress stimuli, causing cavin release inside the cell. Here, McMahon et al. identify cavin interacting proteins using proteomic analyses and reveal functions in stress signaling that can promote apoptosis.

Glioblastoma (GBM) is the most common primary brain cancer. The average survival time for the majority of patients is approximately 15 months after diagnosis. A major feature of GBM that contributes to its poor prognosis is its high invasiveness. Caveolae are plasma membrane subdomains that participate in numerous biological functions. Caveolin-1 and Caveolae Associated Protein 1 (CAVIN1), formerly termed Polymerase I and Transcript Release Factor, are both necessary for caveola formation. We hypothesized that high expression of caveola-forming proteins in GBM promotes invasiveness via modulation of the production of matrix-degrading enzymes. The mRNA expression of caveola-forming proteins and matrix proteases in GBM samples, and survival after stratifying patients according to caveolin-1 or CAVIN1 expression, were analyzed from TCGA and REMBRANDT databases. The proteolytic profile of cell lines expressing or devoid of caveola-forming proteins was investigated using zymography and real-time qPCR. Invasion through basement membrane-like protein was investigated in vitro. Expression of both caveolin-1 and CAVIN1 was increased in GBM compared to normal samples and correlated with expression of urokinase plasminogen activator (uPA) and gelatinases. High expression of caveola-forming proteins was associated with shorter survival time. GBM cell lines capable of forming caveolae expressed more uPA and matrix metalloproteinase-2 (MMP-2) and/or -9 (MMP-9) and were more invasive than GBM cells devoid of caveola-forming proteins. Experimental manipulation of caveolin-1 or CAVIN1 expression in GBM cells recapitulated some, but not all of these features. Caveolae modulate GBM cell invasion in part via matrix protease expression.

Three dimensional engineered culture systems are powerful tools to rapidly expand our knowledge of human biology and identify novel therapeutic targets for disease. Bioengineered skeletal muscle has been recently shown to recapitulate many features of native muscle biology. However, current skeletal muscle bioengineering approaches require large numbers of cells, reagents and labour, limiting their potential for high-throughput studies. Herein, we use a miniaturized 96-well micro-muscle platform to facilitate semi-automated tissue formation, culture and analysis of human skeletal micro muscles (hμMs). Utilising an iterative screening approach we define a serum-free differentiation protocol that drives rapid, directed differentiation of human myoblast to skeletal myofibres. The resulting hμMs comprised organised bundles of striated and functional myofibres, which respond appropriately to electrical stimulation. Additionally, we developed an optogenetic approach to chronically stimulate hμM to recapitulate known features of exercise training including myofibre hypertrophy and increased expression of metabolic proteins. Taken together, our miniaturized approach provides a new platform to enable high-throughput studies of human skeletal muscle biology and exercise physiology.

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

Background Caveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non‐caveolar caveolin‐1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1‐induced metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis‐promoting microRNAs. Results We identify hnRNPK as a CAV1‐regulated microRNA binding protein. In the absence of CAVIN1, non‐caveolar CAV1 drives localisation of hnRPNK to multi‐vesicular bodies (MVBs), recruiting AsUGnA motif‐containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl‐β‐cyclodextrin or by treatment with n‐3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer. Conclusions Taken together, these results support a novel pan‐cancer mechanism for CAV1‐driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment., Highlights 1. Non‐caveolar caveolin‐1 drives non‐canonical localization of hnRNPK to multivesicular bodies (MVB) via membrane raft‐dependent mechanism. 2. MVB localized hnRNPK mediates EV loading of AsUGnA motif‐containing miRNA, which is required for EV‐induced in vitro osteoclastogenesiss. 3. Elevated EV hnRNPK in biofluids from metastatic cancer patients suggest a role as a biomarker.

The aetiology and progression of hypertension involves various endogenous systems, such as the renin angiotensin system, the sympathetic nervous system, and endothelial dysfunction. Recent data suggest that vascular inflammation may also play a key role in the pathogenesis of hypertension. This study sought to determine whether high intraluminal pressure results in vascular inflammation. Leukocyte adhesion was assessed in rat carotid arteries exposed to 1 h of high intraluminal pressure. The effect of intraluminal pressure on signaling mechanisms including reactive oxygen species production (ROS), arginase expression, and NFĸB translocation was monitored. 1 h exposure to high intraluminal pressure (120 mmHg) resulted in increased leukocyte adhesion and inflammatory gene expression in rat carotid arteries. High intraluminal pressure also resulted in a downstream signaling cascade of ROS production, arginase expression, and NFĸB translocation. This process was found to be angiotensin II-independent and mediated by the mechanosensor caveolae, as caveolin-1 (Cav1)-deficient endothelial cells and mice were protected from pressure-induced vascular inflammatory signaling and leukocyte adhesion. Cav1 deficiency also resulted in a reduction in pressure-induced glomerular macrophage infiltration in vivo. These findings demonstrate Cav1 is an important mechanosensor in pressure-induced vascular and renal inflammation.

We integrated quantitative nanoscale lipid mapping with molecular dynamic simulations to define the lipid profile within caveolae. Caveolin-1 and cavin1 individually sort distinct plasma membrane lipids. Intact caveolae containing both caveolin-1 and cavin1 further generate a distinct lipid profile, with headgroup and acyl chain selectivity., Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here, we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin-1 (CAV1) and cavin1 individually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodeling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking, and signaling.

Lonic et al. show that phosphorylation of Y374-PKCδ by FER arrests the maturation of early to late endosomes by inhibiting the release of RAB5 from nascent late endosomes. This promotes EGFR recycling and sustained signaling in triple-negative breast cancer., Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in ∼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.

Retromer core complex is an endosomal scaffold that plays a critical role in orchestrating protein trafficking within the endosomal system. Here we characterized the effect of the Parkinson's disease-linked Vps35 D620N in the endo-lysosomal system using Vps35 D620N rescue cell models. Vps35 D620N fully rescues the lysosomal and autophagy defects caused by retromer knock-out. Analogous to Vps35 knock out cells, the endosome-to-trans-Golgi network transport of cation-independent mannose 6-phosphate receptor (CI-M6PR) is impaired in Vps35 D620N rescue cells because of a reduced capacity to form endosome transport carriers. Cells expressing the Vps35 D620N variant have altered endosomal morphology, resulting in smaller, rounder structures with less tubule-like branches. At the molecular level retromer incorporating Vps35 D620N variant has a decreased binding to retromer associated proteins wiskott-aldrich syndrome protein and SCAR homologue (WASH) and SNX3 which are known to associate with retromer to form the endosome transport carriers. Hence, the partial defects on retrograde protein trafficking carriers in the presence of Vps35 D620N represents an altered cellular state able to cause Parkinson's disease.

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.

CAV1 (caveolin 1) expression and secretion is associated with prostate cancer (PCa) disease progression, but the mechanisms underpinning CAV1 release remain poorly understood. Numerous studies have shown CAV1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) CAV1 topology. Here we show that CAV1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive CAV1 secretion. Characterization of the isolated vesicles by electron microscopy, single-molecule fluorescence microscopy and proteomics reveals they represent a novel class of exosomes ~40 nm in diameter containing ~50-60 copies of CAV1 and, strikingly, are released via a non-canonical secretory macroautophagy/autophagy pathway. This study provides novel insights into a mechanism whereby CAV1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression. Abbreviations: 3-MA: 3-methyladenine; APEX: a modified soybean ascorbate peroxidase; ATG5: autophagy related 5; ATG9A: autophagy related 9A; ATG12: autophagy related 12; BHK: baby hamster kidney; C-exosomes: caveolin-exosomes; CAMKK2/CAMKKβ: calckum/calmodulin dependent protein kinase kinase 2; CAV1: caveolin 1; DAB: 3,3′-diaminobenzidine; DAPK: death associated protein kinase; EEA1: early endosome antigen 1; EM: electron microscopy; FCS: fluorescence correlation spectroscopy; GBP: GFP/YFP-binding peptide; GFP: green fluorescent protein; GOLGA2: golgin A2; ILVs: intralumenal vesicles; LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; MTORC1: mechanistic target of rapamycin kinase complex 1; MVBs: multivesicular bodies; PBS: phosphate-buffered saline; PCa: prostate cancer; PI3K: phosphoinositide 3-kinase; PM: plasma membrane; SFM: serum-free medium; TSG101: tumor susceptibility 101; WCL: whole cell lysates; WT: wild type; YFP: yellow fluorescent protein; βoG: β-octylglucoside

Epithelia must eliminate apoptotic cells to preserve tissue barriers and prevent inflammation.1 Several different mechanisms exist for apoptotic clearance, including efferocytosis2,3 and apical extrusion.4,5 We found that extrusion was the first-line response to apoptosis in cultured monolayers and in zebrafish epidermis. During extrusion, the apoptotic cell elicited active lamellipodial protrusions and assembly of a contractile extrusion ring in its neighbors. Depleting E-cadherin compromised both the contractile ring and extrusion, implying that a cadherin-dependent pathway allows apoptotic cells to engage their neighbors for extrusion. We identify RhoA as the cadherin-dependent signal in the neighbor cells and show that it is activated in response to contractile tension from the apoptotic cell. This mechanical stimulus is conveyed by a myosin-VI-dependent mechanotransduction pathway that is necessary both for extrusion and to preserve the epithelial barrier when apoptosis was stimulated. Earlier studies suggested that release of sphingosine-1-phosphate (S1P) from apoptotic cells might define where RhoA was activated. However, we found that, although S1P is necessary for extrusion, its contribution does not require a localized source of S1P in the epithelium. We therefore propose a unified view of how RhoA is stimulated to engage neighbor cells for apoptotic extrusion. Here, tension-sensitive mechanotransduction is the proximate mechanism that activates RhoA specifically in the immediate neighbors of apoptotic cells, but this also must be primed by S1P in the tissue environment. Together, these elements provide a coincidence detection system that confers robustness on the extrusion response. Refereed/Peer-reviewed

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex., It is unclear how the T-tubule structure of skeletal muscle, which regulates coordinated muscle contraction, forms. Here, the authors develop a four-dimensional quantitative model for T-tubule formation in zebrafish, based on live imaging, proposing a dynamic endocytic capture model.

Caveolin-1 (Cav1) expression and secretion is associated with prostate cancer (PCa) disease progression but the mechanisms underpinning Cav1 release remain poorly understood. Numerous studies have shown Cav1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) Cav1 topology. Here we show that Cav1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive Cav1 secretion. Characterization of the isolated vesicles by electron microscopy, single molecule fluorescent microscopy and proteomics reveals they represent a novel class of exosomes ∼40 nm in diameter containing ∼50-60 copies of Cav1 and strikingly, are released via a non-canonical secretory autophagy pathway. This study provides novel insights into a mechanism whereby Cav1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression.

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction that shows dysmorphology in a number of genetically inherited muscle diseases. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we have used the optically transparent and genetically tractable zebrafish system to probe T-tubule developmentin vivo. By combining live imaging with three-dimensional electron microscopy we derived a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formationin vivowe developed a quantitative screen for proteins that associate with and modulate early T-tubule formation including an overexpression screen of the entire zebrafish Rab protein family. We propose a new endocytic capture model involving i) formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma ii) stabilization by myofibrils/sarcoplasmic reticulum and iii) delivery of membrane from the recycling endosome and Golgi complex.

Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin1 (CAV1) and cavin1 individually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodelling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking and signalling.

The zebrafish is a vertebrate model suited to the exploration of cell biology within a whole organism. Hypotheses in cell mechanics can be tested by using the zebrafish notochord as a manipulable experimental system. Here, the methodologies to prepare, label, and simultaneously induce and image mechanical loading on live zebrafish notochord cells via electrical stimulation are described. This approach investigates membrane mechanics in a live, physiological setting and is thus suited for caveola research where observations within the tissues of an intact organism are increasingly relevant. This chapter also aims to introduce fundamental methodologies for the use of zebrafish in "in vivo cell biology."

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

The plasma membrane of eukaryotic cells is not a simple sheet of lipids and proteins but is differentiated into subdomains with crucial functions. Caveolae, small pits in the plasma membrane, are the most abundant surface subdomains of many mammalian cells. The cellular functions of caveolae have long remained obscure, but a new molecular understanding of caveola formation has led to insights into their workings. Caveolae are formed by the coordinated action of a number of lipid-interacting proteins to produce a microdomain with a specific structure and lipid composition. Caveolae can bud from the plasma membrane to form an endocytic vesicle or can flatten into the membrane to help cells withstand mechanical stress. The role of caveolae as mechanoprotective and signal transduction elements is reviewed in the context of disease conditions associated with caveola dysfunction.

3D computer-animated representations of complex biological systems and environments are often vastly oversimplified. There are a number of key reasons: to highlight a distinct biological mechanism of interest; technical limitations of hardware and software computer graphics (CG) capabilities; and a lack of data regarding cellular environments. This oversimplification perpetuates a naive understanding of fundamental cellular dynamics and topologies. This work attempts to address these challenges through the development of a first-person interactive virtual environment that more authentically depicts molecular scales, densities and interactions in real-time. Driven by a collaboration between scientists, CG developers and 3D computer artists, Nanoscapes utilizes the latest CG advances in real-time pipelines to construct a cinematic 3D environment that better communicates the complexity associated with the cellular surface and nanomedicine delivery to the cell.

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.

The capacity of cells and animals to sense and adapt to fluctuations in the availability of energetic substrates is commonly described as metabolic flexibility. This flexibility allows for example the transition from fed to fasting states and to meet the energy demands of exercise in both states. Flexibility is disrupted in pathological conditions such as the metabolic syndrome but in contrast, it is enhanced in some tumours. Lipid droplets (LDs) and mitochondria are key organelles in bioenergetics. In all eukaryotic cells, LDs store and supply essential lipids to produce signalling molecules, membrane building blocks, and the metabolic energy needed to survive during nutrient poor periods. Highly conserved, robust, and regulated mechanisms ensure these bioenergetic fluxes. Although mitochondria are recognized as the epicentre of metabolic flexibility, the contribution of LDs and LD-proteins is often neglected or considered detrimental. Here, we revisit the key roles of LDs during fasting and the intimate collaboration existing with mitochondria when cells sense and respond to fluctuations in substrate availability.

Caveolae are linked to signaling protein regulation through interactions with caveolins. We describe a cell-free system for the biogenesis of caveolae and show phosphorylated-caveolins preferentially bind signaling proteins. Our validation in vivo shows that phosphorylated CAV1 recruits TRAF2 to the endosome to form a signaling platform., Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae (Lt) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1Y14)–stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1.

Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42 which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negative curvature sensing protein, abolishes CG endocytosis. Furthermore, the identification of ARP2/3 complex at CG endocytic sites, maintained in an inactive state reveals a function for PICK1, an ARP2/3 inhibitor. The spatio-temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin-independent endocytic process. Coincident with the loss of PICK1 by GBF1-activated ARF1, CDC42 recruitment leads to the activation of IRSp53 and the ARP2/3 complex, resulting in a burst of F-actin polymerisation potentially powering scission., Several endocytic pathways operate simultaneously at the cell surface, including the clathrin and dynamin-independent CLIC/GEEC (CG) pathway. Here the authors show that small GTPases and BAR domain proteins regulate branched actin to make clathrin and dynamin-independent endocytic vesicles.

Lipid incorporation from the ER to lipid droplets (LDs) influences LD growth and intracellular lipid homeostasis. Xu et al. identify Rab18 as an important regulator of LD dynamics: activated Rab18 binds to ER-associated proteins such as the NRZ complex and SNAREs. The Rab18-NRZ-SNARE complex tethers LDs to the ER, facilitating lipid incorporation and LD growth., Lipid incorporation from endoplasmic reticulum (ER) to lipid droplet (LD) is important in controlling LD growth and intracellular lipid homeostasis. However, the molecular link mediating ER and LD cross talk remains elusive. Here, we identified Rab18 as an important Rab guanosine triphosphatase in controlling LD growth and maturation. Rab18 deficiency resulted in a drastically reduced number of mature LDs and decreased lipid storage, and was accompanied by increased ER stress. Rab3GAP1/2, the GEF of Rab18, promoted LD growth by activating and targeting Rab18 to LDs. LD-associated Rab18 bound specifically to the ER-associated NAG-RINT1-ZW10 (NRZ) tethering complex and their associated SNAREs (Syntaxin18, Use1, BNIP1), resulting in the recruitment of ER to LD and the formation of direct ER–LD contact. Cells with defects in the NRZ/SNARE complex function showed reduced LD growth and lipid storage. Overall, our data reveal that the Rab18-NRZ-SNARE complex is critical protein machinery for tethering ER–LD and establishing ER–LD contact to promote LD growth.

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum–plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4)P), and act as a PtdIns(4)P/phosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5)P 2, and not PtdIns(4)P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum–plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5)P 2 with little effect on PtdIns(4)P. Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4)P can also serve as co-exchangers for the transport of cargo lipids by ORPs. ORP5/8 are endoplasmic reticulum (ER) membrane proteins implicated in lipid trafficking that localize to ER-plasma membrane (PM) contacts and maintain membrane homeostasis. Here the authors show that PtdIns(4,5)P 2 plays a critical role in the targeting and function of ORP5/8 at the PM.

Lipids are known to influence tumour growth, inflammation and chemoresistance. However, the association of circulating lipids with the clinical outcome of metastatic castration-resistant prostate cancer (CRPC) is unknown. We investigated associations between the plasma lipidome and clinical outcome in CRPC. Lipidomic profiling by liquid chromatography-tandem mass spectrometry was performed on plasma samples from a Phase 1 discovery cohort of 96 CRPC patients. Results were validated in an independent Phase 2 cohort of 63 CRPC patients. Unsupervised analysis of lipidomic profiles (323 lipid species) classified the Phase 1 cohort into two patient subgroups with significant survival differences (HR 2.31, 95% CI 1.44-3.68, p = 0.0005). The levels of 46 lipids were individually prognostic and were predominantly sphingolipids with higher levels associated with poor prognosis. A prognostic three-lipid signature was derived (ceramide d18:1/24:1, sphingomyelin d18:2/16:0, phosphatidylcholine 16:0/16:0) and was also associated with shorter survival in the Phase 2 cohort (HR 4.8, 95% CI 2.06-11.1, p = 0.0003). The signature was an independent prognostic factor when modelled with clinicopathological factors or metabolic characteristics. The association of plasma lipids with CRPC prognosis suggests a possible role of these lipids in disease progression. Further research is required to determine if therapeutic modulation of the levels of these lipids by targeting their metabolic pathways may improve patient outcome.

Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across alpha E-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes a pathway for cadherin-based contractility to contribute to the physiology of established epithelia. Refereed/Peer-reviewed

SummaryTouch, hearing, and blood pressure control require mechanically-gated ion channels that convert mechanical stimuli into electrical currents. Piezo1 and Piezo2 were recently identified as essential eukaryotic mechanically-gated ion channels, yet how they respond to physical forces remains poorly understood. Here we use a multi-disciplinary approach to interrogate the interaction of Piezo1 with its lipid environment. We show that individual Piezo1 channels induce significant local curvature in the membrane that is magnified in a cooperative manner to generate larger curved ‘Piezo1 pits.’ Curvature decreases under lateral membrane tension, consistent with a hypothesis that force detection can involve sensing changes to local curvature. The protein alters its local membrane composition, enriching specific lipids and forming essential binding sites for phosphoinositides and cholesterol that are functionally relevant and often related to Piezo1-mediated pathologies. Finally, we show that Piezo1 alters the expression of lipid-regulating proteins and modifies the cellular lipidome. In short, we find that lipids influence Piezo1 activity and Piezo1 influences the local morphology and composition of the bilayer as well as the cellular lipidome.

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.

Summary A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

International audience; At the first EMBO conference dedicated to caveolae held in Le Pouliguen, France, May 12-16 (http://meetings.embo.org.gate2.inist.fr/event/19-caveolae), round-table discussions were used to address some of the long-standing issues in the field and to decide upon a consensus view regarding key aspects of caveola biology. Here we summarise some of the frequently asked questions (FAQs) posed by cell biologists about caveolae and provide some brief consensual answers based on these discussions. This article is protected by copyright. All rights reserved.