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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.

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection., M.B. acknowledges support from 31/U/2016 from Fundació Marató de TV3. R.K. acknowledges support from an Australian Research Council Discovery Early Career Research Award (DE130100470). B.S. is supported by an Early Postdoc Mobility fellowship from the Swiss National Science Foundation (P2ZHP3_184024). M.J.S. is supported by a National Health and Medical Research Council (NHMRC) Senior Research Fellowship (APP1107914). M.S.-A. was recipient of a CNIC IPP fellowship (COFUND 2014). M.M. is supported by the Instituto de Salud Carlos III (FIS PI19/01410). O.T. is founded by Amgen 2018 Competitive Grant Program. A.P., R.G.P., S.P.G., and P.T.B. have been supported by RGP0020/2015 from the Human Frontier Science Program (HFSP). A.P. is supported by the Ministerio de Ciencia e Innovación (MICINN, RTI2018-098593-B-I00), Fundació Marató de TV3 (31/U/2016), and the CERCA Programme/Generalitat de Catalunya. R.G.P. was supported by the NHMRC of Australia (program grant APP1037320 and Senior Principal Research Fellowship 569452), and the Australian Research Council Centre of Excellence in Convergent Bio-Nanoscience and Technology (CE140100036). P.T.B. is supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) of Brazil and Fundaçao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). M.A.D.P. was funded by MICINN (project grants SAF2014-51876-R and SAF2017-83130-R; and IGP-SO grant MINSEV1512-07-2016) and was a Worldwide Cancer Research Foundation grantee (#15-0404). J.V. is supported by MICINN (BIO2015-67580-P) and from the Carlos III Institute of Health-Fondo de Investigación Sanitaria (PRB2, IPT13/0001—ISCIII-SGEFI/FEDER, ProteoRed). The CNIC is supported by the MICINN and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MICINN award SEV-2015-0505).

Lipid droplets (LDs) are important organelles for cell metabolism. Here, Du et al. show that phosphatidylinositol-4-phosphate produced by PI4K2A can exist on LDs and is used/consumed by ORP5, which localizes to ER–LD contacts during the growth of LDs., Lipid droplets (LDs) are evolutionarily conserved organelles that play important roles in cellular metabolism. Each LD is enclosed by a monolayer of phospholipids, distinct from bilayer membranes. During LD biogenesis and growth, this monolayer of lipids expands by acquiring phospholipids from the endoplasmic reticulum (ER) through nonvesicular mechanisms. Here, in a mini-screen, we find that ORP5, an integral membrane protein of the ER, can localize to ER–LD contact sites upon oleate loading. ORP5 interacts with LDs through its ligand-binding domain, and ORP5 deficiency enhances neutral lipid synthesis and increases the size of LDs. Importantly, there is significantly more phosphatidylinositol-4-phosphate (PI(4)P) and less phosphatidylserine (PS) on LDs in ORP5-deficient cells than in normal cells. The increased presence of PI(4)P on LDs in ORP5-deficient cells requires phosphatidylinositol 4-kinase 2-α. Our results thus demonstrate the existence of PI(4)P on LDs and suggest that LD-associated PI(4)P may be primarily used by ORP5 to deliver PS to LDs.

Parton et al. discuss how the proteins of caveolae, and the architecture of the domain, can concentrate specific lipids in caveolae. They propose that disassembly of caveolae in response to cellular stressors releases specialized lipids to modulate signaling., Caveolae are an abundant and characteristic surface feature of many vertebrate cells. The uniform shape of caveolae is characterized by a bulb with consistent curvature connected to the plasma membrane (PM) by a neck region with opposing curvature. Caveolae act in mechanoprotection by flattening in response to increased membrane tension, and their disassembly influences the lipid organization of the PM. Here, we review evidence for caveolae as a specialized lipid domain and speculate on mechanisms that link changes in caveolar shape and/or protein composition to alterations in specific lipid species. We propose that high membrane curvature in specific regions of caveolae can enrich specific lipid species, with consequent changes in their localization upon caveolar flattening. In addition, we suggest how changes in the association of lipid-binding caveolar proteins upon flattening of caveolae could allow release of specific lipids into the bulk PM. We speculate that the caveolae-lipid system has evolved to function as a general stress-sensing and stress-protective membrane domain.

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.

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule–associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.

Co-polymers of tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform specific manner. Previous work has implicated specific roles for at least 5 different tropomyosin isoforms in stress fibres, as depletion of any of these 5 isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude tropomyosins. In this study we investigate tropomyosin organisation in stress fibres using super resolution light microscopy and electron microscopy with genetically tagged, endogenous tropomyosin. We show that tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. Tpm3.1/3.2 co-localises with non-muscle myosin IIa/IIb heads and are in register but do not overlap with non-muscle myosin IIa/IIb tails. Furthermore, perturbation of Tpm3.1/3.2 results in decreased myosin IIa in stress fibres, which is consistent with a role for Tpm3.1 in maintaining myosin IIa localisation in stress fibres.

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and together with mitochondria key regulators of cell bioenergetics. LDs communicate with mitochondria and other organelles forming “metabolic synapse” contacts to ensure that lipid supply occurs where and when necessary. Although transmission electron microscopy analysis allows an accurate and precise analysis of contacts, the characterization of a large number of cells and conditions can become a long-term process. In order to extend contact analysis to hundreds of cells and multiple conditions, we have combined confocal fluorescence microscopy with advanced image analysis methods. In this work, we have developed the ImageJ macro script ContactJ, a novel and straight image analysis method to identify and quantify contacts between LD and mitochondria in fluorescence microscopy images allowing the automatic analysis. This image analysis workflow combines colocalization and skeletonization methods, enabling the quantification of LD-mitochondria contacts together with a complete characterization of organelles and cellular parameters. The correlation and normalization of these parameters contribute to the complex description of cell behavior under different experimental energetic states. ContactJ is available here: https://github.com/UB-BioMedMicroscopy/ContactJ/tree/1.0

International audience; We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease.

Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4+ and CD8+ T cell proliferation and resulted in an increased proportion of CD4+ central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment.

Annexin A6 (AnxA6) has been implicated in cell signalling by contributing to the organisation of the plasma membrane. Here we examined whether AnxA6 regulates signalling and proliferation in T cells. We used a contact hypersensitivity model to immune challenge wild-type (WT) and AnxA6(-/-) mice and found that the in vivo proliferation of CD4(+) T cells, but not CD8(+) T cells, was impaired in AnxA6(-/-) relative to WT mice. However, T-cell migration and signalling through the T-cell receptor ex vivo was similar between T cells isolated from AnxA6(-/-) and WT mice. In contrast, interleukin-2 (IL-2) signalling was reduced in AnxA6(-/-) compared with WT T cells. Further, AnxA6-deficient T cells had reduced membrane order and cholesterol levels. Taken together, our data suggest that AnxA6 regulates IL-2 homeostasis and sensitivity in T cells by sustaining a lipid raft-like membrane environment.

Caveolae, small bulb-like pits, are the most abundant surface feature of many vertebrate cell types. The relationship of the structure of caveolae to their function has been a subject of considerable scientific interest in view of the association of caveolar dysfunction with human disease. In a recent study Lo et al.1 investigated the organization and function of caveolae in skeletal muscle. Using quantitative 3D electron microscopy caveolae were shown to be predominantly organized into multilobed structures which provide a large reservoir of surface-connected membrane underlying the sarcolemma. These structures were preferentially disassembled in response to changes in membrane tension. Perturbation or loss of caveolae in mouse and zebrafish models suggested that caveolae can protect the muscle sarcolemma against damage in response to excessive membrane activity. Flattening of caveolae to release membrane into the bulk plasma membrane in response to increased membrane tension can allow cell shape...

Munc18-1 heterozygous mutations are associated with developmental diseases, including early infantile epileptic encephalopathy (EIEE). Chai et al. report that Munc18-1 acts as a chaperone for α-synuclein and controls its aggregative propensity. Munc18-1 EIEE-associated mutations promote the aggregation of endogenous α-synuclein in neurons, leading to a neurodegenerative phenotype., Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.

Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

Hedgehog (Hh)-containing exovesicles are mediators of long-range signaling in Drosophila wing imaginal discs. ESCRT-dependent multivesicular body (MVB) biogenesis is essential for generating Hh exovesicles. Oligomerization of Hh at the cell surface is necessary for its endocytic delivery to MVBs and therefore is required for its release as exovesicles., Hedgehog (Hh) is a secreted morphogen involved in both short- and long-range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We previously showed that the long-range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multivesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)–dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Of importance, oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long-range signaling.

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.

The eukaryotic cell surface is composed of many distinct membrane domains that are formed by the cooperative interactions of different proteins and lipids. These domains are important for membrane trafficking and cell signaling and are modulated in turn by changes in the cell environment. Caveolae (“little caves”) are ∼60-nm membrane invaginations (Fig. 1) that are a dominant surface feature of many mammalian cells, including muscle fibers, endothelia, and adipocytes, where they play a role in membrane homeostasis, signaling, and cellular mechanoprotection. Formation of caveolae in vertebrate cells requires two distinct protein families: the membrane-embedded caveolins (CAV1–3) and the peripheral membrane cavins (Cavin1–4). Although the general morphology of caveolae has been known for decades, the atypical structures of the protein subunits has meant that progress has been slow with regards to the high-resolution studies of caveola architecture. By high-resolution scanning electron microscopy (EM) and frozen deep-etch transmission EM, caveolae have been shown to be coated with striations (1, 2) or to possess spike-like structures (3), very different from other well-characterized vesicle coats, such as clathrin (4, 5). In PNAS, Stoeber et al. use a combination of biochemical dissection and EM to provide important insights into the underlying architecture of the caveola protein coat (6). Fig. 1. Proposed model for the assembly of caveolae. ( A ) Electron micrograph of caveolae in an adipocyte cell line. (Scale bar, 100 nm.) ( B ) Schematic diagrams depicting the main features of the CAV1 and Cavin1 proteins required for caveola formation. Cavins possess two regions of α-helical structure, termed HR1 and HR2, which are rich in basic residues. These are linked by disordered acidic sequences DR1–DR3. Caveolins are membrane-integral proteins that are embedded by a central helix–turn–helix. They also possess a conserved sequence suggested to be involved in oligomerization (oligomerization domain, OD) and a sequence with potential … [↵][1]1To whom correspondence may be addressed. Email: r.parton{at}imb.uq.edu.au or b.collins{at}imb.uq.edu.au. [1]: #xref-corresp-1-1

During heart morphogenesis, the cardiac chambers undergo ballooning: a process involving regionalized elongation of cardiomyocytes. Cardiomyocyte shape changes require reorganisation of the actin cytoskeleton however the genetic regulation of this process is not well understood. From a forward genetic screen, we identified the zebrafish uq mutant which manifests chamber ballooning defects. Whole-genome sequencing-mapping identified a truncating mutation in the gene, myo5b. myo5b encodes an atypical myosin required for endosome recycling and, consistent with this, increased vesicles were observed in myo5b mutant cardiomyocytes. Expression of RFP-Rab11a (a recycling endosome marker) confirmed increased recycling endosomes in cardiomyocytes of myo5b mutants. To investigate potential cargo of MyoVb-associated vesicles, we examined the Adherens Junction protein, N-cadherin. N-cadherin appeared mispatterned at cell junctions and an increase in the number of intracellular particles was also apparent. Co-localisation with RFP-Rab11a confirmed increased N-cadherin-positive recycling endosomes, demonstrating N-cadherin trafficking is perturbed in myo5b mutants. Finally, Phalloidin staining showed disorganised F-actin in myo5b cardiomyocytes, suggesting the cytoskeleton fails to remodel, obstructing chamber ballooning. MyoVb is required for cardiomyocyte endosomal recycling and appropriate N-cadherin localisation during the onset of chamber ballooning. Cardiomyocytes lacking MyoVb are unable to reorganize their actin cytoskeleton, resulting in failed chamber ballooning. This article is protected by copyright. All rights reserved.

We uncover how our polymersomes facet through a sphere-to-polyhedron shape transformation pathway that is driven by perylene aggregation confined within a topologically spherical polymersome shell., The creation of “soft” deformable hollow polymeric nanoparticles with complex non-spherical shapes via block copolymer self-assembly remains a challenge. In this work, we show that a perylene-bearing block copolymer can self-assemble into polymeric membrane sacs (polymersomes) that not only possess uncommonly faceted polyhedral shapes but are also intrinsically fluorescent. Here, we further reveal for the first time an experimental visualization of the entire polymersome faceting process. We uncover how our polymersomes facet through a sphere-to-polyhedron shape transformation pathway that is driven by perylene aggregation confined within a topologically spherical polymersome shell. Finally, we illustrate the importance in understanding this shape transformation process by demonstrating our ability to controllably isolate different intermediate polymersome morphologies. The findings presented herein should provide opportunities for those who utilize non-spherical polymersomes for drug delivery, nanoreactor or templating applications, and those who are interested in the fundamental aspects of polymersome self-assembly.

Most cells acquire cholesterol by endocytosis of circulating LDLs. After cholesteryl ester de-esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA-approved compounds in a high content, image-based screen of the endosomal lipids, lysobisphosphatidic acid and LDL-derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann-Pick type C (NPC), as well as in liver ofNpc1−/−mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.

The recent development of 3D electron microscopic techniques for cells and tissues has necessitated the development of new methods for the detection of proteins and protein-complexes in situ. The development of new genetic tags, such as the ascorbate peroxidase, APEX, for electron microscopic detection of tagged proteins has expanded the available toolbox and ushered in a new era in biological electron microscopy. Here, we describe methods for combining conditionally-stable nanobodies to fluorescent protein tags with APEX-based detection. These methods are compatible with detection of low levels of expression of fluorescently-tagged proteins and with detection of protein complexes using split GFP-based complementation methods. We describe a simple protocol for applying these methods to the electron microscopic detection of proteins and protein complexes in cultured cells.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis.

The molecular actions of retromer in the endolysosomal system remain unclear and controversial. Cui et al. demonstrate the essential role of retromer in the selective incorporation of cargo into a specific type of endosome transport carrier and the maintenance of lysosomal function., Retromer is a peripheral membrane protein complex that coordinates multiple vesicular trafficking events within the endolysosomal system. Here, we demonstrate that retromer is required for the maintenance of normal lysosomal morphology and function. The knockout of retromer subunit Vps35 causes an ultrastructural alteration in lysosomal structure and aberrant lysosome function, leading to impaired autophagy. At the whole-cell level, knockout of retromer Vps35 subunit reduces lysosomal proteolytic capacity as a consequence of the improper processing of lysosomal hydrolases, which is dependent on the trafficking of the cation-independent mannose 6-phosphate receptor (CI-M6PR). Incorporation of CI-M6PR into endosome transport carriers via a retromer-dependent process is restricted to those tethered by GCC88 but not golgin-97 or golgin-245. Finally, we show that this retromer-dependent retrograde cargo trafficking pathway requires SNX3, but not other retromer-associated cargo binding proteins, such as SNX27 or SNX-BAR proteins. Therefore, retromer does contribute to the retrograde trafficking of CI-M6PR required for maturation of lysosomal hydrolases and lysosomal function.

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level., Author summary The use of enzymatic tags such as the ascorbate peroxidase (APEX) for electron microscopic detection of proteins is changing electron microscopy (EM) in the same way that the use of GFP and related proteins caused a revolution in light microscopy. We previously developed expression plasmids encoding GFP-binding peptide (or nanobody) fused to APEX, which allows EM localisation of GFP-tagged proteins in vivo. Here, we have generated conditionally stable GFP- and mCherry-binding nanobodies fused to APEX. Using co-transfection of these APEX nanobodies with fluorescent-tagged constructs, we recruit APEX and detect the tagged proteins by electron microscopy. As unbound conditionally stable nanobodies are efficiently degraded by the proteasome, the signal to noise ratio is dramatically reduced. This enables detection of less abundant proteins and eliminates the need to balance expression levels between fluorescent-labelled and APEX nanobody constructs. Furthermore, and perhaps most exciting, is our application of this method to bimolecular fluorescence complementation—in which two tagged proteins interact—allowing the detection and localisation of protein-protein interactions in EM.

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS., eLife digest The inside of a human cell is divided into compartments called organelles, which are surrounded by membranes. Each organelle plays a specific role in keeping the cell healthy and also has unique mix of molecular markers on its surface. These markers allow other molecules to identify the different organelles, meaning that specific organelles can communicate with each other and coordinate their activities. One way that organelles can do this is via so-called membrane contact sites, which are small areas where the compartments’ outer membranes come close together. Mitochondria are organelles that release energy inside human cells. These compartments also work to keep the levels of toxic chemicals called reactive oxygen species in the cell within a safe range. This is important because cells can die if these levels become too high – a state known as oxidative stress. Mitochondria also communicate with other organelles called endosomes, which receive materials from the cell surface, sort and direct them to different destinations throughout the cell. In many diseases affecting the nervous system, the mitochondria and endosomes in nerve cells do not work properly. These cells also have higher than normal levels of oxidative stress. Hsu et al. therefore wanted to find out if mitochondria and endosomes worked together to help cells to cope with this kind of stress. Hsu et al. triggered oxidative stress in human cancer cells by exposing them first to a dye that stained the mitochondria and then to intense light. In stressed cells, a subset of endosomes called early endosomes formed many more membrane contact sites with mitochondria than in non-stressed cells. At the same time, the protein Rab5, usually found on early endosomes, relocated to the surface of mitochondria. Human cells previously engineered to produce larger than normal amounts of Rab5 were also more likely to survive oxidative stress. These experiments suggested that early endosomes cooperate with mitochondria, via Rab5, to protect cells from oxidative stress. So, how is Rab5 relocated to mitochondria? Hsu et al. searched for activators of Rab5 and found that Alsin also migrated to mitochondria in stressed cells. The gene for Alsin is also mutated in amyotrophic lateral sclerosis (ALS), a degenerative nerve disorder that remains poorly understood. Next, Hsu et al. deleted the gene for Alsin from human stem cells growing in the laboratory and coaxed these cells into becoming nerve cells. Experiments with these cells showed that the absence of Alsin prevented Rab5 from moving to the mitochondria. Nerve cells lacking Alsin were also more susceptible to oxidative stress than normal cells. Together, these findings show that early endosomes work with mitochondria to sense and ward off oxidative stress. They also reveal an unexpected connection between this process and a gene mutated in ALS. Further experiments are now needed to explore if problems with endosomes or mitochondria, and specifically with molecules like Alsin and Rab5, are responsible for other neurodegenerative disorders, like Parkinson’s disease and Huntington’s disease.

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.

Studying the interactions between nanoengineered materials and biological systems plays a vital role in the development of biological applications of nanotechnology and the improvement of our fundamental understanding of the bio-nano interface. A significant barrier to progress in this multidisciplinary area is the variability of published literature with regards to characterizations performed and experimental details reported. Here, we suggest a 'minimum information standard' for experimental literature investigating bio-nano interactions. This standard consists of specific components to be reported, divided into three categories: material characterization, biological characterization and details of experimental protocols. Our intention is for these proposed standards to improve reproducibility, increase quantitative comparisons of bio-nano materials, and facilitate meta analyses and in silico modelling. Refereed/Peer-reviewed

Compartmentalization is a functionally important property of the plasma membrane, yet the underlying principles that organize membrane proteins into distinct domains are not well understood. Using single molecule localization microscopy, we assessed the clustering of five model membrane proteins in the plasma membrane of HeLa cells. All five proteins formed discrete and distinct nano-scaled clusters. The extent of clustering of the five proteins, independent of their membrane anchors, increased significantly when the fluorescent protein mEOS2 was employed, suggesting that protein-protein interactions are a key driver for clustering. Further, actin depolymerization or reduction of membrane order had a greater, and in some instances opposing effects on the clustering of membrane proteins fused to mEOS2 compared to PS-CFP2-fusion proteins. The data propose that protein interactions can override the lateral organization imposed by membrane anchors to provide an exquisite regulation of the mosaic-like compartmentalization of the plasma membrane.

Background and Purpose Monocyte-derived macrophages are critical in the development of atherosclerosis and can adopt a wide range of functional phenotypes depending on their surrounding milieu. High-density lipoproteins (HDLs) have many cardio-protective properties including potent anti-inflammatory effects. We investigated the effects of HDL on human macrophage phenotype and the mechanisms by which these occur. Experimental Approach Human blood monocytes were differentiated into macrophages in the presence or absence of HDL and were then induced to either an inflammatory macrophage (M1) or anti-inflammatory macrophage (M2) phenotype using LPS and IFN-γ or IL-4, respectively. Key Results HDL inhibited the induction of macrophages to an M1-phenotype, as evidenced by a decrease in the expression of M1-specific cell surface markers CD192 and CD64, as well as M1-associated inflammatory genes TNF-α, IL-6 and MCP-1 (CCL2). HDL also inhibited M1 function by reducing the production of ROS. In contrast, HDL had no effect on macrophage induction to the M2-phenotype. Similarly, methyl-β-cyclodextrin, a non-specific cholesterol acceptor also suppressed the induction of M1 suggesting that cholesterol efflux is important in this process. Furthermore, HDL decreased membrane caveolin-1 in M1 macrophages. We confirmed that caveolin-1 is required for HDL to inhibit M1 induction as bone marrow-derived macrophages from caveolin-1 knockout mice continued to polarize into M1-phenotype despite the presence of HDL. Moreover, HDL decreased ERK1/2 and STAT3 phosphorylation in M1 macrophages. Conclusions and Implications We concluded that HDL reduces the induction of macrophages to the inflammatory M1-phenotype via redistribution of caveolin-1, preventing the activation of ERK1/2 and STAT3. Linked Articles This article is part of a themed section on Inflammation: maladies, models, mechanisms and molecules. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2016.173.issue-4