RNF26 binds perinuclear vimentin filaments to integrate ER and endolysosomal responses to proteotoxic stress.

Proteotoxic stress causes profound endoplasmic reticulum (ER) membrane remodeling into a perinuclear quality control compartment (ERQC) for the degradation of misfolded proteins. Subsequent return to homeostasis involves clearance of the ERQC by endolysosomes. However, the factors that control perinuclear ER integrity and dynamics remain unclear. Here, we identify vimentin intermediate filaments as perinuclear anchors for the ER and endolysosomes. We show that perinuclear vimentin filaments engage the ER-embedded RING finger protein 26 (RNF26) at the C-terminus of its RING domain. This restricts RNF26 to perinuclear ER subdomains and enables the corresponding spatial retention of endolysosomes through RNF26-mediated membrane contact sites (MCS). We find that both RNF26 and vimentin are required for the perinuclear coalescence of the ERQC and its juxtaposition with proteolytic compartments, which facilitates efficient recovery from ER stress via the Sec62-mediated ER-phagy pathway. Collectively, our findings reveal a scaffolding mechanism that underpins the spatiotemporal integration of organelles during cellular proteostasis.

Oleic acid enhances proliferation and calcium mobilization of CD3/CD28 activated CD4+ T cells through incorporation into membrane lipids.

Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function.

The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER.

Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.

Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of the peptide. The aim of this study was to investigate the physical and functional characteristics of SAAP-148 NPs and to compare the selectivity index of the formulated peptide with that of the peptide in solution. SAAP-148 NPs displayed favorable physiochemical properties [size = 94.1 ± 23 nm, polydispersity index (PDI) = 0.08 ± 0.1, surface charge = 1.65 ± 0.1 mV, and encapsulation efficiency (EE) = 86.7 ± 0.3%] and sustained release of peptide for up to 21 days in PBS at 37 °C. The antibacterial and cytotoxicity studies showed that the selectivity index for SAAP-148 NPs was drastically increased, by 10-fold, regarding AMR Staphylococcus aureus and 20-fold regarding AMR Acinetobacter baumannii after 4 h. Interestingly, the antibiofilm activity of SAAP-148 NPs against AMR S. aureus and A. baumannii gradually increased overtime, suggesting a dose-effect relationship based on the peptide's in vitro release profile. Using 3D human skin equivalents (HSEs), dual drug SAAP-148 NPs and the novel antibiotic halicin NPs provided a stronger antibacterial response against planktonic and cell-associated bacteria than SAAP-148 NPs but not halicin NPs after 24 h. Confocal laser scanning microscopy revealed the presence of SAAP-148 NPs on the top layers of the skin models in close proximity to AMR S. aureus at 24 h. Overall, SAAP-148 NPs present a promising yet challenging approach for further development as treatment against bacterial infections.

Localization of active endogenous and exogenous ß-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts.

ß-Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor-targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol-derived activity-based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre-labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme.

Distinct antigen uptake receptors route to the same storage compartments for cross-presentation in dendritic cells.

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Ultrastructural Imaging of Salmonella-Host Interactions Using Super-resolution Correlative Light-Electron Microscopy of Bioorthogonal Pathogens.

The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM-CLEM approach thus presents a new approach to understand these pathogens during infection.

Restricted immune activation and internalisation of anti-idiotype complexes between drug and antidrug antibodies.

OBJECTIVES: Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. METHODS: Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. RESULTS: Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. CONCLUSIONS: Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.

Vitrification of Tokuyasu-style immuno-labelled sections for correlative cryo light microscopy and cryo electron tomography.

We present an approach for the preparation of immuno-labelled ultrathin sections from cells or tissue that are compatible with both fluorescence and transmission electron microscopy. Our approach is inspired by a method of Sabanay et al. (1991) that is based on the Tokuyasu technique for immunogold labelling of sections from aldehyde-fixed samples. The difference of this method with the original Tokuyasu technique is that the immuno-labelled sections are stabilized in a thin layer of vitreous water by plunge-freezing prior to electron microscopical observation. The vitrification step allows for phase contrast-based imaging at cryogenic conditions. We show that this immuno-labelling method is well-suited for imaging cellular ultrastructure in three dimensions (tomography) at cryogenic conditions, and that fluorescence associated with the sections is retained. This method is a valuable tool for Correlative Light and Electron Microscopy (CLEM), and we refer to this method in combination with CLEM as VOS (vitrification of sections). We provide examples for the application of VOS using dendritic cells and neurons, and show specifically that this method enables the researcher to navigate to lysosomes and synapses.

Encapsulation of SAAP-148 in Octenyl Succinic Anhydride-Modified Hyaluronic Acid Nanogels for Treatment of Skin Wound Infections.

Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204-253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37-41% in 72 h) upon reconstitution. Lyophilized SAAP-148-loaded nanogels showed similar antimicrobial activity as SAAP-148 against planktonic and biofilm-residing AMR Staphylococcus aureus and Acinetobacter baumannii. Importantly, formulated SAAP-148 showed reduced cytotoxicity against human erythrocytes, primary human skin fibroblasts and human keratinocytes. Additionally, lyophilized SAAP-148-loaded nanogels eradicated AMR S. aureus and A. baumannii colonizing a 3D human epidermal model, without inducing any cytotoxicity in contrast to SAAP-148. These findings indicate that OSA-HA nanogels increase SAAP-148's therapeutic potential for treatment of skin wound infections.

Life stage-specific glycosylation of extracellular vesicles from Schistosoma mansoni schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL.

Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells via C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcß1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host.

VISTA Expression on Cancer-Associated Endothelium Selectively Prevents T-cell Extravasation.

Cancers evade T-cell immunity by several mechanisms such as secretion of anti-inflammatory cytokines, down regulation of antigen presentation machinery, upregulation of immune checkpoint molecules, and exclusion of T cells from tumor tissues. The distribution and function of immune checkpoint molecules on tumor cells and tumor-infiltrating leukocytes is well established, but less is known about their impact on intratumoral endothelial cells. Here, we demonstrated that V-domain Ig suppressor of T-cell activation (VISTA), a PD-L1 homolog, was highly expressed on endothelial cells in synovial sarcoma, subsets of different carcinomas, and immune-privileged tissues. We created an ex vivo model of the human vasculature and demonstrated that expression of VISTA on endothelial cells selectively prevented T-cell transmigration over endothelial layers under physiologic flow conditions, whereas it does not affect migration of other immune cell types. Furthermore, endothelial VISTA correlated with reduced infiltration of T cells and poor prognosis in metastatic synovial sarcoma. In endothelial cells, we detected VISTA on the plasma membrane and in recycling endosomes, and its expression was upregulated by cancer cell-secreted factors in a VEGF-A-dependent manner. Our study reveals that endothelial VISTA is upregulated by cancer-secreted factors and that it regulates T-cell accessibility to cancer and healthy tissues. This newly identified mechanism should be considered when using immunotherapeutic approaches aimed at unleashing T cell-mediated cancer immunity.

An ACAP1-containing clathrin coat complex for endocytic recycling.

Whether coat proteins play a widespread role in endocytic recycling remains unclear. We find that ACAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF) 6, is part of a novel clathrin coat complex that is regulated by ARF6 for endocytic recycling in two key physiological settings, stimulation-dependent recycling of integrin that is critical for cell migration and insulin-stimulated recycling of glucose transporter type 4 (Glut4), which is required for glucose homeostasis. These findings not only advance a basic understanding of an early mechanistic step in endocytic recycling but also shed key mechanistic insights into major physiological events for which this transport plays a critical role.

Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield.

In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources.

A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography.

Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample.

Super-resolution correlative light-electron microscopy using a click-chemistry approach for studying intracellular trafficking.

Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.

The adapter protein Myd88 plays an important role in limiting mycobacterial growth in a zebrafish model for tuberculosis.

Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.

Intracellular Dynamic Assembly of Deep-Red Emitting Supramolecular Nanostructures Based on the Pt Pt Metallophilic Interaction.

Many drug delivery systems end up in the lysosome because they are built from covalent or kinetically inert supramolecular bonds. To reach other organelles, nanoparticles hence need to either be made from a kinetically labile interaction that allows re-assembly of the nanoparticles inside the cell following endocytic uptake, or, be taken up by a mechanism that short-circuits the classical endocytosis pathway. In this work, the intracellular fate of nanorods that self-assemble via the Pt…Pt interaction of cyclometalated platinum(II) compounds, is studied. These deep-red emissive nanostructures (638 nm excitation, ≈700 nm emission) are stabilized by proteins in cell medium. Once in contact with cancer cells, they cross the cell membrane via dynamin- and clathrin-dependent endocytosis. However, time-dependent confocal colocalization and cellular electron microscopy demonstrate that they directly move to mitochondria without passing by the lysosomes. Altogether, this study suggests that Pt…Pt interaction is strong enough to generate emissive, aggregated nanoparticles inside cells, but labile enough to allow these nanostructures to reach the mitochondria without being trapped in the lysosomes. These findings open new venues to the development of bioimaging nanoplatforms based on the Pt…Pt interaction.