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Contains fulltext : 292891.pdf (Publisher’s version ) (Open Access) Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.

The use of colloid supported lipid bilayers (CSLBs) for assembling colloidal structures has been of recent interest. Here, we use multi-component lipid bilayer membranes formed around anisotropic colloids and show that the curvature anisotropy of the colloids drives a sorting of the lipids in the membrane along the colloids. We then exploit this curvature-sensitive lipid sorting to create "shape-anisotropic patchy colloids" - specifically, we use colloids with six rods sticking out of a central cubic core, "hexapods", for this purpose and demonstrate that membrane patches self-assemble at the tip of each of the six colloidal rods. The membrane patches are rendered sticky using biotinylated lipids in complement with a biotin-binding streptavidin protein. Finally, using these "shape-anisotropic patchy colloids", we demonstrate the directed assembly of colloidal links, paving the way for the creation of heterogeneous and flexible colloidal structures.

Localized flux, production and/or degradation coupled to limited diffusion are well-known to result in stable spatial concentration gradients of biomolecules in the cell. In this study, we demonstrate that this also holds true for small ions, since we found that the close membrane apposition between the membrane of a phagosome and the surface of the cargo particle it encloses, suffices for stable gradients of protons and iron cations within the lumen of the phagosome. Our data show that in phagosomes containing hexapod-shaped silica colloid particles, the phagosomal membrane is ruptured at the positions of the tips of the rods, but not at other positions. This results in the confined leakage at these positions of protons and iron from the lumen of the phagosome into the cytosol. In contrast, acidification and iron accumulation still occur at the positions of the phagosomes of the cores of the particles. Our study strengthens the concept that the coupling of metabolic and signaling reaction cascades can be spatially confined by localized limited diffusion., ST, SS and GB are funded by a Young Investigator Grant from the Human Frontier Science Program (HFSP; RGY0080/2018). GB has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. 862137) and the Netherlands Organization for Scientific Research (Vidi grant NWO-ALW VIDI 864.14.001). ST further acknowledges support from the Department of Atomic Energy, Government of India, under project no. 12-R&D-TFR-5.04-0800 and 12-D&D-TFR-5.10-1100, the Simons Foundation (Grant No. 287975) and the Max Planck Society through a Max-Planck Partner-Group.

Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most haplotypes of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHCI and MHC-II. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells respond more to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs therefore are less prone to escape mutations that enable pathogens to evade T cell responses.

Since their invention about two decades ago, super-resolution microscopes have become a method of choice in cell biology. Owing to a spatial resolution below 50 nm, smaller than the size of most organelles, and an order of magnitude better than the diffraction limit of conventional light microscopes, super-resolution microscopy is a powerful technique for resolving intracellular trafficking. In this review we discuss discoveries in endocytosis and phagocytosis that have been made possible by super-resolution microscopy - from uptake at the plasma membrane, endocytic coat formation, and cytoskeletal rearrangements to endosomal maturation. The detailed visualization of the diverse molecular assemblies that mediate endocytic uptake will provide a better understanding of how cells ingest extracellular material.

Summary Antigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal “clickable” antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer., Graphical Abstract, Highlights • PIKfyve converts PI(3)P into PI(3,5)P2 on the surface of endosomes and phagosomes • PIKfyve of DCs modulates phagosomal maturation, acidification, and ROS production • PIKfyve defects disrupt cathepsin S trafficking and activity • Inactive cathepin S slows Ii processing for MHC class II trafficking and antigen loading, Molecular Mechanism of Gene Regulation; Immunology; Immune Response

The PIKfyve inhibitor apilimod is currently undergoing clinical trials for treatment of COVID-19. However, although apilimod might prevent viral invasion by inhibiting host cell proteases, the same proteases are critical for antigen presentation leading to T cell activation and there is good evidence from both in vitro studies and the clinic that apilimod blocks antiviral immune responses. We therefore warn that the immunosuppression observed in many COVID-19 patients might be aggravated by apilimod.

We recently identified a key role for SWAP70 as the tethering factor stabilizing F-actin filaments on the surface of phagosomes in human dendritic cells by interacting both with Rho-family GTPases and the lipid phosphatidylinositol (3,4)-bisphosphate. In this study, we aimed to investigate whether this role of SWAP70 was general among immune phagocytes. Our data reveal that SWAP70 is recruited to early phagosomes of macrophages and dendritic cells from both human and mouse. The putative inhibitor of SWAP70 sanguinarine blocked phagocytosis and F-actin polymerization, supporting a key role for SWAP70 in phagocytosis as demonstrated previously with knock-down. Moreover, SWAP70 was recently shown to sequester the F-actin severing protein cofilin and we investigated this relationship in phagocytosis. Our data show an increased activation of cellular cofilin upon siRNA knockdown of SWAP70. Finally, we explored whether SWAP70 would be recruited to the immune synapse between dendritic cells and T cells required for antigen presentation, as the formation of such synapses depends on F-actin. However, we observed that SWAP70 was depleted at immune synapses and specifically was recruited to phagosomes. Our data support an essential and specific role for SWAP70 in tethering and stabilizing F-actin to the phagosomal surface in a wide range of phagocytes.

Since chloroquine (CQ) and hydroxychloroquine (HCQ) can inhibit the invasion and proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cultured cells, the repurposing of these antimalarial drugs was considered a promising strategy for treatment and prevention of coronavirus disease (COVID-19). However, despite promising preliminary findings, many clinical trials showed neither significant therapeutic nor prophylactic benefits of CQ and HCQ against COVID-19. Here, we aim to answer the question of why these drugs are not effective against the disease by examining the cellular working mechanisms of CQ and HCQ in prevention of SARS-CoV-2 infections.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by Forster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-) quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.

The review considers different experimental and theoretical approaches to the investigation of RNA folding and identification of nucleotides that critically affect the folding of molecules, such as tRNA, and several classes of ribozymes. For instance, it has been shown that nucleotides of the D- and T-loop regions are the last to be involved in the tRNA structure, or, rather, they are not included in the tRNA folding nucleus. A specially developed SHAPE method was used to show that the long-recognized hierarchical folding model does not hold true for tRNA folding. In the second part of the review, algorithms and programs used for the prediction of RNA secondary structures, as well as for modeling RNA folding, are considered.

Contains fulltext : 136538.pdf (Publisher’s version ) (Open Access) Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here, we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for the podosomes of dendritic cells.

The role of exopolyphosphatase PPN1 in polyphosphate metabolism in fungi has been studied in strains of Saccharomyces cerevisiae transformed by the yeast PPN1 gene and its ortholog of the fungus Acremonium chrysogenum producing cephalosporin C. The PPN1 genes were expressed under a strong constitutive promoter of the gene of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae in the vector pMB1. The yeast strain with inactivated PPN1 gene was transformed by the above vectors containing the PPN1 genes of S. cerevisiae and A. chrysogenum. Exopolyphosphatase activity in the transformant with the yeast PPN1 increased 28- and 11-fold compared to the mutant and parent PPN1 strains. The amount of polyphosphate in this transformant decreased threefold. Neither the increase in exopolyphosphatase activity nor the decrease in polyphosphate content was observed in the transformant with the orthologous PPN1 gene of A. chrysogenum, suggesting the absence of the active form of PPN1 in this transformant.

Prediction of folding nuclei in RNA molecules allows one to look in a new way at the problem of possible RNA base sequence folding and at problems associated with incorrect RNA folding, as well as at RNA structure stability. We have chosen a model and energy parameters for description of RNA structure. The algorithm for studying processes including protein folding/unfolding was successfully applied to calculations on tRNA. Four tRNA molecules were considered whose structures were obtained in the free state (tRNA(Phe), tRNA(Asp), tRNA(fMet), and tRNA(Lys)). The calculated Φ-values for tRNA molecules correlate with experimental data showing that nucleotide residues in the D and T hairpin regions are involved in tRNA structure last, or more exactly, they are not included in the tRNA folding nucleus. High Φ-values in the anticodon hairpin region show that the nucleus of tRNA folding is localized just in that place.

We recently identified a novel role for podosomes in antigen sampling. Podosomes are dynamic cellular structures that consist of point-like concentrations of actin surrounded by integrins and adaptor proteins such as vinculin and talin. Podosomes establish cellular contact with the extracellular matrix (ECM) and facilitate cell migration via ECM degradation. In our recent paper, we studied podosomes of human dendritic cells (DCs), major antigen presenting cells (APC) that take-up, process, and present foreign antigen to naive T-cells. We employed gelatin-impregnated porous polycarbonate filters to demonstrate that the mechanosensitive podosomes of DCs selectively localize to regions of low-physical resistance such as the filter pores. After degradation of the gelatin, podosomes increasingly protrude into the lumen of these pores. These protrusive podosome-derived structures contain several endocytic and early endosomal markers such as clathrin, Rab5, and VAMP3, and, surprisingly, also contain C-type lectins, a type of pathogen recognition receptors (PRRs). Finally, we performed functional uptake experiments to demonstrate that these PRRs facilitate uptake of antigen from the opposite side of the filter. Our data provide mechanistic insight in how dendritic cells sample for antigen across epithelial barriers for instance from the lumen of the lung and gut.

Summary Actin plays a critical role during the early stages of pathogenic microbe internalization by immune cells. In this study, we identified a key mechanism of actin filament tethering and stabilization to the surface of phagosomes in human dendritic cells. We found that the actin-binding protein SWAP70 is specifically recruited to nascent phagosomes by binding to the lipid phosphatidylinositol (3,4)-bisphosphate. Multi-color super-resolution stimulated emission depletion (STED) microscopy revealed that the actin cage surrounding early phagosomes is formed by multiple concentric rings containing SWAP70. SWAP70 colocalized with and stimulated activation of RAC1, a known activator of actin polymerization, on phagosomes. Genetic ablation of SWAP70 impaired actin polymerization around phagosomes and resulted in a phagocytic defect. These data show a key role for SWAP70 as a scaffold for tethering the peripheral actin cage to phagosomes., Graphical Abstract, Highlights • SWAP70 is transiently recruited to the nascent cup of emerging phagosomes • Phagosomal recruitment of SWAP70 is driven by RAC1 and phosphoinositides • SWAP70 tethers concentric rings of F-actin on the phagosomal membrane • Absence of SWAP70 results in phagocytic defects, Baranov et al. find a key role for SWAP70 in tethering concentric rings of F-actin to emerging phagosomes. Specific phagosomal recruitment of SWAP70 is achieved by interactions with the GTPase RAC1 and phosphatidylinositol (3,4)-bisphosphate. Absence of SWAP70 leads to disruption of phagosomal actin and impaired phagocytosis.