RESUMO
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.
Assuntos
Coloides , Bicamadas Lipídicas , Anisotropia , Coloides/química , Bicamadas Lipídicas/química , Transporte ProteicoRESUMO
In this study, interactions of Fe3O4 magnetic nanoparticles with serum albumin biomolecules in aqueous solutions were considered. The studies were conducted with the laser correlation spectroscopy and optical analysis of dehydrated films. It was shown that the addition of magnetite to an albumin solution at low concentrations of up to 10-6 g/L led to the formation of aggregates with sizes of up to 300 nm in the liquid phase and an increase in the number of spiral structures in the dehydrated films, which indicated an increase in their stability. With a further increase in the magnetite concentration in the solution (from 10-4 g/L), the magnetic particles stuck together and to albumin, thus forming aggregates with sizes larger than 1000 nm. At the same time, the formation of morphological structures in molecular films was disturbed, and a characteristic decrease in their stability occurred. Most stable films were formed at low concentrations of magnetic nanoparticles (less than 10-4 g/L) when small albumin-magnetic nanoparticle aggregates were formed. These results are important for characterizing the interaction processes of biomolecules with magnetic nanoparticles and can be useful for predicting the stability of biomolecular films with the inclusion of magnetite particles.
Assuntos
Fenômenos Magnéticos , Agregados Proteicos , Albumina Sérica/química , Albumina Sérica/metabolismo , Algoritmos , Nanopartículas de Magnetita/química , Modelos Teóricos , Tamanho da Partícula , Ligação Proteica , Análise EspectralRESUMO
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.
Assuntos
Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Fagossomos/metabolismo , Compartimento Celular , Membrana Celular/metabolismo , Grupo dos Citocromos b/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Membranas Intracelulares/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Modelos Biológicos , NADPH Oxidase 2 , Oxirredução , Fosfatidilinositóis/metabolismo , Proteínas Qa-SNARE , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismoRESUMO
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.
Assuntos
Apresentação de Antígeno , Extensões da Superfície Celular/metabolismo , Células Dendríticas/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Endocitose , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
It is not well understood why severe acute respiratory syndrome (SARS)-CoV-2 spreads much faster than other ß-coronaviruses such as SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. In a previous publication, we predicted the binding of the N-terminal domain (NTD) of SARS-CoV-2 spike to sialic acids (SAs). Here, we experimentally validate this interaction and present simulations that reveal a second possible interaction between SAs and the spike protein via a binding site located in the receptor-binding domain (RBD). The predictions from molecular-dynamics simulations and the previously-published 2D-Zernike binding-site recognition approach were validated through flow-induced dispersion analysis (FIDA)âwhich reveals the capability of the SARS-CoV-2 spike to bind to SA-containing (glyco)lipid vesicles, and flow-cytometry measurementsâwhich show that spike binding is strongly decreased upon inhibition of SA expression on the membranes of angiotensin converting enzyme-2 (ACE2)-expressing HEK cells. Our analyses reveal that the SA binding of the NTD and RBD strongly enhances the infection-inducing ACE2 binding. Altogether, our work provides in silico, in vitro, and cellular evidence that the SARS-CoV-2 virus utilizes a two-receptor (SA and ACE2) strategy. This allows the SARS-CoV-2 spike to use SA moieties on the cell membrane as a binding anchor, which increases the residence time of the virus on the cell surface and aids in the binding of the main receptor, ACE2, via 2D diffusion.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2 , Ligação Proteica , Sítios de LigaçãoRESUMO
To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-µm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.
Assuntos
Fatores de Despolimerização de Actina , Actomiosina , Fatores de Despolimerização de Actina/metabolismo , Movimento Celular/fisiologia , Actomiosina/metabolismo , Citocinese , Cofilina 1/metabolismo , Matriz Extracelular/metabolismo , Células Dendríticas/metabolismoRESUMO
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.
Assuntos
Actinas , Complexos Endossomais de Distribuição Requeridos para Transporte , Integrinas , Actinas/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Integrinas/genética , Integrinas/metabolismo , Transporte Proteico , Fosfolipídeos/química , Membrana Celular , Macrófagos , Células Dendríticas , Fibroblastos , Humanos , Conformação ProteicaRESUMO
Localized fluxes, 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, together with localized membrane rupture, suffice 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 nearer to the cores of the particles. Our study strengthens the concept that coupling metabolic and signaling reaction cascades can be spatially confined by localized limited diffusion.
RESUMO
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.
Assuntos
Tratamento Farmacológico da COVID-19 , Cloroquina/uso terapêutico , Hidroxicloroquina/uso terapêutico , Antivirais/uso terapêutico , COVID-19/virologia , Proliferação de Células/efeitos dos fármacos , Cloroquina/efeitos adversos , Reposicionamento de Medicamentos , Humanos , Hidroxicloroquina/efeitos adversos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidadeRESUMO
Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most alleles 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 predicted over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHC-I and MHC-II, and confirmed by re-analysis of epitope databases. 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 are more tuned to respond to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs are less prone to mutations that enable pathogens to evade T cell responses.
Assuntos
Apresentação de Antígeno/genética , Epitopos de Linfócito T/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Estrutura Secundária de Proteína/genética , Alelos , Apresentação de Antígeno/imunologia , Cromossomos/genética , Cromossomos/imunologia , Citoplasma/genética , Citoplasma/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Proteínas de Membrana/imunologia , Peptídeos/genética , Peptídeos/imunologia , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
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.
Assuntos
Antivirais/efeitos adversos , Tratamento Farmacológico da COVID-19 , Hidrazonas/efeitos adversos , Morfolinas/efeitos adversos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/efeitos adversos , Pirimidinas/efeitos adversos , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , COVID-19/imunologia , Humanos , Hidrazonas/farmacologia , Morfolinas/farmacologia , Peptídeo Hidrolases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Proteases/farmacologia , Pirimidinas/farmacologia , Serina Endopeptidases/metabolismoRESUMO
The immune system has to cope with a wide range of irregularly shaped pathogens that can actively move (e.g., by flagella) and also dynamically remodel their shape (e.g., transition from yeast-shaped to hyphal fungi). The goal of this review is to draw general conclusions of how the size and geometry of a pathogen affect its uptake and processing by phagocytes of the immune system. We compared both theoretical and experimental studies with different cells, model particles, and pathogenic microbes (particularly fungi) showing that particle size, shape, rigidity, and surface roughness are important parameters for cellular uptake and subsequent immune responses, particularly inflammasome activation and T cell activation. Understanding how the physical properties of particles affect immune responses can aid the design of better vaccines.
Assuntos
Fungos/patogenicidade , Sistema Imunitário/microbiologia , Animais , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citoesqueleto/microbiologia , Endocitose , Fungos/imunologia , Interações Hospedeiro-Patógeno , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Inflamassomos/metabolismo , Ativação Linfocitária , Modelos Teóricos , Tamanho da Partícula , Fagocitose , Propriedades de Superfície , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologia , Vírion/imunologia , Vírion/patogenicidade , Vírus/imunologia , Vírus/patogenicidadeRESUMO
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.
Assuntos
Endocitose , Endossomos/metabolismo , Microscopia/métodos , Fagocitose , Membrana Celular/fisiologia , Fluorescência , Humanos , Transporte ProteicoRESUMO
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.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Nucleares/metabolismo , Fagossomos/metabolismo , Animais , Benzofenantridinas/farmacologia , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Humanos , Isoquinolinas/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Sinapses/metabolismoRESUMO
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.
RESUMO
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 Förster 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.
Assuntos
Microscopia de Fluorescência/métodos , Proteínas SNARE/metabolismo , Células HeLa , Humanos , Fusão de Membrana/fisiologia , Transporte ProteicoRESUMO
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.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Nucleares/metabolismo , Fagocitose , Actinas/metabolismo , Células Dendríticas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Polimerização , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
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.