ABSTRACT
A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.
Subject(s)
Clathrin , Endocytosis , Endocytosis/physiology , Humans , Clathrin/metabolism , Microscopy, Fluorescence/methods , Animals , AlgorithmsABSTRACT
The early phases of clathrin mediated endocytosis are organized through a highly complex interaction network mediated by clathrin associated sorting proteins (CLASPs) that comprise long intrinsically disordered regions (IDRs). AP180 is a CLASP exclusively expressed in neurons and comprises a long IDR of around 600 residues, whose function remains partially elusive. Using NMR spectroscopy, we discovered an extended and strong interaction site within AP180 with the major adaptor protein AP2, and describe its binding dynamics at atomic resolution. We find that the 70 residue-long site determines the overall interaction between AP180 and AP2 in a dynamic equilibrium between its bound and unbound states, while weaker binding sites contribute to the overall affinity at much higher concentrations of AP2. Our data suggest that this particular interaction site might play a central role in recruitment of adaptors to the clathrin coated pit, whereas more transient and promiscuous interactions allow reshaping of the interaction network until cargo uptake inside a coated vesicle.
Subject(s)
Adaptor Protein Complex 2 , Clathrin , Endocytosis , Monomeric Clathrin Assembly Proteins , Protein Binding , Adaptor Protein Complex 2/metabolism , Clathrin/metabolism , Binding Sites , Monomeric Clathrin Assembly Proteins/metabolism , Monomeric Clathrin Assembly Proteins/genetics , Humans , Animals , Magnetic Resonance Spectroscopy , Clathrin-Coated Vesicles/metabolism , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/geneticsSubject(s)
Clathrin , Endocytosis , GTP-Binding Proteins , Hantaan virus , Virus Internalization , Humans , Endocytosis/drug effects , Virus Internalization/drug effects , Clathrin/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Hantaan virus/physiology , Hantaan virus/drug effects , Host-Pathogen Interactions , AnimalsABSTRACT
Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.
Subject(s)
Cell Differentiation , Endocytosis , Epithelial Cells , Nanoparticles , Humans , Endocytosis/physiology , Caco-2 Cells , Nanoparticles/chemistry , Cell Differentiation/drug effects , Epithelial Cells/metabolism , Dynamins/metabolism , Cholesterol/metabolism , Cholesterol/chemistry , Clathrin/metabolismABSTRACT
Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.
Subject(s)
Blood Platelets , Clathrin , Dynamins , Pinocytosis , Humans , Blood Platelets/metabolism , Dynamins/metabolism , Clathrin/metabolism , EndocytosisABSTRACT
A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.
Subject(s)
Endocytosis , Humans , Cell Line , Cell Membrane Permeability , Cell-Penetrating Peptides/metabolism , Clathrin/metabolism , Oligonucleotides/metabolism , Peptides/metabolism , Hydrocarbons, Chlorinated/chemistry , Hydrocarbons, Chlorinated/metabolismABSTRACT
Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.
Subject(s)
Biliary Atresia , Capsid Proteins , Disease Models, Animal , Endocytosis , Rotavirus Infections , Rotavirus , Biliary Atresia/metabolism , Biliary Atresia/virology , Animals , Mice , Rotavirus Infections/metabolism , Rotavirus Infections/virology , Humans , Capsid Proteins/metabolism , Toll-Like Receptor 3/metabolism , Binding Sites , HSC70 Heat-Shock Proteins/metabolism , HSC70 Heat-Shock Proteins/genetics , Mice, Knockout , NF-kappa B/metabolism , Signal Transduction , Clathrin/metabolism , Mice, Inbred C57BL , Chemokine CXCL10ABSTRACT
Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), which are then sorted for recycling or degradation. We investigated how differential activation of the same GPCR affects its endocytic trafficking in vivo using rhodopsin as a model in pupal photoreceptors of flies expressing mCherry-tagged rhodopsin 1 (Rh1-mC) or GFP-tagged arrestin 1 (Arr1-GFP). Upon blue light stimulation, activated Rh1 recruited Arr1-GFP to the rhabdomere, which became co-internalized and accumulated in cytoplasmic vesicles of photoreceptors. This internalization was eliminated in shits1 mutants affecting dynamin. Moreover, it was blocked by either rdgA or rdgB mutations affecting the PIP2 biosynthesis. Together, the blue light-initiated internalization of Rh1 and Arr1 belongs to CME. Green light stimulation also triggered the internalization and accumulation of activated Rh1-mC in the cytoplasm but with faster kinetics. Importantly, Arr1-GFP was also recruited to the rhabdomere but not co-internalized with Rh1-mC. This endocytosis was not affected in shits1 nor rdgA mutants, indicating it is not CME. We explored the fate of internalized Rh1-mC following CME and observed it remained in cytoplasmic vesicles following 30 min of dark adaptation. In contrast, in the non-CME Rh1-mC appeared readily recycled back to the rhabdomere within five min of dark treatment. This faster recycling may be regulated by rhodopsin phosphatase, RdgC. Together, we demonstrate two distinct endocytic and recycling mechanisms of Rh1 via two light stimulations. It appears that each stimulation triggers a distinct conformation leading to different phosphorylation patterns of Rh1 capable of recruiting Arr1 to rhabdomeres. However, a more stable interaction leads to the co-internalization of Arr1 that orchestrates CME. A stronger Arr1 association appears to impede the recycling of the phosphorylated Rh1 by preventing the recruitment of RdgC. We conclude that conformations of activated rhodopsin determine the downstream outputs upon phosphorylation that confers differential protein-protein interactions.
Subject(s)
Endocytosis , Rhodopsin , Rhodopsin/metabolism , Rhodopsin/genetics , Animals , Phosphorylation , Protein Transport , Light , Mutation , Photoreceptor Cells, Invertebrate/metabolism , Drosophila melanogaster/metabolism , Clathrin/metabolismABSTRACT
Influenza virus infection is initiated by the attachment of the viral haemagglutinin (HA) protein to sialic acid receptors on the host cell surface. Most virus particles enter cells through clathrin-mediated endocytosis (CME). However, it is unclear how viral binding signals are transmitted through the plasma membrane triggering CME. Here we found that metabotropic glutamate receptor subtype 2 (mGluR2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the initiation and completion of CME of influenza virus using an siRNA screen approach. Influenza virus HA directly interacted with mGluR2 and used it as an endocytic receptor to initiate CME. mGluR2 interacted and activated KCa1.1, leading to polymerization of F-actin, maturation of clathrin-coated pits and completion of the CME of influenza virus. Importantly, mGluR2-knockout mice were significantly more resistant to different influenza subtypes than the wild type. Therefore, blocking HA and mGluR2 interaction could be a promising host-directed antiviral strategy.
Subject(s)
Endocytosis , Mice, Knockout , Receptors, Metabotropic Glutamate , Animals , Receptors, Metabotropic Glutamate/metabolism , Receptors, Metabotropic Glutamate/genetics , Mice , Humans , Virus Internalization , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Clathrin/metabolism , Orthomyxoviridae Infections/virology , Orthomyxoviridae Infections/metabolism , HEK293 Cells , Actins/metabolism , Dogs , Madin Darby Canine Kidney Cells , Receptors, Virus/metabolism , Receptors, Virus/genetics , Influenza, Human/virology , Influenza, Human/metabolism , Orthomyxoviridae/physiology , Orthomyxoviridae/genetics , Orthomyxoviridae/metabolismABSTRACT
OBJECTIVE: The objective of this study is to determine whether inhibition of mitophagy affects seizures through Clathrin-mediated endocytosis (CME). METHODS: Pentylenetetrazol (PTZ) was intraperitoneally injected daily to establish a chronic PTZ-kindled seizure. The Western blot (WB) was used to compare the differences in Parkin protein expression between the epilepsy group and the control group. Immunofluorescence was used to detect the expression of MitoTracker and LysoTracker. Transferrin-Alexa488 (Tf-A488) was injected into the hippocampus of mice. We evaluated the effect of 3-methyladenine (3-MA) on epilepsy behavior through observation in PTZ-kindled models. RESULTS: The methylated derivative of adenine, known as 3-MA, has been extensively utilized in the field of autophagy research. The transferrin protein is internalized from the extracellular environment into the intracellular space via the CME pathway. Tf-A488 uses a fluorescent marker to track CME. Western blot showed that the expression of Parkin was significantly increased in the PTZ-kindled model (p < 0.05), while 3-MA could reduce the expression (p < 0.05). The fluorescence uptake of MitoTracker and LysoTracker was increased in the primary cultured neurons induced by magnesium-free extracellular fluid (p < 0.05); the fluorescence uptake of Tf-A488 was significantly decreased in the 3-MA group compared with the control group (p < 0.05). Following hippocampal injection of Tf-A488, both the epilepsy group and the 3-MA group exhibited decreased fluorescence uptake, with a more pronounced effect observed in the 3-MA group. Inhibition of mitophagy by 3-MA from day 3 to day 9 progressively exacerbated seizure severity and shortened latency. SIGNIFICANCE: It is speculated that the aggravation of seizures by 3-MA may be related to the failure to remove damaged mitochondria in time and effectively after inhibiting mitochondrial autophagy, affecting the vesicle endocytosis function of CME and increasing the susceptibility to epilepsy. SUMMARY: Abnormal mitophagy was observed in a chronic pentylenetetrazol-induced seizure model and a Mg2+-free-induced spontaneous recurrent epileptiform discharge model. A fluorescent transferrin marker was utilized to track clathrin-mediated endocytosis. Using an autophagy inhibitor (3-methyladenine) on primary cultured neurons, we discovered that inhibition of autophagy led to a reduction in fluorescent transferrin uptake, while impairing clathrin-mediated endocytosis function mediated by mitophagy. Finally, we examined the effects of 3-methyladenine in an animal model of seizures showing that it exacerbated seizure severity. Ultimately, this study provides insights into potential mechanisms through which mitophagy regulates clathrin-mediated endocytosis in epilepsy.
Subject(s)
Autophagy , Clathrin , Endocytosis , Epilepsy , Mitochondria , Mitophagy , Animals , Mice , Epilepsy/chemically induced , Epilepsy/metabolism , Endocytosis/physiology , Endocytosis/drug effects , Autophagy/drug effects , Autophagy/physiology , Mitochondria/metabolism , Mitochondria/drug effects , Mitophagy/drug effects , Mitophagy/physiology , Clathrin/metabolism , Male , Pentylenetetrazole , Adenine/analogs & derivatives , Adenine/pharmacology , Hippocampus/metabolism , Hippocampus/drug effects , Disease Models, Animal , Ubiquitin-Protein Ligases/metabolismABSTRACT
Clathrin-mediated endocytosis (CME) is an essential process of cargo uptake operating in all eukaryotes. In animals and yeast, BAR-SH3 domain proteins, endophilins and amphiphysins, function at the conclusion of CME to recruit factors for vesicle scission and uncoating. Arabidopsis thaliana contains the BAR-SH3 domain proteins SH3P1-SH3P3, but their role is poorly understood. Here, we identify SH3Ps as functional homologs of endophilin/amphiphysin. SH3P1-SH3P3 bind to discrete foci at the plasma membrane (PM), and SH3P2 recruits late to a subset of clathrin-coated pits. The SH3P2 PM recruitment pattern is nearly identical to its interactor, a putative uncoating factor, AUXILIN-LIKE1. Notably, SH3P1-SH3P3 are required for most of AUXILIN-LIKE1 recruitment to the PM. This indicates a plant-specific modification of CME, where BAR-SH3 proteins recruit auxilin-like uncoating factors rather than the uncoating phosphatases, synaptojanins. SH3P1-SH3P3 act redundantly in overall CME with the plant-specific endocytic adaptor TPLATE complex but not due to an SH3 domain in its TASH3 subunit.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Clathrin , Endocytosis , Clathrin/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Auxilins/metabolism , Cell Membrane/metabolism , Clathrin-Coated Vesicles/metabolism , src Homology Domains , Protein BindingABSTRACT
CED-1 (cell death abnormal) is a transmembrane receptor involved in the recognition of "eat-me" signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in Caenorhabditis elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter include the adapter protein CED-6/GULP and the ATP-binding cassette family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the Adaptor protein 2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Cell Membrane , Clathrin , Endocytosis , Animals , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/genetics , Clathrin/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Apoptosis , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/genetics , Adaptor Protein Complex 2/metabolism , Protein Transport , Apoptosis Regulatory ProteinsABSTRACT
Fungal pathogens deliver effector proteins into living plant cells to suppress plant immunity and control plant processes that are needed for infection. During plant infection, the devastating rice blast fungus, Magnaporthe oryzae, forms the specialized biotrophic interfacial complex (BIC), which is essential for effector translocation. Cytoplasmic effectors are first focally secreted into BICs, and subsequently packaged into dynamic membranous effector compartments (MECs), then translocated via clathrin-mediated endocytosis (CME) into the host cytoplasm. This study demonstrates that clathrin-heavy chain inhibitors endosidin-9 (ES9) and endosidin-9-17 (ES9-17) blocked the internalization of the fluorescently labeled effectors Bas1 and Pwl2 in rice cells, leading to swollen BICs lacking MECs. In contrast, ES9-17 treatment had no impact on the localization pattern of the apoplastic effector Bas4. This study provides further evidence that cytoplasmic effector translocation occurs by CME in BICs, suggesting a potential role for M. oryzae effectors in co-opting plant endocytosis.
Subject(s)
Endocytosis , Oryza , Oryza/microbiology , Oryza/metabolism , Plant Diseases/microbiology , Ascomycota , Host-Pathogen Interactions , Protein Transport , Fungal Proteins/metabolism , Clathrin/metabolismABSTRACT
The vitellogenin receptor (VgR) is essential for the uptake and transport of the yolk precursor, vitellogenin (Vg). Vg is synthesized in the fat body, released in the hemolymph, and absorbed in the ovaries, via receptor-mediated endocytosis. Besides its important role in the reproductive pathway, Vg occurs in nonreproductive worker honey bee, suggesting its participation in other pathways. The objective was to verify if the VgR occurs in the hypopharyngeal glands of Apis mellifera workers and how Vg is internalized by these cells. VgR occurrence in the hypopharyngeal glands was evaluated by qPCR analyses of VgR and immunohistochemistry in workers with different tasks. The VgR gene is expressed in the hypopharyngeal glands of workers with higher transcript levels in nurse honey bees. VgR is more expressed in 11-day-old workers from queenright colonies, compared to orphan ones. Nurse workers with developed hypopharyngeal glands present higher VgR transcripts than those with poorly developed glands. The immunohistochemistry results showed the co-localization of Vg, VgR and clathrin (protein that plays a major role in the formation of coated vesicles in endocytosis) in the hypopharyngeal glands, suggesting receptor-mediated endocytosis. The results demonstrate that VgR performs the transport of Vg to the hypopharyngeal glands, supporting the Ovary Ground Plan Hypothesis and contributing to the understanding of the role of this gland in the social context of honey bees.
Subject(s)
Egg Proteins , Hypopharynx , Insect Proteins , Receptors, Cell Surface , Animals , Bees/metabolism , Bees/genetics , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/genetics , Insect Proteins/metabolism , Insect Proteins/genetics , Egg Proteins/metabolism , Egg Proteins/genetics , Hypopharynx/metabolism , Female , Vitellogenins/metabolism , Vitellogenins/genetics , Clathrin/metabolismABSTRACT
The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN's involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.
Subject(s)
Endothelial Cells , Vascular Endothelial Growth Factor A , Humans , Endothelial Cells/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Sialomucins/metabolism , Endocytosis , Clathrin/metabolismABSTRACT
Clathrin assembles into honeycomb-like lattices at the plasma membrane but also on internal membranes, such as at the Golgi and tubular endosomes. Clathrin assemblies primarily regulate the intracellular trafficking of different cargoes, but clathrin also has non-endocytic functions in cell adhesion through interactions with specific integrins, contributes to intraluminal vesicle formation by forming flat bilayered coats on endosomes and even assembles on kinetochore k-fibers during mitosis. In this Cell Science at a Glance article and the accompanying poster, we review our current knowledge on the different types of canonical and non-canonical membrane-associated clathrin assemblies in mammalian cells, as observed by thin-section or platinum replica electron microscopy in various cell types, and discuss how the structural plasticity of clathrin contributes to its functional diversity.
Subject(s)
Clathrin , Animals , Humans , Cell Membrane/metabolism , Clathrin/metabolism , Endosomes/metabolism , Golgi Apparatus/metabolismABSTRACT
Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.
Subject(s)
Extracellular Vesicles , Genistein , Sulfonamides , Thiazolidines , Female , Animals , Mice , Genistein/pharmacology , Granulosa Cells , Clathrin , MammalsABSTRACT
Infectious salmon anaemia virus (ISAV) is a pathogen that causes disease and large mortality in farm-raised Salmo salar L., being considered as a major problem in the salmon industry. However, despite its relevance, there are still numerous knowledge gaps on virus entry and early stages of infection. Previous studies suggested that virus entry into cells occurs via endocytosis, with no description of specific mechanisms. However, it remains unknown if the endocytosis induced by ISAV is a clathrin-dependent or clathrin-independent process. This study aimed to identify cellular mechanisms allowing ISAV entry into Atlantic Salmon head kidney (ASK) cells. Our results showed that ISAV can be found in coated pits and membrane ruffles, the latter being induced by a rearrangement of actin filaments promoted by ISAV infection. Additionally, it was determined that ISAV stimulate the uptake of extracellular fluid in a multiplicity of infection (MOI)-dependent manner. When the clathrin-mediated endocytic pathway was pharmacologically inhibited, ISAV infection was significantly reduced but not entirely inhibited. Similarly, when the Na+/H+ exchanger (NHE), a key component of macropinocytosis, was inhibited, ISAV infection was negatively affected. Our results suggest that ISAV enters cells via both clathrin-mediated endocytosis and most likely macropinocytosis.
Subject(s)
Fish Diseases , Isavirus , Orthomyxoviridae Infections , Animals , Endocytosis , Clathrin , Orthomyxoviridae Infections/veterinaryABSTRACT
Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.
Subject(s)
Arrestin , Histamine , Animals , Cricetinae , Humans , Arrestin/metabolism , Arrestins/metabolism , beta-Arrestins/metabolism , CHO Cells , Clathrin/metabolism , Cricetulus , Extracellular Signal-Regulated MAP Kinases/metabolism , G-Protein-Coupled Receptor Kinases/metabolism , GTP-Binding Proteins/metabolism , Histamine/pharmacology , Histamine/metabolism , Phosphorylation , Protein Kinase C/metabolism , Receptors, Histamine H1/genetics , Receptors, Histamine H1/metabolism , Signal TransductionABSTRACT
Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.