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Journal of Virology Nov 2021Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species...
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na/H exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection . Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.
Topics: Animals; Cell Line; Cell Survival; Clathrin; Coronavirus; Coronavirus Infections; Deltacoronavirus; Dynamins; Endocytosis; Epithelial Cells; Hydrogen-Ion Concentration; Ileum; Kidney; Phosphatidylinositol 3-Kinases; Pinocytosis; RNA, Small Interfering; Swine; Swine Diseases; Virus Internalization; rab5 GTP-Binding Proteins
PubMed: 34586858
DOI: 10.1128/JVI.01345-21 -
Journal of Visualized Experiments : JoVE Aug 2021Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris,...
Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.
Topics: Endocytosis; Endosomes; Microscopy, Fluorescence; Pinocytosis; Reproducibility of Results
PubMed: 34515683
DOI: 10.3791/62828 -
The Journal of Cell Biology Sep 2021In this issue, Le et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202012114) describe a new role for the recently discovered protein CYRI in controlling the...
In this issue, Le et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202012114) describe a new role for the recently discovered protein CYRI in controlling the protrusions that allow cells to engulf extracellular fluid by macropinocytosis. This study helps explain how these structures are disassembled, but also uncovers a new mechanism linking the ability of cells to drink and their capacity for invasive migration.
Topics: Pinocytosis
PubMed: 34402856
DOI: 10.1083/jcb.202108041 -
Frontiers in Immunology 2021Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore...
Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore a pillar of innate immunity, whereby foreign particles are engulfed and degraded in lysolitic vesicles. In hexacorallians, phagocytic mechanisms are poorly understood, though putative anthozoan phagocytic cells (amoebocytes) have been identified histologically. We identify and characterize phagocytes from the coral and the sea anemone . Using fluorescence-activated cell sorting and microscopy, we show that distinct populations of phagocytic cells engulf bacteria, fungal antigens, and beads. In addition to pathogenic antigens, we show that phagocytic cells engulf self, damaged cells. We show that target antigens localize to low pH phagolysosomes, and that degradation is occurring within them. Inhibiting actin filament rearrangement interferes with efficient particle phagocytosis but does not affect small molecule pinocytosis. We also demonstrate that cellular markers for lysolitic vesicles and reactive oxygen species (ROS) correlate with hexacorallian phagocytes. These results establish a foundation for improving our understanding of hexacorallian immune cell biology.
Topics: Animals; Anthozoa; Biomarkers; Cytokines; Cytoplasmic Vesicles; Flow Cytometry; Hydrogen-Ion Concentration; Immunity, Innate; Phagocytes; Phagocytosis; Phagosomes; Sea Anemones
PubMed: 34381444
DOI: 10.3389/fimmu.2021.662803 -
Nature Communications Aug 2021Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner....
Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.
Topics: Animals; Cell Membrane; Cells, Cultured; Chromones; Enzyme Inhibitors; HEK293 Cells; Humans; Macrophages; Mice; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Morpholines; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Pinocytosis; RAW 264.7 Cells
PubMed: 34376698
DOI: 10.1038/s41467-021-25187-1 -
Journal of Nanobiotechnology Aug 2021Based on the concept of "multimodal analgesia", a novel dual drug delivery system was designed to achieve synergistic analgesia between najanajaatra venom protein (αCT)...
BACKGROUND
Based on the concept of "multimodal analgesia", a novel dual drug delivery system was designed to achieve synergistic analgesia between najanajaatra venom protein (αCT) and resveratrol (Res). In order to meet the joint loading of two drugs with different physicochemical properties without affecting each other, an oral Janus nanoparticle (JNP) with a unique cavity structure and synergistic drug delivery was constructed using an improved double emulsion solvent evaporation method, and combined with low-molecular-weight chitosan/sodium alginate and PLGA to achieve its pH-responsive.
RESULTS
The synthesized αCT/Res-JNPs are homogeneous in shape, with a two-compartment structure, approximately 230 nm in size, and zeta potential of 23.6 mV. Drug release assayed in vitro show that JNP was stable in simulated gastric juice (pH = 1.2) but was released in phosphate buffer saline (pH = 7.4). After intragastric administration in rats, PK evaluation showed that αCT/Res-JNPs could significantly improve the oral bioavailability, and the simultaneous encapsulation of the two drugs had no significant interaction on PK parameters. An obvious synergistic analgesic effects of αCT/Res-JNPs was confirmed in a spinal cord injury and acute pain model. Confocal laser scanning microscopy and single-pass intestinal perfusion model provided strong evidence that αCT/Res-JNPs could pass through intestinal epithelial cells, and the endocytosis pathway was mainly involved in the mediation and pinocytosis of reticulin. The concentrations of αCT and Res from αCT/Res-JNP in lymphatic transport were only about 8.72% and 6.08% of their blood concentrations at 1 h, respectively, which indicated that lymphatic transport in the form of JNP has limited advantages in improving the oral bioavailability of Res and αCT. Cellular uptake efficiency at 4 h was about 10-15% in Caco-2 cell lines for αCT/Res-JNP, but was reduced to 7% in Caco-2/HT29-MTX co-culture models due to the hindrance by the mucus layers. Approximately 12-17% of αCT/Res-JNP were transported across Caco-2/HT29-MTX/Raji monolayers. The cumulative absorption of JNP in three cell models was higher than that of free drug.
CONCLUSIONS
This study investigated the contribution of Janus nanoparticles in oral absorption, and provide a new perspective for oral administration and analgesic treatment of dual drug delivery system containing peptide drugs.
Topics: Administration, Oral; Alginates; Analgesia; Animals; Biological Availability; Caco-2 Cells; Chitosan; Drug Carriers; Drug Delivery Systems; Drug Liberation; Female; Humans; Hydrogen-Ion Concentration; Male; Multifunctional Nanoparticles; Nanoparticles; Rats; Rats, Sprague-Dawley; Resveratrol
PubMed: 34362394
DOI: 10.1186/s12951-021-00974-6 -
Molecules (Basel, Switzerland) Jul 2021Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered...
Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy--methyl-,-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.
Topics: Animals; Caveolae; Caveolin 1; Cell Line, Transformed; Cholesterol; Clathrin; DNA; Endocytosis; Endothelial Cells; Filipin; Gene Expression; Liposomes; Membrane Microdomains; Nystatin; Phosphatidylethanolamines; Pinocytosis; Plasmids; RNA, Small Interfering; Rats; Transfection
PubMed: 34361779
DOI: 10.3390/molecules26154626 -
Foods (Basel, Switzerland) Jul 2021Casticin has wide-ranging functional activities, but its water solubility is poor in food products. Here, a nanoemulsion stabilized by Maillard whey protein isolate...
Casticin has wide-ranging functional activities, but its water solubility is poor in food products. Here, a nanoemulsion stabilized by Maillard whey protein isolate conjugates (MWPI) was fabricated to encapsulate casticin. The nanoemulsion, with an average diameter of 200 nm, possessed the capability to load 700 μg/g casticin. MWPI-stabilized nanoemulsion showed better stability than that of the WPI nanoemulsion during 4 weeks of storage. Both the inhibition effects of the casticin-loaded nanoemulsion on cancer cells and the process of cellular uptake were studied. Results revealed that the casticin-loaded nanoemulsion had better inhibitory activity in HepG2 and MCF-7 cells than free casticin. Cellular uptake of the nanoemulsion displayed a time-dependent manner. After the nanoemulsion passed into HepG2 and MCF-7 cells, it would locate in the lysosome but not in the nucleus. The main pathway for the nanoemulsion to enter HepG2 cells was pinocytosis, whereas, it entered MCF-7 predominantly through the clathrin-mediated pit. This work implies that MWPI-stabilized nanoemulsions could be utilized as an effective delivery system to load casticin and have the potential to be applied in the food and pharmaceutical industries.
PubMed: 34359510
DOI: 10.3390/foods10071640 -
Molecular Aspects of Medicine Feb 2022Endocytosis mechanisms are one of the methods that cells use to interact with their environments. Endocytosis mechanisms vary from the clathrin-mediated endocytosis to... (Review)
Review
Endocytosis mechanisms are one of the methods that cells use to interact with their environments. Endocytosis mechanisms vary from the clathrin-mediated endocytosis to the receptor independent macropinocytosis. Macropinocytosis is a niche of endocytosis that is quickly becoming more relevant in various fields of research since its discovery in the 1930s. Macropinocytosis has several distinguishing factors from other receptor-mediated forms of endocytosis, including: types of extracellular material for uptake, signaling cascade, and niche uses between cell types. Nanoparticles (NPs) are an important tool for various applications, including drug delivery and disease treatment. However, surface engineering of NPs could be tailored to target them inside the cells exploiting different endocytosis pathways, such as endocytosis versus macropinocytosis. Such surface engineering of NPs mainly, size, charge, shape and the core material will allow identification of new adapter molecules regulating different endocytosis process and provide further insight into how cells tweak these pathways to meet their physiological need. In this review, we focus on the description of macropinocytosis, a lesser studied endocytosis mechanism than the conventional receptor mediated endocytosis. Additionally, we will discuss nanoparticle endocytosis (including macropinocytosis), and how the physio-chemical properties of the NP (size, charge, and surface coating) affect their intracellular uptake and exploiting them as tools to identify new adapter molecules regulating these processes.
Topics: Biological Transport; Clathrin; Endocytosis; Humans; Nanoparticles; Pinocytosis
PubMed: 34281720
DOI: 10.1016/j.mam.2021.100993 -
Journal of Visualized Experiments : JoVE Jun 2021Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance,...
Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 10 to 10 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled. These authors' laboratories' contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.
Topics: Adenosine Triphosphate; Animals; Cell Line, Tumor; Endocytosis; Humans; Mice; Microscopy, Fluorescence; Pinocytosis
PubMed: 34279488
DOI: 10.3791/62768