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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 -
Environmental Pollution (Barking, Essex... Jul 2020Civilization development is associated with the use of plastic. When plastic was introduced to the market, it was assumed that it was less toxic than glass. Recently, it... (Review)
Review
Civilization development is associated with the use of plastic. When plastic was introduced to the market, it was assumed that it was less toxic than glass. Recently, it is known that plastics are serious ecological problem they, do not degrade and remain in the environment for hundreds of years. Plastic may be degraded into micro-particles < 5000 nm in diameter, and further into nanoparticles (NPs) < 100 nm in diameter. NPs have been detected in air, soil, water and sludge. One of the most commonly used plastics is polystyrene (PS) - a product of polymerization of styrene monomers. It is used for the production of styrofoam and other products like toys, CDs and cup covers. In vivo and in vitro studies have suggested that polystyrene nanoparticles (PS-NPs) may penetrate organisms through several routes i.e. skin, respiratory and digestive tracts. They can be deposited in living organisms and accumulate further along the food chain. NPs are surrounded by "protein corona" that allows them penetrating cellular membranes and interacting with cellular structures. Depending on the cell type, NPs may be transported through pinocytosis, phagocytosis, or be transported passively. Currently there are no studies that would indicate a carcinogenic potential of PS-NPs. On the other hand, the PS monomer (styrene) was classified by the International Agency for Research on Cancer (IARC) as a potentially carcinogenic substance (carcinogenicity class B2). Despite of the widespread use of plastics and the presence of plastic NPs of secondary or primary nature, there are no studies that would assess the effect of those substances on human organism. This study was aimed at the review of the literature data concerning the formation of PS-NPs in the environment, their accumulation along the food chain, and their potential adverse effects on organisms on living various organization levels.
Topics: Food Chain; Humans; Nanoparticles; Plastics; Polystyrenes; Water Pollutants, Chemical
PubMed: 32155552
DOI: 10.1016/j.envpol.2020.114297 -
Toxins Jul 2020, the causative agent of inhalation anthrax, is a serious concern as a bioterrorism weapon. The vegetative form produces two exotoxins: Lethal toxin (LT) and edema toxin... (Comparative Study)
Comparative Study
, the causative agent of inhalation anthrax, is a serious concern as a bioterrorism weapon. The vegetative form produces two exotoxins: Lethal toxin (LT) and edema toxin (ET). We recently characterized and compared six human airway and alveolar-resident phagocyte (AARP) subsets at the transcriptional and functional levels. In this study, we examined the effects of LT and ET on these subsets and human leukocytes. AARPs and leukocytes do not express high levels of the toxin receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). Less than 20% expressed surface TEM8, while less than 15% expressed CMG2. All cell types bound or internalized protective antigen, the common component of the two toxins, in a dose-dependent manner. Most protective antigen was likely internalized via macropinocytosis. Cells were not sensitive to LT-induced apoptosis or necrosis at concentrations up to 1000 ng/mL. However, toxin exposure inhibited spore internalization. This inhibition was driven primarily by ET in AARPs and LT in leukocytes. These results support a model of inhalation anthrax in which spores germinate and produce toxins. ET inhibits pathogen phagocytosis by AARPs, allowing alveolar escape. In late-stage disease, LT inhibits phagocytosis by leukocytes, allowing bacterial replication in the bloodstream.
Topics: Adolescent; Adult; Aged; Animals; Antigens, Bacterial; Apoptosis; Bacillus anthracis; Bacterial Toxins; Dose-Response Relationship, Drug; Female; Humans; Leukocytes; Macrophages, Alveolar; Male; Mice; Microfilament Proteins; Middle Aged; Necrosis; Phagocytosis; Pinocytosis; RAW 264.7 Cells; Receptors, Cell Surface; Receptors, Peptide; Spores, Bacterial; Young Adult
PubMed: 32698436
DOI: 10.3390/toxins12070464 -
Bioconjugate Chemistry May 2022Macropinocytosis is a form of endocytosis that allows massive uptake of extracellular materials and is a promising route for intracellular delivery of biofunctional...
Macropinocytosis is a form of endocytosis that allows massive uptake of extracellular materials and is a promising route for intracellular delivery of biofunctional macromolecules and nanoparticles. Our laboratory developed a potent macropinocytosis-inducing peptide named P4A. However, the ability of this peptide is not apparent in the presence of serum. This study aims to endow P4A and related peptides with the ability to induce macropinocytosis in the presence of serum by N-terminal acylation with long-chain fatty acids (i.e., decanoic, myristic, and stearic acids). Stearylated P4A (stearyl-P4A) had the highest effect on stimulating macropinocytotic uptake. Moreover, the intramolecularly disulfide-bridged analogue, stearyl-oxP4A, showed an even higher ability. The effect of stearyl-oxP4A to facilitate the intracellular delivery of small extracellular vesicles (sEVs) was evaluated in terms of (i) cellular uptake using sEVs labeled with an enhanced green fluorescent protein (EGFP) and (ii) cytosolic liberation and expression of sEV-encapsulated luciferase mRNA in recipient cells. The two- to threefold uptake of both sEVs in the presence of stearyl-oxP4A suggests the potential of the peptide for sEV delivery in the presence of serum.
Topics: Biological Transport; Endocytosis; Extracellular Vesicles; Peptides; Pinocytosis
PubMed: 35506582
DOI: 10.1021/acs.bioconjchem.2c00113 -
Nature Communications Feb 2020Macropinocytic cancer cells scavenge amino acids from extracellular proteins. Here, we show that consuming necrotic cell debris via macropinocytosis (necrocytosis)...
Macropinocytic cancer cells scavenge amino acids from extracellular proteins. Here, we show that consuming necrotic cell debris via macropinocytosis (necrocytosis) offers additional anabolic benefits. A click chemistry-based flux assay reveals that necrocytosis provides not only amino acids, but sugars, fatty acids and nucleotides for biosynthesis, conferring resistance to therapies targeting anabolic pathways. Indeed, necrotic cell debris allow macropinocytic breast and prostate cancer cells to proliferate, despite fatty acid synthase inhibition. Standard therapies such as gemcitabine, 5-fluorouracil (5-FU), doxorubicin and gamma-irradiation directly or indirectly target nucleotide biosynthesis, creating stress that is relieved by scavenged nucleotides. Strikingly, necrotic debris also render macropinocytic, but not non-macropinocytic, pancreas and breast cancer cells resistant to these treatments. Selective, genetic inhibition of macropinocytosis confirms that necrocytosis both supports tumor growth and limits the effectiveness of 5-FU in vivo. Therefore, this study establishes necrocytosis as a mechanism for drug resistance.
Topics: Animals; Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Fatty Acid Synthases; Female; Fluorouracil; Humans; Metabolic Flux Analysis; Mice; Microfilament Proteins; Mutation; Nutrients; Pinocytosis
PubMed: 32111826
DOI: 10.1038/s41467-020-14928-3 -
PloS One 2021In the present study, we evaluated adjuvant potential of Poria cocos polysaccharide (PCP) on the Th1-type immune responses of C57/BL6 mice against ovalbumin (OVA). We...
In the present study, we evaluated adjuvant potential of Poria cocos polysaccharide (PCP) on the Th1-type immune responses of C57/BL6 mice against ovalbumin (OVA). We first determined the effect of PCP on maturation of murine bone marrow derived dendritic cells (BMDCs), PCP significantly upregulated surface expression of MHCII, CD40, CD80, CD86 and enhanced production of IL-6 and IL-12p40. In addition, PCP affected receptor-mediated endocytosis, but not pinocytosis in BMDCs. Furthermore, OVA + PCP immunization induced specific cytotoxic CD8+ T cell killing of OVA (257-264) peptide pulsed cell. When mice were immunized subcutaneously in a week interval with OVA + PCP. Serum were collected for measuring OVA-specific antibody and splenocytes were harvested for analyzing CD69, IFN-γ ELISpot and cytokines production. The result indicated that OVA-specific IgG, IgG2a and IgG1 antibody levels in serum were significantly elevated by PCP compared with control. PCP increased OVA-specific IFN-γ-secreting CD8+, CD4+ T cells, promoted CD8+ T cell proliferation and up-regulated Th-1 type (IFN-γ, IL-2) cytokine production. In conclusion, data suggest that PCP enhanced cellular immune response and possess potential as a vaccine adjuvant for Th1 immune response.
Topics: Animals; Antibody Formation; Bone Marrow Cells; CD8-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Female; Fungal Polysaccharides; Immunity, Cellular; Immunoglobulin G; Mice; Ovalbumin; Th1 Cells; Wolfiporia
PubMed: 33411807
DOI: 10.1371/journal.pone.0245207 -
Frontiers in Nutrition 2022From the earliest times, manna has been widely used as a tasty local sweet or folk medicine. The type of manna being investigated in the present study is called...
BACKGROUND
From the earliest times, manna has been widely used as a tasty local sweet or folk medicine. The type of manna being investigated in the present study is called Gaz-alafi, a mixture of insect and leaves secretions from oak forests in the north of Iraq and west of Iran.
METHODS
Aqueous and ethanol extracts were prepared as decoction. Various phytochemical tests were conducted to analyze manna composition, including total phenolic contents using the Folin-Ciocalteu method and LC-MS. Gallic acid and catechin were detected in both extracts, in addition to tiliroside presence in ethanol extract, which added more value to the phenolic content of ethanol extract. Cytotoxic activities of Gaz alafi were evaluated against breast cancer cell lines and compared to normal cell lines and doxorubicin using the MTT assay. Antimicrobial properties were assessed against , and using the dilution method of the micro-titer plate. Serum levels of IFN-γ, interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) were measured using ELISA. The effect of extracts on splenocyte proliferation was evaluated using the lymphocytes proliferation assay. Macrophage function was evaluated using the nitro blue tetrazolium assay, whereas pinocytosis was evaluated using the neutral red uptake assay. Ten days after tumor inoculation, changes in tumor size, survival rates, levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine were measured.
RESULTS
The growth of cancer cells was inhibited by Gaz alafi ethanol extract. An alteration in IFN- γ, IL-2, and IL-4 levels toward antiproliferation immune response were reported for both extracts. The aqueous extract efficiently stimulated lymphocyte proliferation, phagocytosis, and pinocytosis, followed by the ethanol extracts with moderate activity. After treating the mice with ethanol extracts, a significant reduction in tumor size and several undetected tumors were recorded.
CONCLUSIONS
Gaz alafi extracts (aqueous and ethanol) are promising sources for anticancer and immunostimulatory agents. Further studies are needed to fully identify the chemical composition of Gaz alafi extracts.
PubMed: 35845806
DOI: 10.3389/fnut.2022.900506 -
Current Biology : CB Aug 2020Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialized cup-shaped protrusions. To effectively...
Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialized cup-shaped protrusions. To effectively capture and internalize their targets, these cups are organized into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR, and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac, and phospholipid interactions to localize at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, we propose RGBARG shapes the protrusion by expanding Rac activation at the rim while suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of coordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.
Topics: Bacteria; Cell Cycle Proteins; Dictyostelium; Phagocytosis; Pinocytosis; rac GTP-Binding Proteins; ras Proteins
PubMed: 32531280
DOI: 10.1016/j.cub.2020.05.049 -
Pharmaceutics Sep 2020Macrophages are hosts for intracellular pathogens involved in numerous diseases including leishmaniasis. They express surface receptors that may be exploited for...
Macrophages are hosts for intracellular pathogens involved in numerous diseases including leishmaniasis. They express surface receptors that may be exploited for specific drug-targeting. Recently, we developed a PEGylated dendritic polyglycerol-based conjugate (PG-PEG) that colocalizes with intracellular parasite. We hereby study the effect of surface decoration with mannose units on the conjugates' targeting ability toward leishmania intracellular parasites. Murine and human macrophages were exposed to fluorescently labeled mannosylated PG-PEG and uptake was quantified by flow cytometry analysis. Nanocarriers bearing five mannose units showed the highest uptake, which varied between 30 and 88% in the population in human and murine macrophages, respectively. The uptake was found to be dependent on phagocytosis and pinocytosis (80%), as well as clathrin-mediated endocytosis (79%). Confocal microscopy showed that mannosylated PG-PEGs target acidic compartments in macrophages. In addition, when both murine and human macrophages were infected and treated, colocalization between parasites and mannosylated nanoconjugates was observed. Leishmania-infected bone marrow-derived macrophages (BMM) showed avidity by mannosylated PG-PEG whereas non-infected macrophages rarely accumulated conjugates. Moreover, the antileishmanial activity of Amphotericin B was kept upon conjugation to mannosylated PG-PEG through a pH-labile linker. This study demonstrates that leishmania infected macrophages are selectively targeted by mannosylated PEGylated dendritic conjugates.
PubMed: 32987800
DOI: 10.3390/pharmaceutics12100915 -
Cells May 2020Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric...
Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. single, double, and triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.
Topics: Amino Acid Sequence; Autophagy; Autophagy-Related Proteins; Cell Proliferation; Cell Survival; Conserved Sequence; Dictyostelium; Mutation; Phagocytosis; Phenotype; Pinocytosis; Proteostasis; Protozoan Proteins; Reproducibility of Results
PubMed: 32397394
DOI: 10.3390/cells9051179