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Proceedings of the National Academy of... Dec 2021In fast-moving cells such as amoeba and immune cells, dendritic actin filaments are spatiotemporally regulated to shape large-scale plasma membrane protrusions. Despite...
In fast-moving cells such as amoeba and immune cells, dendritic actin filaments are spatiotemporally regulated to shape large-scale plasma membrane protrusions. Despite their importance in migration, as well as in particle and liquid ingestion, how their dynamics are affected by micrometer-scale features of the contact surface is still poorly understood. Here, through quantitative image analysis of on microfabricated surfaces, we show that there is a distinct mode of topographical guidance directed by the macropinocytic membrane cup. Unlike other topographical guidance known to date that depends on nanometer-scale curvature sensing protein or stress fibers, the macropinocytic membrane cup is driven by the Ras/PI3K/F-actin signaling patch and its dependency on the micrometer-scale topographical features, namely PI3K/F-actin-independent accumulation of Ras-GTP at the convex curved surface, PI3K-dependent patch propagation along the convex edge, and its actomyosin-dependent constriction at the concave edge. Mathematical model simulations demonstrate that the topographically dependent initiation, in combination with the mutually defining patch patterning and the membrane deformation, gives rise to the topographical guidance. Our results suggest that the macropinocytic cup is a self-enclosing structure that can support liquid ingestion by default; however, in the presence of structured surfaces, it is directed to faithfully trace bent and bifurcating ridges for particle ingestion and cell guidance.
Topics: Cell Membrane; Chemotaxis; Computer Simulation; Dictyostelium; Models, Biological; Movement; Phosphatidylinositol 3-Kinases; Pinocytosis; Signal Transduction
PubMed: 34876521
DOI: 10.1073/pnas.2110281118 -
Cancer Science Feb 2022Various cell types secrete exosomes into their surrounding extracellular space, which consequently affect the function and activity of recipient cells. Numerous studies...
Epidermal growth factor/epidermal growth factor receptor signaling blockage inhibits tumor cell-derived exosome uptake by oral squamous cell carcinoma through macropinocytosis.
Various cell types secrete exosomes into their surrounding extracellular space, which consequently affect the function and activity of recipient cells. Numerous studies have showed that tumor cell-derived exosomes play important roles in tumor growth and progression. Although a variety of endocytic pathways are reportedly involved in the cellular uptake of exosomes, detailed mechanisms remain unknown. The present study demonstrated that treatment with recombinant epidermal growth factor (EGF) time- and dose-dependently promoted cellular uptake of oral squamous cell carcinoma (OSCC) cell-derived exosomes into OSCC cells themselves. Conversely, EGF receptor (EGFR) knockdown and treatment with EGFR inhibitors, including erlotinib and cetuximab, abrogated OSCC cell uptake of exosomes. The macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked the effects of active EGF/EGFR signaling on uptake of OSCC cell-derived exosomes. These EGFR inhibitors also suppressed OSCC cell-derived exosome-induced proliferation, migration, invasion, stemness, and chemoresistance of OSCC cells. Taken together, the data presented herein suggest that EGFR inhibitors might inhibit the malignant potential of OSCC cells through direct inhibition of not only EGFR downstream signaling pathway but also cellular uptake of OSCC cell-derived exosomes through macropinocytosis.
Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Exosomes; Humans; Mouth Neoplasms; Neoplastic Stem Cells; Pinocytosis; Protein Kinase Inhibitors; Signal Transduction; Squamous Cell Carcinoma of Head and Neck
PubMed: 34874595
DOI: 10.1111/cas.15225 -
Cells Nov 2021DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine...
DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell-cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.
Topics: Allogeneic Cells; Antigen-Presenting Cells; CD47 Antigen; Cancer Vaccines; Cell Differentiation; Cell Membrane; Chemokines; Dendritic Cells; Humans; Inflammation; Models, Biological; Phagocytosis; Phenotype; Phosphatidylserines; Pinocytosis; Signal Transduction
PubMed: 34831455
DOI: 10.3390/cells10113233 -
Nature Communications Nov 2021Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is...
Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.
Topics: Actins; Animals; Biological Transport; Cell Line; Cell Membrane; Endocytosis; Endosomes; Humans; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pinocytosis; Salmonella; Transcriptome; Vesicular Transport Proteins
PubMed: 34772942
DOI: 10.1038/s41467-021-26775-x -
Drug Delivery Dec 2021Cisplatin (DDP) is a first-line chemotherapeutic drug applied for the treatment of oral squamous cell carcinoma (OSCC). The anticancer activity of DDP is tightly linked...
Cisplatin (DDP) is a first-line chemotherapeutic drug applied for the treatment of oral squamous cell carcinoma (OSCC). The anticancer activity of DDP is tightly linked to its intracellular uptake. It is unwise to increase the DDP intake by increasing the dose or shortening the dosing interval because of the severe systemic toxicity (nephrotoxicity, ototoxicity and neurotoxicity) in DDP application. The main uptake pathways of DDP include passive diffusion and active transporter transport. Therefore, finding additional uptake pathways that can improve the effective intracellular concentration of DDP is critical. Macropinocytosis, an endocytic mechanism for extracellular material absorption, contributes to the intracellular uptake of anticancer drugs. No research has been conducted to determine whether macropinocytosis can augment the intracellular uptake of DDP in OSCC cells or not. Based on that, we proved for the first time that silmitasertib (previously CX-4945) could trigger macropinocytosis, which may increase the intracellular uptake of DDP and enhance apoptosis via and experiments. We hope that our findings will inspire a new approach for the application of DDP in cancer treatment.
Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Drug Liberation; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Mouth Neoplasms; Naphthyridines; Phenazines; Pinocytosis; Xenograft Model Antitumor Assays
PubMed: 34766543
DOI: 10.1080/10717544.2021.2000677 -
Frontiers in Pharmacology 2021Qingfei Paidu decoction (QPD) and Xuanfei Baidu decoction (XBD) are two typical traditional Chinese medicines with proven efficacy for the treatment of SARS-CoV-2,...
Traditional Chinese Medicine, Qingfei Paidu Decoction and Xuanfei Baidu Decoction, Inhibited Cytokine Production NF-κB Signaling Pathway in Macrophages: Implications for Coronavirus Disease 2019 (COVID-19) Therapy.
Qingfei Paidu decoction (QPD) and Xuanfei Baidu decoction (XBD) are two typical traditional Chinese medicines with proven efficacy for the treatment of SARS-CoV-2, although the underlying mechanism is not well defined. Blunted immune response and enhanced production of pro-inflammatory cytokines (cytokine storm) are two main features observed in patients infected with SARS-CoV-2. Analysis based on network pharmacology has revealed that both QPD and XBD played an important role in the regulation of host immunity. We therefore investigated the role of QPD and XBD in the modulation of innate immunity , focusing on the type 1 interferon (IFN) signaling pathway in A549 cells and pro-inflammatory cytokine production in macrophages. A549 cells were treated with QPD or XBD and the production of endogenous IFNα and IFNβ as well as the expression levels of some interferon-stimulated genes (ISGs) were detected by reverse transcriptase-quantitative PCR (RT-qPCR). Macrophages derived from THP-1 cells were treated with QPD or XBD and their pro-inflammatory cytokine expression levels were measured by RT-qPCR, 6 h post LPS stimulation. In addition, the expression levels of some pro-inflammatory cytokines were further analyzed by ELISA. The effect of QPD and XBD on the NF-κB signaling pathway and the pinocytosis activity of THP-1-derived macrophages were evaluated by Western blot and neutral red uptake assay, respectively. Although QPD and XBD showed very little effect on the type 1 IFN signaling pathway in A549 cells, either QPD or XBD markedly inhibited the production of pro-inflammatory markers including interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1, and chemokine ligand 10 in THP-1-derived M1 macrophages. In addition, the phosphorylation of IκBα and NF-κB p65 during the process of macrophage polarization was significantly suppressed following QPD or XBD treatment. QPD and XBD also suppressed the pinocytosis activity of macrophages. QPD and XBD have been shown to have robust anti-inflammatory activities . Our study demonstrated that both QPD and XBD decreased pro-inflammatory cytokine expression, inhibited the activation of the NF-κB signaling pathway, and blunted pinocytosis activity in THP-1-derived macrophages.
PubMed: 34764867
DOI: 10.3389/fphar.2021.722126 -
Trends in Cancer Jan 2022Macropinocytosis, an evolutionarily conserved endocytic mechanism that mediates non-specific fluid-phase uptake, is potently upregulated by various oncogenic pathways.... (Review)
Review
Macropinocytosis, an evolutionarily conserved endocytic mechanism that mediates non-specific fluid-phase uptake, is potently upregulated by various oncogenic pathways. It is now well appreciated that high macropinocytic activity is a hallmark of many human tumors, which use this adaptation to scavenge extracellular nutrients for fueling cell growth. In the context of the nutrient-scarce tumor microenvironment, this process provides tumor cells with metabolic flexibility. However, dependence on this scavenging mechanism also illuminates a potential metabolic vulnerability. As such, there is a great deal of interest in understanding the molecular underpinnings of macropinocytosis. In this review, we will discuss the most recent advances in characterizing macropinocytosis: the pathways that regulate it, its contribution to the metabolic fitness of cancer cells, and its therapeutic potential.
Topics: Carcinogenesis; Humans; Neoplasms; Oncogenes; Pinocytosis; Tumor Microenvironment
PubMed: 34649835
DOI: 10.1016/j.trecan.2021.09.004 -
Frontiers in Immunology 2021
Topics: Neoplasms; Phagocytes; Phagocytosis; Pinocytosis; Receptors, IgG
PubMed: 34630442
DOI: 10.3389/fimmu.2021.772256 -
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