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Current Biology : CB Aug 2020Macropinocytic cups enable cells to take up droplets of medium into internal vesicles. These cups are formed by the actin cytoskeleton around signaling patches of Ras,...
Macropinocytic cups enable cells to take up droplets of medium into internal vesicles. These cups are formed by the actin cytoskeleton around signaling patches of Ras, Rac and the phosphoinositide PIP3 in the plasma membrane. New work now describes a Ras regulator that controls both the size and efficiency of these patches.
Topics: Bacteria; Cell Membrane Structures; Endocytosis; Phagocytosis; Pinocytosis
PubMed: 32750349
DOI: 10.1016/j.cub.2020.06.050 -
Journal of Visualized Experiments : JoVE May 2021Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form...
Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.
Topics: Actin Cytoskeleton; Cell Membrane; Cell Surface Extensions; Microscopy, Electron, Scanning; Pinocytosis
PubMed: 34125102
DOI: 10.3791/62658 -
Journal of Virology Apr 2023Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its...
Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.
Topics: Virus Internalization; Alphacoronavirus; rab GTP-Binding Proteins; Endosomes; Coronavirus Infections; Hydrogen-Ion Concentration; Dynamins; Caveolae; Cholesterol; Clathrin; Pinocytosis; Vero Cells; Chlorocebus aethiops; Animals
PubMed: 36975780
DOI: 10.1128/jvi.00210-23 -
Journal of Extracellular Vesicles Apr 2024Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical...
Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.
Topics: Proton Pump Inhibitors; Extracellular Vesicles; Endocytosis; Pinocytosis; Adenosine Triphosphatases
PubMed: 38532609
DOI: 10.1002/jev2.12426 -
Traffic (Copenhagen, Denmark) Jul 2002Whether the endocytic uptake of a given molecule is mediated through clathrin-coated pits or not is a classical criterion used to characterize its endocytic pathway(s).... (Review)
Review
Whether the endocytic uptake of a given molecule is mediated through clathrin-coated pits or not is a classical criterion used to characterize its endocytic pathway(s). Hence, clathrin-dependent endocytosis has been associated with highly selective and efficient uptake, whereas clathrin-independent endocytosis appeared to be confined to bulk uptake of fluid-phase markers. This scholastic view has recently been challenged using newly developed molecular tools that allow for the first time a functional and mechanistic analysis of these less well-characterized clathrin-independent pathways, including caveolar uptake and macropinocytosis. Furthermore, several studies point to a critical role of lateral lipid asymmetry--lipid rafts/microdomains--in membrane sorting. We will discuss the potential role of these structures in endocytosis and the possibility that differential sorting at the plasma membrane predisposes the ensuing intracellular fate of a given molecule as well as its physiological function.
Topics: Animals; Caveolae; Clathrin; Clathrin-Coated Vesicles; Coated Pits, Cell-Membrane; Endocytosis; Endosomes; Humans; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Models, Biological; Pinocytosis
PubMed: 12047552
DOI: 10.1034/j.1600-0854.2002.30701.x -
Autophagy May 2021Poorly vascularized tumors embedded within a thick desmoplastic stroma, like pancreatic ductal adenocarcinoma (PDAC), are nutritionally stressed. Such tumors are also...
Poorly vascularized tumors embedded within a thick desmoplastic stroma, like pancreatic ductal adenocarcinoma (PDAC), are nutritionally stressed. Such tumors are also hypoxic and rely on a number of adaptive responses, including macroautophagy/autophagy and macropinocytosis (MP), to support their bioenergetic needs. Whereas autophagy enables starved cells to recycle intracellular macromolecules via lysosomal degradation and use the liberated amino acids (AA) to fuel their metabolism, MP allows cells to take up extracellular proteins via fluid-phase endocytosis and use them as an energy source. However, how any MP-enabled organism, including the prototypical cancer cell, coordinately regulates and balances autophagy and MP is not fully understood. We recently found that inhibition of autophagy results in upregulation of MP, which enables cancer cells to overcome autophagy deficiency and continue to support their bioenergetic demands. The NFE2L2/NRF2-driven induction of MP-related genes (MRGs) is responsible for the upregulation of MP in autophagy inhibited, hypoxic, and oxidatively stressed-exposed cancer cells. Concurrent autophagy and MP blockade effectively cuts off the cancer cell's nutrient and supplies, leading to rapid tumor regression. These findings suggest MP to be an important target in cancer treatment and that shutting off the energy spigot is a promising therapeutic strategy. AA: amino acids; ADCs: autophagy deficient-cells; AI: autophagy inhibition; ALB: albumin; CHUK/IKKα: component of inhibitor of nuclear factor kappa B kinase complex; CQ: chloroquine; ECM: extracellular matrix; HCQ: hydroxychloroquine; MI: MP inhibition; MP: macropinocytosis; MRGs: MP-related genes; MRPs: MP-related proteins; PDAC: pancreatic ductal adenocarcinoma.
Topics: Autophagy; Carcinoma, Pancreatic Ductal; Humans; NF-E2-Related Factor 2; Pancreatic Neoplasms; Pinocytosis
PubMed: 33879021
DOI: 10.1080/15548627.2021.1919969 -
Current Opinion in Lipidology Oct 2011Because early findings indicated that native low-density lipoprotein (LDL) did not substantially increase macrophage cholesterol content during in-vitro incubations,... (Review)
Review
PURPOSE OF REVIEW
Because early findings indicated that native low-density lipoprotein (LDL) did not substantially increase macrophage cholesterol content during in-vitro incubations, investigators presumed that LDL must be modified in some way to trigger its uptake by the macrophage. The purpose of this review is to discuss recent findings showing that native unmodified LDL can induce massive macrophage cholesterol accumulation mimicking macrophage foam cell formation that occurs within atherosclerotic plaques.
RECENT FINDINGS
Macrophages that show high rates of fluid-phase pinocytosis also show similar high rates of uptake of native unmodified LDL through nonreceptor mediated uptake within both macropinosomes and micropinosomes. Nonsaturable fluid-phase uptake of LDL by macrophages converts the macrophages into foam cells. Different macrophage phenotypes demonstrate either constitutive fluid-phase pinocytosis or inducible fluid-phase pinocytosis. Fluid-phase pinocytosis has been demonstrated by macrophages within mouse atherosclerotic plaques indicating that this pathway contributes to plaque macrophage cholesterol accumulation.
SUMMARY
Contrary to what has been believed previously, macrophages can take up large amounts of native unmodified LDL by receptor-independent, fluid-phase pinocytosis converting these macrophages into foam cells. Thus, targeting macrophage fluid-phase pinocytosis should be considered when investigating strategies to limit macrophage cholesterol accumulation in atherosclerotic plaques.
Topics: Animals; Cholesterol; Foam Cells; Humans; Lipoproteins, LDL; Macrophages; Pinocytosis
PubMed: 21881499
DOI: 10.1097/MOL.0b013e32834adadb -
JCI Insight Jun 2021Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated...
Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated that a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first-pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, not only did we measure the effect of altering the intestinal microbiota on lipid accumulation in coronary arteries, but we also examined macrophage lipid uptake and handling pathways in response to uPC. We found that atherosclerosis-prone mice fed a high-fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, and hepatic and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated that uPC exposure increased apoptosis whereas PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate LDL uptake and only uPC induced endocytosis- and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate that uPC itself had a distinct effect on macrophage biology that might have contributed to increased cardiovascular risk in patients with CKD.
Topics: Animals; Aorta; Cholesterol; Cholesterol, LDL; Coronary Artery Disease; Coronary Vessels; Cresols; Diet, High-Fat; Fecal Microbiota Transplantation; Gastrointestinal Microbiome; Kidney Failure, Chronic; Liver; Macrophages; Mice; Pinocytosis; Renal Insufficiency, Chronic; Triglycerides
PubMed: 33914709
DOI: 10.1172/jci.insight.144410 -
Philosophical Transactions of the Royal... Feb 2019Nucleic acids are a rapidly emerging therapeutic modality with the potential to become the third major drug modality alongside antibodies and small molecules. Owing to... (Review)
Review
Nucleic acids are a rapidly emerging therapeutic modality with the potential to become the third major drug modality alongside antibodies and small molecules. Owing to the unfavourable physico-chemical characteristics of nucleic acids, such as large size and negative charge, intracellular delivery remains a fundamental challenge to realizing this potential. Delivery technologies such as lipids, polymers and peptides have been used to facilitate delivery, with many of the most successful technologies using macropinocytosis to gain cellular entry; mostly by default rather than design. Fundamental knowledge of macropinocytosis is rapidly growing, presenting opportunities to better tailor design strategies to target this pathway. Furthermore, certain types of tumour cells have been observed to have high levels of macropinocytic activity and traffic cargo to favourable destinations within the cell for endosomal release, providing unique opportunities to further use this entry route for drug delivery. In this article, we review the delivery systems reported to be taken up by macropinocytosis and what is known about the mechanisms for regulating macropinocytosis in tumour cells. From this analysis, we identify new opportunities for exploiting this pathway for the intracellular delivery of nucleic acids to tumour cells. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.
Topics: Drug Delivery Systems; Nucleic Acids; Pinocytosis; Tumor Cells, Cultured
PubMed: 30967005
DOI: 10.1098/rstb.2018.0156 -
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