-
The Journal of Cell Biology Mar 1985Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at... (Comparative Study)
Comparative Study
Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.
Topics: Animals; Exocytosis; Female; Horseradish Peroxidase; Isoquinolines; Macrophages; Mice; Mice, Inbred ICR; Peroxidases; Phorbols; Pinocytosis; Tetradecanoylphorbol Acetate
PubMed: 3972898
DOI: 10.1083/jcb.100.3.851 -
Current Opinion in Cell Biology Jun 2024Macropinocytosis (MP), the actin-dependent bulk uptake of extracellular fluids, plays a central role in nutrient scavenging, allowing cancer cells to sustain their... (Review)
Review
Macropinocytosis (MP), the actin-dependent bulk uptake of extracellular fluids, plays a central role in nutrient scavenging, allowing cancer cells to sustain their growth in the hypoxic and nutrient-deprived microenvironment often found in solid tumours. The lack of soluble nutrients and several oncogenic signalling pathways, with RAS being the most studied, push MP-dependent internalisation of extracellular proteins, which are then digested in the lysosomes, replenishing the intracellular nutrient pools. This review will highlight recent advances in understanding how MP is regulated in hypoxic cancers, how it impinges on chemoresistance, and how different MP cargos facilitate tumour growth. Finally, I will highlight the crosstalk between MP and extracellular matrix receptors.
Topics: Humans; Pinocytosis; Neoplasms; Animals; Nutrients; Tumor Microenvironment; Signal Transduction
PubMed: 38626703
DOI: 10.1016/j.ceb.2024.102359 -
MBio Aug 2014Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East...
Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. Importance: Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.
Topics: Animals; Cell Line, Tumor; Coronavirus; Mice; Pinocytosis; Pseudopodia; Severe acute respiratory syndrome-related coronavirus; Virus Replication
PubMed: 25096879
DOI: 10.1128/mBio.01340-14 -
Molecular Biology of the Cell Mar 2024Cells rely on a diverse array of engulfment processes to sense, exploit, and adapt to their environments. Among these, macropinocytosis enables indiscriminate and rapid...
Cells rely on a diverse array of engulfment processes to sense, exploit, and adapt to their environments. Among these, macropinocytosis enables indiscriminate and rapid uptake of large volumes of fluid and membrane, rendering it a highly versatile engulfment strategy. Much of the molecular machinery required for macropinocytosis has been well established, yet how this process is regulated in the context of organs and organisms remains poorly understood. Here, we report the discovery of extensive macropinocytosis in the outer epithelium of the cnidarian . Exploiting 's relatively simple body plan, we developed approaches to visualize macropinocytosis over extended periods of time, revealing constitutive engulfment across the entire body axis. We show that the direct application of planar stretch leads to calcium influx and the inhibition of macropinocytosis. Finally, we establish a role for stretch-activated channels in inhibiting this process. Together, our approaches provide a platform for the mechanistic dissection of constitutive macropinocytosis in physiological contexts and highlight a potential role for macropinocytosis in responding to cell surface tension.
Topics: Animals; Hydra; Pinocytosis
PubMed: 38265917
DOI: 10.1091/mbc.E22-02-0065 -
Current Pharmaceutical Design 2013Circulating low-density lipoprotein (LDL) that enters the blood vessel wall is the main source of cholesterol that accumulates within atherosclerotic plaques. Much of... (Review)
Review
Circulating low-density lipoprotein (LDL) that enters the blood vessel wall is the main source of cholesterol that accumulates within atherosclerotic plaques. Much of the deposited cholesterol accumulates within plaque macrophages converting these macrophages into cholesterol-rich foamy looking cells. Cholesterol accumulation in macrophages contributes to cholesterol retention within the vessel wall, and promotes vessel wall inflammation and thrombogenicity. Thus, how macrophages accumulate cholesterol and become foam cells has been the subject of intense investigation. It is generally believed that macrophages accumulate cholesterol only through scavenger receptor-mediated uptake of modified LDL. However, an alternative mechanism for macrophage foam cell formation that does not depend on LDL modification or macrophage receptors has been elucidated. By this alternative mechanism, macrophages show receptor-independent uptake of unmodified native LDL that is mediated by fluid-phase pinocytosis. In receptor-independent, fluid-phase pinocytosis, macrophages take up LDL as part of the fluid that they ingest during micropinocytosis within small vesicles called micropinosomes, and by macropinocytosis within larger vacuoles called macropinosomes. This produces cholesterol accumulation in macrophages to levels characteristic of macrophage foam cells in atherosclerotic plaques. Fluid-phase pinocytosis of LDL is a plausible mechanism that can explain how macrophages accumulate cholesterol and become disease-causing foam cells. Fluid-phase pinocytosis of LDL is a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis. Recent studies show that phosphoinositide 3-kinase (PI3K), liver X receptors (LXRs), the macrophage colony-stimulating factor (M-CSF) receptor, and protein kinase C (PKC) mediate macrophage macropinocytosis of LDL, and thus, these may be relevant targets to inhibit macrophage cholesterol accumulation in atherosclerosis.
Topics: Animals; Cholesterol; Foam Cells; Humans; Lipoproteins, LDL; Macrophages; Molecular Targeted Therapy; Pinocytosis; Plaque, Atherosclerotic
PubMed: 23438954
DOI: 10.2174/1381612811319330005 -
The Journal of Experimental Medicine Aug 1995
Review
Topics: Animals; Antigen-Presenting Cells; Dendritic Cells; Histocompatibility Antigens Class II; Humans; Pinocytosis
PubMed: 7629494
DOI: 10.1084/jem.182.2.283 -
British Journal of Pharmacology Nov 1984We investigated whether differences in induction or stimulation of pinocytosis by six amino-glycosides reflected reported differences in their nephrotoxicity....
We investigated whether differences in induction or stimulation of pinocytosis by six amino-glycosides reflected reported differences in their nephrotoxicity. Pinocytosis induced by antibiotics, Na+, K+ or Ca2+ was quantified by the number of pinocytotic channels in Amoeba proteus, a cell suitable for the study of the pinocytotic process. The aminoglycosides were potent inducers of pinocytosis. They were effective in the order of their cationic charge: neomycin greater than gentamicin greater than netilmicin = tobramycin greater than kanamycin greater than streptomycin. Factors which reduced the charge of the molecules, i.e. alkaline pH and combination with carbenicillin or heparin, diminished pinocytosis. Like La3+ the antibiotics inhibited Na+ -induced pinocytosis. The order of efficacy was netilmicin greater than gentamicin greater than neomycin. A similar rank order, which is the reverse of the order of nephrotoxicity, was observed for inhibition of Ca2+ -stimulated, Na+ -induced pinocytosis. Netilmicin was also the most potent inhibitor of the Ca2+-induced pinocytosis in cells treated with concanavalin A. Inhibition of Ca2+ -stimulated pinocytosis by netilmicin was reversed by Ca2+, the calcium ionophore A 23187, or 4-aminopyridine. We have shown that several nephrotoxic cations are strong inducers of pinocytosis in the amoeba, that aminoglycosides in Ringer solution induce pinocytosis in the approximate order of their nephrotoxicity and that factors which are known to diminish toxicity reduce pinocytosis. It, therefore, appears that the mechanism of aminoglycoside nephrotoxicity is related to their ability to induce pinocytosis in the amoeba. Low inducing potency and strong Ca2+ -antagonism, as for netilmicin, are qualities which may reduce the tendency of polycationic compounds to damage proximal tubular cells.
Topics: Aminoglycosides; Amoeba; Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Calcium Channel Blockers; Concanavalin A; Egtazic Acid; Hydrogen-Ion Concentration; Pinocytosis; Sodium
PubMed: 6439268
DOI: 10.1111/j.1476-5381.1984.tb16215.x -
Genes & Development Oct 2020Cancer cells must adapt metabolism to thrive despite nutrient limitations in the tumor microenvironment. In this issue of , King and colleagues (pp. 1345-1358) report a... (Review)
Review
Cancer cells must adapt metabolism to thrive despite nutrient limitations in the tumor microenvironment. In this issue of , King and colleagues (pp. 1345-1358) report a role for transcriptional regulators of the Hippo pathway to facilitate protein scavenging and support proliferation under some nutrient-deprived conditions.
Topics: Nutrients; Pinocytosis; Protein Serine-Threonine Kinases; Signal Transduction; Transcriptional Activation
PubMed: 33004484
DOI: 10.1101/gad.343632.120 -
Nature Communications Aug 2020Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory...
Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa.
Topics: Animals; Chemokine CXCL1; Cytoskeleton; Enzyme-Linked Immunosorbent Assay; Female; Intercellular Signaling Peptides and Proteins; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; NF-kappa B; Nerve Tissue Proteins; Phagocytes; Pinocytosis; Receptors, Immunologic; Signal Transduction; rhoA GTP-Binding Protein; Roundabout Proteins
PubMed: 32807784
DOI: 10.1038/s41467-020-17651-1 -
Blood Jun 1999Lipopolysaccharide (LPS) is a mediator of inflammation and septic shock during bacterial infection. Although monocytes and macrophages are highly responsive to LPS, the...
Lipopolysaccharide (LPS) is a mediator of inflammation and septic shock during bacterial infection. Although monocytes and macrophages are highly responsive to LPS, the biological effects of LPS in these cell types are only partially understood. We decided, therefore, to investigate the influence of LPS on macrophage pinocytosis and Fc receptor-mediated endocytosis, two prominent and related macrophage effector functions. We observed that LPS did not greatly influence endocytosis in either macrophages or monocytes, but did exert a dual action on pinocytosis: at lower concentrations (0.1 to 100 ng/mL), LPS caused a decrease in pinocytosis in both macrophages and monocytes, whereas at higher LPS concentrations, enhanced pinocytosis in macrophages was observed. Detoxified LPS was two orders of magnitude less potent in producing these effects. After inhibition of the LPS receptor CD14, the LPS-induced decrease in pinocytosis was absent, and stimulation of pinocytosis at lower LPS concentrations was unmasked. We conclude that LPS can influence pinocytosis via CD14-dependent and CD14-independent signaling pathways. Furthermore, as addition of LPS to macrophages effected pinocytosis but not Fc receptor-mediated endocytosis, these two processes are independently regulated in macrophages.
Topics: Animals; Cells, Cultured; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Mice; Pinocytosis; Signal Transduction
PubMed: 10339511
DOI: No ID Found