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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 -
Frontiers in Cellular Neuroscience 2020Microglia are constantly surveying their microenvironment and rapidly react to impairments by changing their morphology, migrating toward stimuli and adopting gene...
Microglia are constantly surveying their microenvironment and rapidly react to impairments by changing their morphology, migrating toward stimuli and adopting gene expression profiles characterizing their activated state. The increased expression of the M2-like marker (), which is also referred to as CD206, in microglia has been reported after M2-like activation and . is a 175-kDa transmembrane pattern recognition receptor which binds a variety of carbohydrates and is involved in the pinocytosis and the phagocytosis of immune cells, including microglia, and thought to contribute to a neuroprotective microglial phenotype. Here we analyzed the effects of TGFβ signaling on expression in microglia and . Using C57BL/6 wild type and mice-derived microglia, we show that the silencing of TGFβ signaling results in the upregulation of , whereas recombinant TGFβ1 induced the delayed downregulation of . Furthermore, chromatin immunoprecipitation experiments provided evidence that is not a direct Smad2/Smad4 target gene in microglia. Altogether our data indicate that the changes in expression after the activation or the silencing of microglial TGFβ signaling are likely to be mediated by modifications of the secondary intracellular signaling events influenced by TGFβ signaling.
PubMed: 32296307
DOI: 10.3389/fncel.2020.00066 -
The Journal of Cell Biology Jan 2023Macropinocytosis is a nonspecific endocytic process that may enhance cancer cell survival under nutrient-poor conditions. Ataxia-Telangiectasia mutated (ATM) is a tumor...
Macropinocytosis is a nonspecific endocytic process that may enhance cancer cell survival under nutrient-poor conditions. Ataxia-Telangiectasia mutated (ATM) is a tumor suppressor that has been previously shown to play a role in cellular metabolic reprogramming. We report that the suppression of ATM increases macropinocytosis to promote cancer cell survival in nutrient-poor conditions. Combined inhibition of ATM and macropinocytosis suppressed proliferation and induced cell death both in vitro and in vivo. Supplementation of ATM-inhibited cells with amino acids, branched-chain amino acids (BCAAs) in particular, abrogated macropinocytosis. Analysis of ATM-inhibited cells in vitro demonstrated increased BCAA uptake, and metabolomics of ascites and interstitial fluid from tumors indicated decreased BCAAs in the microenvironment of ATM-inhibited tumors. These data reveal a novel basis of ATM-mediated tumor suppression whereby loss of ATM stimulates protumorigenic uptake of nutrients in part via macropinocytosis to promote cancer cell survival and reveal a potential metabolic vulnerability of ATM-inhibited cells.
Topics: Humans; Adaptation, Physiological; Ataxia Telangiectasia Mutated Proteins; Cellular Reprogramming; Neoplasms; Pinocytosis; Tumor Microenvironment; Amino Acids, Branched-Chain; Metabolomics; Animals; Mice; Cell Line, Tumor
PubMed: 36399181
DOI: 10.1083/jcb.202007026 -
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 -
Frontiers in Cell and Developmental... 2022MFSD8 is a transmembrane protein that has been reported to transport chloride ions across the lysosomal membrane. Mutations in are associated with a subtype of Batten...
MFSD8 is a transmembrane protein that has been reported to transport chloride ions across the lysosomal membrane. Mutations in are associated with a subtype of Batten disease called CLN7 disease. Batten disease encompasses a family of 13 inherited neurodegenerative lysosomal storage diseases collectively referred to as the neuronal ceroid lipofuscinoses (NCLs). Previous work identified an ortholog of human MFSD8 in the social amoeba (gene: , protein: Mfsd8) reported its localization to endocytic compartments, and demonstrated its involvement in protein secretion. In this study, we further characterized the effects of loss during growth and early stages of multicellular development. During growth, cells displayed increased rates of proliferation, pinocytosis, and expansion on bacterial lawns. Loss of also increased cell size, inhibited cytokinesis, affected the intracellular and extracellular levels of the quorum-sensing protein autocrine proliferation repressor A, and altered lysosomal enzyme activity. During the early stages of development, loss of delayed aggregation, which we determined was at least partly due to impaired cell-substrate adhesion, defects in protein secretion, and alterations in lysosomal enzyme activity. Overall, these results show that Mfsd8 plays an important role in modulating a variety of processes during the growth and early development of .
PubMed: 35756993
DOI: 10.3389/fcell.2022.930235 -
Journal of Virology Dec 2022Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology...
Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology pathogenesis is crucial for preventing and controlling this disease, the molecular mechanisms for the entry and post-internalization of SVV, which represent crucial steps in viral infection, are not well characterized. In this study, specific inhibitors, Western blotting, and immunofluorescence detection revealed that SVV entry into PK-15 cells depends on low-pH conditions and dynamin. Furthermore, results showed that caveolae-mediated endocytosis (CavME) contributes crucially to the internalization of SVV, as evidenced by cholesterol depletion, downregulation of caveolin-1 expression by small interfering RNA knockdown, and overexpression of a caveolin-1 dominant negative (caveolin-1-DN) in SVV-infected PK-15 cells. However, SVV entry into PK-15 cells did not depend on clathrin-mediated endocytosis (CME). Furthermore, treatment with specific inhibitors demonstrated that SVV entry into PK-15 cells via macropinocytosis depended on the Na+/H+ exchanger (NHE), p21-activated kinase 1 (Pak1), and actin rearrangement, but not phosphatidylinositol 3-kinase (PI3K). Electron microscopy showed that SVV particles or proteins were localized in CavME and macropinocytosis. Finally, knockdown of GTPase Rab5 and Rab7 by siRNA significantly inhibited SVV replication, as determined by measuring viral genome copy numbers, viral protein expression, and viral titers. In this study, our results demonstrated that SVV utilizes caveolae-mediated endocytosis and macropinocytosis to enter PK-15 cells, dependent on low pH, dynamin, Rab5, and Rab7. Entry of virus into cells represents the initiation of a successful infection. As an emerging pathogen of porcine vesicular disease, clarification of the process of SVV entry into cells enables us to better understand the viral life cycle and pathogenesis. In this study, patterns of SVV internalization and key factors required were explored. We demonstrated for the first time that SVV entry into PK-15 cells via caveolae-mediated endocytosis and macropinocytosis requires Rab5 and Rab7 and is independent of clathrin-mediated endocytosis, and that low-pH conditions and dynamin are involved in the process of SVV internalization. This information increases our understanding of the patterns in which all members of the family enter host cells, and provides new insights for preventing and controlling SVV infection.
Topics: Animals; Caveolae; Caveolin 1; Clathrin; Dynamins; Endocytosis; Picornaviridae; RNA, Small Interfering; Swine; Swine Vesicular Disease; Virus Internalization; rab5 GTP-Binding Proteins; Pinocytosis; Cell Line
PubMed: 36472440
DOI: 10.1128/jvi.01446-22 -
Plants (Basel, Switzerland) Mar 2021L. (family Hyacinthaceae) is traditionally used to treat different diseases including cancer. In this study, the anticancer and immunomodulatory effects of this plant...
L. (family Hyacinthaceae) is traditionally used to treat different diseases including cancer. In this study, the anticancer and immunomodulatory effects of this plant were evaluated. Hydroalcoholic extract was prepared, and different solvent fractions were obtained using solvent-solvent extraction. In the anticancer part, MTT assay and caspase-3 ELISA kits were used to measure the antiproliferative and apoptosis induction ability for each extract, respectively. In the immunomodulatory part, lymphocyte proliferation assay and cytokines detection kit were used to measure the effect of extracts of acquired immunity. Phagocytosis and pinocytosis induction were used to evaluate the effect of extracts on the innate immunity. GC-MS, LC-MS, and Foline-Ciocalteu assays were used to identify the chemical composition of the plant. Balb/C mice were inoculated with breast cancer and treated with hydroalcoholic extract of L. Results showed that hydroalcoholic extract and -hexane fraction were highly effective in apoptosis induction. Both extract and fraction were also effective in stimulating lymphocytes proliferation and phagocytosis. Significant reduction in tumor size was achieved after treating tumor-bearing mice with hydroalcoholic extract. Additionally, high cure percentages (50%) were obtained in treated mice. Results of this study showed that L. has promising anticancer and immunomodulatory activities. However, further studies are needed to explore more details of apoptosis induction ability and other mechanisms of action and to measure different signaling pathways responsible for the anticancer and immunomodulatory response.
PubMed: 33805000
DOI: 10.3390/plants10040617 -
Frontiers in Cellular and Infection... 2020, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite. can invade and multiply inside any nucleated cell of a wide range of...
, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite. can invade and multiply inside any nucleated cell of a wide range of homeothermic hosts. The canonical process of internalization involves several steps: an initial recognition of the host cell surface and a sequential secretion of proteins from micronemes followed by rhoptries that assemble a macromolecular complex constituting a specialized and transient moving junction. The parasite is then internalized via an endocytic process with the establishment of a parasitophorous vacuole (PV), that does not fuse with lysosomes, where the parasites survive and multiply. This process of host cell invasion is usually referred to active penetration. Using different cell types and inhibitors of distinct endocytic pathways, we show that treatment of host cells with compounds that interfere with clathrin-mediated endocytosis (hypertonic sucrose medium, chlorpromazine hydrochloride, and pitstop 2 inhibited the internalization of tachyzoites). In addition, treatments that interfere with macropinocytosis, such as incubation with amiloride or IPA-3, increased parasite attachment to the host cell surface but significantly blocked parasite internalization. Immunofluorescence microscopy showed that markers of macropinocytosis, such as the Rab5 effector rabankyrin 5 and Pak1, are associated with parasite-containing cytoplasmic vacuoles. These results indicate that entrance of into mammalian cells can take place both by the well-characterized interaction of parasite and host cell endocytic machinery and other processes, such as the clathrin-mediated endocytosis, and macropinocytosis.
Topics: Animals; Endocytosis; Host-Parasite Interactions; Pinocytosis; Toxoplasma; Toxoplasmosis; Vacuoles
PubMed: 32714877
DOI: 10.3389/fcimb.2020.00294 -
International Journal of Nanomedicine 2019The family of oncogenes () are the most frequent mutations in cancers and regulate key signaling pathways that drive tumor progression. As a result, drug delivery...
BACKGROUND
The family of oncogenes () are the most frequent mutations in cancers and regulate key signaling pathways that drive tumor progression. As a result, drug delivery targeting -driven tumors has been a long-standing challenge in cancer therapy. Mutant activates cancer cells to actively take up nutrients, including glucose, lipids, and albumin, via macropinocytosis to fulfill their energetic requirements to survive and proliferate.
PURPOSE
We exploit macropinocytosis pathway to deliver nanoparticles (NPs) in cancer cells harboring activating mutations.
METHODS
NPs were synthesized by the desolvation method. The physicochemical properties and stability of NPs were characterized by dynamic light scattering and transmission electron microscopy. Uptake of fluorescently labelled NPs in wild-type and mutant cells were quantitively determined by flow cytometry and qualitatively by fluorescent microscopy. NP uptake by -driven macropinocytosis was confirmed by pharmacological inhibition and genetic knockdown.
RESULTS
We have synthesized stable albumin NPs that demonstrate significantly greater uptake in cancer cells with activating mutations of than monomeric albumin (ie, dissociated form of clinically used nab-paclitaxel). From pharmacological inhibition and semi-quantitative fluorescent microscopy studies, these NPs exhibit significantly increased uptake in mutant cancer cells than wild-type S cells by macropinocytosis.
CONCLUSIONS
The uptake of albumin nanoparticles is driven by . This NP-based strategy targeting -driven macropinocytosis is a facile approach toward improved delivery into -driven cancers.
Topics: Cell Line, Tumor; Chemical Phenomena; Humans; Intracellular Space; Nanoparticles; Oncogenes; Particle Size; Pinocytosis; Proto-Oncogene Proteins p21(ras); Serum Albumin, Bovine
PubMed: 31496700
DOI: 10.2147/IJN.S212861