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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 -
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 -
The Journal of Neuroscience : the... Feb 2015Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually...
Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually survey the CNS microenvironment and scavenge cell debris and aberrant proteins by phagocytosis and pinocytosis, and that reactive microglia are capable to present antigens to T cells and initiate immune responses. However, how microglia process the endocytosed contents and evoke an immune response remain unclear. Here we report that a size-dependent selective transport of small soluble contents from the pinosomal lumen into lysosomes is critical for the antigen processing in microglia. Using fluorescent probes and water-soluble magnetic nanobeads of defined sizes, we showed in cultured rodent microglia, and in a cell-free reconstructed system that pinocytosed proteins become degraded immediately following pinocytosis and the resulting peptides are selectively delivered to major histocompatibility complex class II (MHC-II) containing lysosomes, whereas undegraded proteins are retained in the pinosomal lumen. This early size-based sorting of pinosomal contents relied on the formation of transient tunnel between pinosomes and lysosomes in a Rab7- and dynamin II-dependent manner, which allowed the small contents to pass through but restricted large ones. Inhibition of the size-based sorting markedly reduced proliferation and cytokine release of cocultured CD4(+) T cells, indicating that the size-based sorting is required for efficient antigen presentation by microglial cells. Together, these findings reveal a novel early sorting mechanism for pinosomal luminal contents in microglial cells, which may explain how microglia efficiently process protein antigens and evoke an immune response.
Topics: Animals; Antigen-Presenting Cells; Antigens; Cell Fusion; Cell Separation; Cell Size; Female; In Vitro Techniques; Lysosomes; Macrophage Activation; Male; Mice; Microglia; Pinocytosis; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Receptors, Interleukin-8A
PubMed: 25673858
DOI: 10.1523/JNEUROSCI.4389-14.2015 -
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 -
The Journal of Cell Biology Feb 2014In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol...
In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been particularly controversial. PIP₃ has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP₃ is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP₃. PIP₃ patches in these cells show no directional bias, and overall only PIP₃-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP₃ patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP₃ promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.
Topics: Cell Movement; Chemotaxis; Dictyostelium; Folic Acid; Mutation; Phosphatidylinositol Phosphates; Pinocytosis; Protozoan Proteins; Pseudopodia; Signal Transduction
PubMed: 24535823
DOI: 10.1083/jcb.201309081 -
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 -
The Journal of Physiological Sciences :... Mar 2019Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse...
Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2β, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)-dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2β were required for clathrin-mediated-but not clathrin-non-mediated-FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2β were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2β, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2β play differential, indispensable roles in clathrin-mediated pinocytosis.
Topics: Actin Cytoskeleton; Animals; Cells, Cultured; Class II Phosphatidylinositol 3-Kinases; Clathrin; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Phosphatidylinositol 3-Kinases; Pinocytosis; RNA, Small Interfering
PubMed: 30374841
DOI: 10.1007/s12576-018-0644-2 -
Journal of Cell Science Jan 2019Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism...
Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism In , macropinocytic cups are organized by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3, and direct actin polymerization to their periphery. We show that a Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PIP3 and, together, are nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA and PkbR1 target, and show that it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.
Topics: Dictyostelium; Pinocytosis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Protozoan Proteins
PubMed: 30617109
DOI: 10.1242/jcs.224998 -
The Journal of Experimental Medicine Feb 1967The pinocytosis-inducing effect of a number of molecular species was studied in cultures of mouse macrophages. Agents were added to a basal medium containing 1% NBCS-No....
The pinocytosis-inducing effect of a number of molecular species was studied in cultures of mouse macrophages. Agents were added to a basal medium containing 1% NBCS-No. 199 and allowed to interact with cells for 150 min. Vesicle counts were then performed and compared to control cells in the basal medium. Certain proteins, i.e. albumin and fetuin, with isoelectric points of five and below were found to be potent stimulators of vesicle formation. Basic proteins including lysozyme, histone, and protamine had little influence at sublethal concentrations. The pinocytosis-stimulating activity of bovine plasma albumin could be markedly depressed by removal of bound fatty acids. The addition of either oleic or linoleic acid to de-fatted albumin restored its inducing properties to initial levels. The activity of fetuin could be abolished by either mild acid hydrolysis or neuraminidase digestion. Both procedures removed the majority of the sialic acid content of fetuin. The D and L isomers of polyglutamic acid were found to produce a marked increase in pinosome production. In contrast, poly-DL-lysine was not effective. Neutral and basic amino acids were without significant effect on pinocytosis, whereas aspartic and glutamic acids were stimulatory. The amides of glutamic and aspartic acid did not induce pinocytosis. The unnatural D isomers of glutamic, aspartic, leucine, and phenylalanine inhibited pinocytosis. The inhibition by D-glutamic acid could be reversed with the L isomer. A number of acid mucopolysaccharides, including heparin, hyaluronic acid, and chondroitin sulfate, were excellent inducers. High molecular weight dextran was without significant stimulatory effect whereas dextran sulfate was very active. Both desoxyribonucleic acid and ribonucleic acid enhanced pinosome formation. A number of low molecular weight anions including N-acetylneuraminic acid were found to enhance vesicle formation. In general, anionic molecules were better inducers than either neutral or cationic species. The minimum effective dose of macroanions was a function of molecular weight and their activity appeared unrelated to specific chemical groupings.
Topics: Amino Acids; Animals; Glutamates; Glycosaminoglycans; Macrophages; Mice; Neuraminic Acids; Nucleic Acids; Pinocytosis; Polysaccharides; Proteins
PubMed: 4225263
DOI: 10.1084/jem.125.2.213