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The Journal of Cell Biology Nov 2018Macropinocytosis or "cell drinking" involves the elaboration of membrane ruffles that enclose and internalize extracellular fluids. Using lattice light sheet microscopy,...
Macropinocytosis or "cell drinking" involves the elaboration of membrane ruffles that enclose and internalize extracellular fluids. Using lattice light sheet microscopy, Condon et al. (2018. https://doi.org/10.1083/jcb.201804137) reveal the presence of parallel membrane protrusions termed "tent poles" that flank and direct membrane ruffle formation.
Topics: Cell Membrane Structures; Endosomes; Macrophages; Membranes; Pinocytosis
PubMed: 30305313
DOI: 10.1083/jcb.201810022 -
Journal of Cell Science Feb 2022The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to...
The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to growth factors and other stimuli. To test the hypothesis that nutrient environments modulate solute uptake by macropinocytosis, this study analyzed the effects of extracellular amino acids on the accumulation of fluorescent fluid-phase probes in murine macrophages. Nine amino acids, added individually or together, were capable of suppressing macropinocytosis in murine bone marrow-derived macrophages stimulated with the growth factors colony stimulating factor 1 (CSF1) or interleukin 34, both ligands of the CSF1 receptor (CSF1R). The suppressive amino acids did not inhibit macropinocytosis in response to lipopolysaccharide, the chemokine CXCL12, or the tumor promoter phorbol myristate acetate. Suppressive amino acids promoted release of CSF1R from cells and resulted in the formation of smaller macropinosomes in response to CSF1. This suppression of growth factor-stimulated macropinocytosis indicates that different nutrient environments modulate CSF1R levels and bulk ingestion by macropinocytosis, with likely consequences for macrophage growth and function.
Topics: Amino Acids; Animals; Endosomes; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Pinocytosis; Receptor Protein-Tyrosine Kinases
PubMed: 35107133
DOI: 10.1242/jcs.259284 -
Molecular Biology of the Cell Jan 2018Macropinocytosis mediates the uptake of antigens and of nutrients that dictate the regulation of cell growth by mechanistic target of rapamycin complex 1 (mTORC1)....
Macropinocytosis mediates the uptake of antigens and of nutrients that dictate the regulation of cell growth by mechanistic target of rapamycin complex 1 (mTORC1). Because these functions differ in proinflammatory and anti-inflammatory macrophages, we compared the macropinocytic ability of two extreme polarization states. We found that anti-inflammatory macrophages perform vigorous macropinocytosis constitutively, while proinflammatory cells are virtually inactive. The total cellular content of Rho-family GTPases was higher in anti-inflammatory cells, but this disparity failed to account for the differential macropinocytic activity. Instead, reduced activity of Rac/RhoG was responsible for the deficient macropinocytosis of proinflammatory macrophages, as suggested by the stimulatory effects of heterologously expressed guanine nucleotide-exchange factors or of constitutively active (but not wild-type) forms of these GTPases. Similarly, differences in the activation state of phosphatidylinositol 3-kinase (PtdIns3K) correlated with the macropinocytic activity of pro- and anti-inflammatory macrophages. Differences in PtdIns3K and Rho-GTPase activity were attributable to the activity of calcium-sensing receptors (CaSRs), which appear to be functional only in anti-inflammatory cells. However, agonists of PtdIns3K, including cytokines, chemokines, and LPS, induced macropinocytosis in proinflammatory cells. Our findings revealed a striking difference in the macropinocytic ability of pro- and anti-inflammatory macrophages that correlates with their antigen-presenting and metabolic activity.
Topics: Biomarkers; Cell Membrane; Cell Polarity; Cells, Cultured; Cytokines; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Lipopolysaccharides; Macrophages; Phenotype; Phosphatidylinositol 3-Kinases; Pinocytosis; Receptors, Calcium-Sensing; rho GTP-Binding Proteins
PubMed: 29093026
DOI: 10.1091/mbc.E17-06-0419 -
MSphere Sep 2018Nutrient limitation restricts bacterial growth in privileged sites such as the middle ear. Transient heme-iron restriction of nontypeable (NTHI), the major causative...
Nutrient limitation restricts bacterial growth in privileged sites such as the middle ear. Transient heme-iron restriction of nontypeable (NTHI), the major causative agent of chronic and recurrent otitis media (OM), promotes new and diverse phenotypes that can influence planktonic, biofilm, and intracellular lifestyles of NTHI. However, the bacterial responses to nutrient restriction that impact intracellular fate and survival of NTHI are unknown. In this work, we provide evidence for the role of transient heme-iron restriction in promoting the formation of intracellular bacterial communities (IBCs) of NTHI both and in a preclinical model of OM. We show that transient heme-iron restriction of NTHI results in significantly increased invasion and intracellular populations that escape or evade the endolysosomal pathway for increased intracellular survival. In contrast, NTHI continuously exposed to heme-iron traffics through the endolysosomal pathway for degradation. The use of pharmacological inhibitors revealed that prior heme-iron status does not appear to influence NTHI internalization through endocytic pathways. However, inhibition of macropinocytosis altered the intracellular fate of transiently restricted NTHI for degradation in the endolysosomal pathway. Furthermore, prevention of macropinocytosis significantly reduced the number of IBCs in cultured middle ear epithelial cells, providing evidence for the feasibility of this approach to reduce OM persistence. These results reveal that microenvironmental cues can influence the intracellular fate of NTHI, leading to new mechanisms for survival during disease progression. Otitis media is the most common bacterial infection in childhood. Current therapies are limited in the prevention of chronic or recurrent otitis media which leads to increased antibiotic exposure and represents a significant socioeconomic burden. In this study, we delineate the effect of nutritional limitation on the intracellular trafficking pathways used by nontypeable (NTHI). Moreover, transient limitation of heme-iron led to the development of intracellular bacterial communities that are known to contribute to persistence and recurrence in other diseases. New approaches for therapeutic interventions that reduce the production of intracellular bacterial communities and promote trafficking through the endolysosomal pathway were revealed through the use of pharmacological inhibition of macropinocytosis. This work demonstrates the importance of an intracellular niche for NTHI and provides new approaches for intervention for acute, chronic, and recurring episodes of otitis media.
Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cell Line; Chinchilla; Cytoplasm; Disease Models, Animal; Ear, Middle; Haemophilus Infections; Haemophilus influenzae; Heme; Humans; Iron; Otitis Media; Pinocytosis; Protein Transport
PubMed: 30209128
DOI: 10.1128/mSphere.00286-18 -
Proceedings of the National Academy of... Jan 2019Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles,...
Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT has been, for the most part, attributed to the ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.
Topics: Amino Acid Sequence; Cytokinesis; Dictyostelium; Endocytosis; Humans; Phagocytosis; Phagosomes; Pinocytosis; Protozoan Proteins; Sequence Alignment; Signal Transduction; ras GTPase-Activating Proteins; ras Proteins
PubMed: 30622175
DOI: 10.1073/pnas.1810268116 -
Leep1 interacts with PIP3 and the Scar/WAVE complex to regulate cell migration and macropinocytosis.The Journal of Cell Biology Jul 2021Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to...
Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.
Topics: Actins; Cell Movement; Cell Polarity; Chemotaxis; Cytoplasm; Dictyostelium; Pinocytosis; Protozoan Proteins; Pseudopodia; Signal Transduction
PubMed: 33978708
DOI: 10.1083/jcb.202010096 -
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 -
Cancer Research Apr 2021Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In...
Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2 mouse embryonic fibroblasts (MEF), with no impact on Tsc2 MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2 MEFs compared with Tsc2 MEFs, and treatment of Tsc2 MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2 MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.
Topics: Angiolipoma; Animals; Autophagy; Cell Proliferation; Chloroquine; Diacylglycerol Kinase; Down-Regulation; Drug Synergism; Female; Fibroblasts; Gene Expression; Kidney Neoplasms; Lung Neoplasms; Lymphangioleiomyomatosis; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Nutrients; Phosphatidic Acids; Phospholipids; Pinocytosis; Ritanserin; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein
PubMed: 33593821
DOI: 10.1158/0008-5472.CAN-20-2218 -
BioMed Research International 2018is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. survives in macrophages...
is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. survives in macrophages in membrane-bound vacuoles; however, the mechanism by which gains entry into macrophages remains unknown. After macrophage internalization, survival of within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of to enter macrophages through the induction of macropinocytosis.
Topics: Animals; Bacterial Secretion Systems; Burkholderia cenocepacia; Female; Macrophages; Mice, Inbred BALB C; Pinocytosis
PubMed: 29850514
DOI: 10.1155/2018/4271560 -
The Journal of Organic Chemistry May 2019Extrapolating from lessons learnt with previous low-molecular-weight β-(1→3)-glucan mimetics, we designed a series of minimal 2,4-dideoxy-thioether-linked carbacyclic...
Extrapolating from lessons learnt with previous low-molecular-weight β-(1→3)-glucan mimetics, we designed a series of minimal 2,4-dideoxy-thioether-linked carbacyclic β-(1→3)-glucan mimetics and synthesized di-, tri-, and tetramers in an enantiomerically pure form by an iterative sequence based on a simple building block readily available from commercial ( S)-(-)-3-cyclohexenecarboxylic acid. These substances were screened for their ability to inhibit anti-CR3-fluorescein isothiocyanate (FITC) staining of human neutrophils and anti-Dectin-1-FITC staining of mouse macrophages as well as for their ability to stimulate phagocytosis and pinocytosis. In each assay, the synthetic compounds displayed comparable activity to the corresponding native β-(1→3)-glucans, laminaritriose, and tetraose, suggesting that the exploitation of hydrophobic patches in the lectin-binding domains of CR3 and Dectin-1 is a promising strategy for the development of small-molecule analogues of β-(1→3)-glucans.
Topics: Animals; Biomimetic Materials; Chemistry Techniques, Synthetic; Ethers; Glucans; Humans; Mice; Phagocytosis; Pinocytosis; Polymerization; RAW 264.7 Cells
PubMed: 30933504
DOI: 10.1021/acs.joc.9b00504