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The Journal of Cell Biology Sep 2024Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of...
Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.
Topics: Dictyostelium; Pinocytosis; Protozoan Proteins; ras GTPase-Activating Proteins; ras Proteins; Signal Transduction
PubMed: 38888895
DOI: 10.1083/jcb.202401110 -
International Journal of Molecular... Apr 2024Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To...
Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.
Topics: Quantum Dots; Endocytosis; Saccharomyces cerevisiae; Cadmium Compounds; Selenium Compounds; Sulfides; Zinc Compounds; Vacuoles; Saccharomyces cerevisiae Proteins; Green Fluorescent Proteins; Cell Membrane
PubMed: 38731933
DOI: 10.3390/ijms25094714 -
MAbs 2024Off-target biodistribution of biologics bears important toxicological consequences. Antibody fragments intended for use as vectors of cytotoxic payloads (e.g....
Off-target biodistribution of biologics bears important toxicological consequences. Antibody fragments intended for use as vectors of cytotoxic payloads (e.g. antibody-drug conjugates, radiotherapy) can accumulate at clearance organs like kidneys and liver, where they can cause dose-limiting toxicities. Renal and hepatic uptakes are known to be affected by protein electrostatics, which promote protein internalization through pinocytosis. Using minibodies as a model of an antibody fragment lacking FcRn recycling, we compared the biodistributions of leads with different degrees of accumulation at the kidney and liver. We identified a positive electrostatic patch highly conserved in a germline family very commonly used in the humanization of approved biologics. Neutralization of this patch led to a drastic reduction in the kidney uptake, leading to a biodistribution more favorable to the delivery of highly cytotoxic payloads. Next, we conducted a high throughput study of the electrostatic properties for all combinations of VH and VL germlines. This analysis shows how different VH/VL combinations exhibit varying tendencies to create electrostatic patches, resulting in Fv variants with different isoelectric points. Our work emphasizes the importance of carefully selecting germlines for humanization with optimal electrostatic properties in order to control the unspecific tissue uptake of low molecular weight biologics.
Topics: Humans; Tissue Distribution; Biological Products; Static Electricity; Kidney; Immunoglobulin Fragments; Germ Cells
PubMed: 38334129
DOI: 10.1080/19420862.2024.2311991 -
Cells May 2024Mutations in p53 and KRAS are seen in most cases of colon cancer. The impact of these mutations on signaling pathways related to cancer growth has been studied in depth,...
Impact of Oncogenic Changes in p53 and KRAS on Macropinocytosis and Ferroptosis in Colon Cancer Cells and Anticancer Efficacy of Niclosamide with Differential Effects on These Two Processes.
Mutations in p53 and KRAS are seen in most cases of colon cancer. The impact of these mutations on signaling pathways related to cancer growth has been studied in depth, but relatively less is known on their effects on amino acid transporters in cancer cells. This represents a significant knowledge gap because amino acid nutrition in cancer cells profoundly influences macropinocytosis and ferroptosis, two processes with opposing effects on tumor growth. Here, we used isogenic colon cancer cell lines to investigate the effects of p53 deletion and KRAS activation on two amino acid transporters relevant to macropinocytosis (SLC38A5) and ferroptosis (SLC7A11). Our studies show that the predominant effect of p53 deletion is to induce SLC7A11 with the resultant potentiation of antioxidant machinery and protection of cancer cells from ferroptosis, whereas KRAS activation induces not only SLC7A11 but also SLC38A5, thus offering protection from ferroptosis as well as improving amino acid nutrition in cancer cells via accelerated macropinocytosis. Niclosamide, an FDA-approved anti-helminthic, blocks the functions of SLC7A11 and SLC38A5, thus inducing ferroptosis and suppressing macropinocytosis, with the resultant effective reversal of tumor-promoting actions of oncogenic changes in p53 and KRAS. These findings underscore the potential of this drug in colon cancer treatment.
Topics: Humans; Ferroptosis; Pinocytosis; Colonic Neoplasms; Tumor Suppressor Protein p53; Proto-Oncogene Proteins p21(ras); Cell Line, Tumor; Niclosamide; Antineoplastic Agents; Amino Acid Transport System y+; Mutation
PubMed: 38891084
DOI: 10.3390/cells13110951 -
The Journal of Cell Biology Jul 2024Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has...
Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.
Topics: Animals; Actin Cytoskeleton; Bone Morphogenetic Proteins; Cyclic AMP; Guanine Nucleotide Exchange Factors; Neuronal Plasticity; Presynaptic Terminals; Proto-Oncogene Proteins c-vav; rap1 GTP-Binding Proteins; Shelterin Complex; Signal Transduction; Pinocytosis; Drosophila
PubMed: 38748250
DOI: 10.1083/jcb.202309095 -
The multifunction effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery.Proceedings of the National Academy of... Jun 2024Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. , the causative agent of Q fever, secretes...
Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. , the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a -containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and found that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine and lysobisphosphatidic acid. Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We found that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery. Collectively, our results reveal that Vice is an essential effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche.
Topics: Endosomal Sorting Complexes Required for Transport; Pinocytosis; Bacterial Proteins; Coxiella burnetii; Vacuoles; Humans; HeLa Cells; Cell Membrane; Animals; Phosphatidylinositols
PubMed: 38870060
DOI: 10.1073/pnas.2315481121 -
Biology Open May 2024Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside...
Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.
Topics: Wnt Signaling Pathway; Pinocytosis; Colonic Neoplasms; Sodium-Potassium-Exchanging ATPase; Animals; Humans; Ouabain; Cell Line, Tumor; Xenopus
PubMed: 38713004
DOI: 10.1242/bio.060269 -
Cancer Research Communications Jan 2024Extracellular vesicles (EV) have emerged as critical effectors in the cross-talk between cancer and normal cells by transferring intracellular materials between adjacent...
UNLABELLED
Extracellular vesicles (EV) have emerged as critical effectors in the cross-talk between cancer and normal cells by transferring intracellular materials between adjacent or distant cells. Previous studies have begun to elucidate how cancer cells, by secreting EVs, adapt normal cells at a metastatic site to facilitate cancer cell metastasis. In this study, we utilized a high-content microscopic screening platform to investigate the mechanisms of EV uptake by primary lung fibroblasts. A selected library containing 90 FDA-approved anticancer drugs was screened for the effect on fibroblast uptake of EVs from MDA-MB-231 breast cancer cells. Among the drugs identified to inhibit EV uptake without exerting significant cytotoxicity, we validated the dose-dependent effect of Trametinib (a MEK1/2 inhibitor) and Copanlisib (a PI3K inhibitor). Trametinib suppressed macropinocytosis in lung fibroblasts and inhibited EV uptake with a higher potency comparing with Copanlisib. Gene knockdown and overexpression studies demonstrated that uptake of MDA-MB-231 EVs by lung fibroblasts required MEK2. These findings provide important insights into the mechanisms underlying lung fibroblast uptake of breast cancer cell-derived EVs, which could play a role in breast cancer metastasis to the lungs and suggest potential therapeutic targets for preventing or treating this deadly disease.
SIGNIFICANCE
Through a phenotypic screen, we found that MEK inhibitor Trametinib suppressed EV uptake and macropinocytosis in lung fibroblasts, and that EV uptake is mediated by MEK2 in these cells. Our results suggest that MEK2 inhibition could serve as a strategy to block cancer EV uptake by lung fibroblasts.
Topics: Biological Transport; Extracellular Vesicles; Fibroblasts; Lung; Phosphatidylinositol 3-Kinases; Humans; MDA-MB-231 Cells; MAP Kinase Kinase 2; Pinocytosis; Breast Neoplasms
PubMed: 38259097
DOI: 10.1158/2767-9764.CRC-23-0316 -
Biology of the Cell May 2024Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca from acidic intracellular stores and are ubiquitously present in both animals...
BACKGROUND INFORMATION
Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy.
RESULTS
We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2 cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2 cells showed higher intracellular cAMP levels as compared to the tpc2 cells while pinocytosis was found to be higher in the tpc2 cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2 cells showed a bias towards the prestalk/stalk region while tpc2 cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions.
CONCLUSION
TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning.
SIGNIFICANCE
Decreased calcium due to deletion of tpc2 inhibit autophagic flux.
Topics: Dictyostelium; Autophagy; Protozoan Proteins; Gene Deletion; Calcium Channels; Calcium; Cell Differentiation
PubMed: 38537110
DOI: 10.1111/boc.202300067 -
Genes To Cells : Devoted To Molecular &... Jun 2024Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate...
Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca remains unclear. Here, we find that both intracellular and extracellular Ca are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca dynamics.
Topics: Pinocytosis; Humans; Epidermal Growth Factor; Plasma Membrane Calcium-Transporting ATPases; Calcium; Cell Line, Tumor
PubMed: 38597132
DOI: 10.1111/gtc.13118