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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 -
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 -
The Science of the Total Environment Jun 2024Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat...
Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat posed by MP, the possible uptake and trophic transfer in lower trophic levels of freshwater ecosystems remains understudied. This study aims to investigate the internalization and potential trophic transfer of fluorescent polystyrene (PS) beads (0.5 μm, 3.6 × 10 particles/mL; 6 μm, 2.1 × 10 particles/mL) and fragments (<30 μm, 5 × 10 particles/mL) in three unicellular eukaryotes. This study focuses on the size-dependent uptake of MP by two freshwater Ciliophora, Tetrahymena pyriformis, Paramecium caudatum and one Amoebozoa, Amoeba proteus, serving also as predator for experiments on potential trophic transfer. Size-dependent uptake of MP in all three unicellular eukaryotes was shown. P. caudatum is able to take up MP fragments up to 27.7 μm, while T. pyriformis ingests particles up to 10 μm. In A. proteus, small MP (PS and PS) were taken up via pinocytosis and were detected in the cytoplasm for up to 14 days after exposure. Large PS-MP (PS) were detected in A. proteus only after predation on MP-fed Ciliophora. These results indicate that A. proteus ingests larger MP via predation on Ciliophora (PS), which would not be taken up otherwise. This study shows trophic transfer of MP at the base of the aquatic food web and serves as basis to study the impact of MP in freshwater ecosystems.
Topics: Microplastics; Water Pollutants, Chemical; Food Chain; Polystyrenes; Fresh Water; Environmental Monitoring; Tetrahymena pyriformis; Amoeba; Paramecium caudatum; Particle Size
PubMed: 38621530
DOI: 10.1016/j.scitotenv.2024.172470 -
Haematologica Apr 2024Belantamab mafodotin (belantamab) is a first-in-class anti-BCMA antibody-drug conjugate approved for the treatment of triple-class refractory multiple myeloma. It...
Belantamab mafodotin (belantamab) is a first-in-class anti-BCMA antibody-drug conjugate approved for the treatment of triple-class refractory multiple myeloma. It provides a unique therapeutic option for patients ineligible for CAR-T and bispecific antibody therapy, and/or patients progressing on anti-CD38 treatment where CAR-T and bispecifics might be kept in reserve. Wider use of the drug can be challenged by its distinct ocular side effect profile, including corneal microcysts and keratopathy. While dose reduction has been the most effective way to reduce these toxicities, the underlying mechanism of this BCMA off-target effect remains to be characterized. In this study, we provide the first evidence for soluble BCMA (sBCMA) in lacrimal fluid and report on its correlation with tumor burden in myeloma patients. We confirm that corneal cells do not express BCMA, and show that sBCMA-belantamab complexes may rather be internalized by corneal epithelial cells through receptor-ligand independent pinocytosis. Using an hTcEpi corneal cell-line model, we show that the pinocytosis inhibitor EIPA significantly reduces belantamab-specific cell killing. As a proof of concept, we provide detailed patient profiles demonstrating that, after belantamab-induced cell killing, sBCMA is released into circulation, followed by a delayed increase of sBCMA in the tear fluid and subsequent onset of keratopathy. Based on the proposed mechanism, pinocytosis-induced keratopathy can be prevented by lowering the entry of sBCMA into the lacrimal fluid. Future therapeutic concepts may therefore consist of belantamab-free debulking therapy prior to belantamab consolidation and/or concomitant use of gamma-secretase inhibition as currently evaluated for belantamab and nirogacestat in ongoing studies.
PubMed: 38572568
DOI: 10.3324/haematol.2024.285205 -
Frontiers in Immunology 2024Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality but lacks specific therapeutic options. Diverse endocytic processes play a key... (Review)
Review
Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality but lacks specific therapeutic options. Diverse endocytic processes play a key role in all phases of acute lung injury (ALI), including the initial insult, development of respiratory failure due to alveolar flooding, as a consequence of altered alveolar-capillary barrier function, as well as in the resolution or deleterious remodeling after injury. In particular, clathrin-, caveolae-, endophilin- and glycosylphosphatidyl inositol-anchored protein-mediated endocytosis, as well as, macropinocytosis and phagocytosis have been implicated in the setting of acute lung damage. This manuscript reviews our current understanding of these endocytic pathways and subsequent intracellular trafficking in various phases of ALI, and also aims to identify potential therapeutic targets for patients with ARDS.
Topics: Humans; Respiratory Distress Syndrome; Endocytosis; Acute Lung Injury; Pinocytosis; Phagocytosis
PubMed: 38533500
DOI: 10.3389/fimmu.2024.1360370 -
Journal of Extracellular Vesicles Apr 2024Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical...
Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.
Topics: Proton Pump Inhibitors; Extracellular Vesicles; Endocytosis; Pinocytosis; Adenosine Triphosphatases
PubMed: 38532609
DOI: 10.1002/jev2.12426 -
F1000Research 2023Heterozygous variants in the gene cause the complex multisystem disorder, MIRAGE syndrome. Patients are characterised by myelodysplasia, infections, growth... (Observational Study)
Observational Study
BACKGROUND
Heterozygous variants in the gene cause the complex multisystem disorder, MIRAGE syndrome. Patients are characterised by myelodysplasia, infections, growth restriction, adrenal insufficiency, gonadal dysfunction and enteropathies. Pathogenic variants in SAMD9 are gain-of-function and enhance its role as a growth repressor, leading to growth restriction of many tissues. Two studies have reported changes in skin fibroblasts derived from MIRAGE patients, more specifically identifying enlarged endosomes. We have also previously shown subtle changes in endosome size in patients' fibroblasts compared to controls. However, these variations in endosomes were not as marked as those described in the literature.
METHODS
We have performed an observational study using transmission electron microscopy (TEM) in a larger number of cells derived from three patients' fibroblasts to assess ultrastructure morphology compared to control images.
RESULTS
Consistent changes were observed in cell organelles in all patient samples. In particular, increased endosomal activity was detected, characterised by augmented pinocytosis and vesicle budding, increased endosome number, as well as by large lysosomes and endosomes. Endoplasmic reticulum was also prominent. Mitochondria appeared enlarged in selected cells, possibly due to cellular stress. Cell nuclei did not display major differences compared to controls.
CONCLUSIONS
TEM is a powerful tool to investigate morphological features of tissues and cell organelles, although TEM data could be affected by sample preparation methodology, therefore potentially explaining the variability between independent studies, and its analysis can be dependent on the experience of the researcher. The increased endosomal activity we have observed in patients' fibroblasts could indicate that SAMD9 regulates endocytosis of receptors, acting as an endosome fusion facilitator, or in lysosomal activation. However, the precise mechanism(s) by which SAMD9 regulates cell growth is still not fully understood, and further studies are needed to elucidate its pathogenic pathway and develop therapeutic approaches to support patients.
Topics: Humans; Fibroblasts; Cell Nucleus; Endocytosis; Lysosomes; Cell Cycle; Intracellular Signaling Peptides and Proteins
PubMed: 38434662
DOI: 10.12688/f1000research.129559.2 -
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 -
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 Cardiovascular Medicine 2023Endocytosis constitutes a cellular process in which cells selectively encapsulate surface substances into endocytic vesicles, also known as endosomes, thereby modulating... (Review)
Review
Endocytosis constitutes a cellular process in which cells selectively encapsulate surface substances into endocytic vesicles, also known as endosomes, thereby modulating their interaction with the environment. Platelets, as pivotal hematologic elements, play a crucial role not only in regulating coagulation and thrombus formation but also in facilitating tumor invasion and metastasis. Functioning as critical components in the circulatory system, platelets can internalize various endosomal compartments, such as surface receptors, extracellular proteins, small molecules, and pathogens, from the extracellular environment through diverse endocytic pathways, including pinocytosis, phagocytosis, and receptor-mediated endocytosis. We summarize recent advancements in platelet endocytosis, encompassing the catalog of cargoes, regulatory mechanisms, and internal trafficking routes. Furthermore, we describe the influence of endocytosis on platelet regulatory functions and related physiological and pathological processes, aiming to offer foundational insights for future research into platelet endocytosis.
PubMed: 38264257
DOI: 10.3389/fcvm.2023.1308170