scholarly journals Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Emeline Bonsergent ◽  
Eleonora Grisard ◽  
Julian Buchrieser ◽  
Olivier Schwartz ◽  
Clotilde Théry ◽  
...  

AbstractExtracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.

1991 ◽  
Vol 280 (1) ◽  
pp. 211-218 ◽  
Author(s):  
F Authier ◽  
B Desbuquois

Endosomes have recently been identified as one major site of glucagon degradation in intact rat liver. In this study, a cell-free system has been used to assess the role of ATP-dependent acidification in endosomal glucagon degradation and identify the glucagon products generated. Percoll gradient fractionation of Golgi-endosomal fractions prepared 10-30 min after injection of [125I]iodoglucagon showed a time-dependent shift of the radioactivity towards high densities. Regardless of time, the radioactivity was less precipitable by trichloroacetic acid (Cl3Ac) at high densities than at low densities. Chloroquine treatment slightly increased the density shift of the radioactivity and decreased its Cl3Ac-precipitability throughout the gradient. Incubation of endosomal fractions containing [125I]iodoglucagon in 0.15 M-KCl at 30 degrees C resulted in a time- and pH-dependent generation of Cl3Ac-soluble radioactivity, with a maximum at pH 4 (t1/2, 7 min). At pH 5, 1,10-phenanthroline, bacitracin and p-chloromercuribenzoic acid partially inhibited [125I]iodoglucagon degradation. At pH 6-7, ATP stimulated [125I]iodoglucagon degradation by 5-10-fold and caused endosomal acidification as judged from Acridine Orange uptake. The effects of ATP were inhibited by chloroquine, monensin, N-ethylmaleimide and dansylcadaverine. Poly(ethylene glycol) (PEG) precipitation of the radioactivity associated with endosomes showed that lowering the pH below 5.5 caused dissociation of the glucagon-receptor complex, and that, regardless of incubation conditions, all degraded [125I]iodoglucagon diffused extraluminally. On h.p.l.c., at least three products less hydrophobic than [125I]iodoglucagon were identified in incubation mixtures along with monoiodotyrosine. Radiosequence analysis of the products revealed one major cleavage located C-terminally to Tyr-13 and two minor cleavages affecting Thr-5-Phe-6 and Phe-6-Thr-7 bonds. It is concluded that glucagon degradation in liver endosomes is functionally linked to ATP-dependent endosomal acidification and involves several cleavages in the glucagon sequence.


2002 ◽  
Vol 50 (5) ◽  
pp. 697-708 ◽  
Author(s):  
Riyan Chen ◽  
Veronica H. Kang ◽  
Jian Chen ◽  
Joseph C. Shope ◽  
Javad Torabinejad ◽  
...  

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsPn) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsPn quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsPns. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P3 IgM, and immunodetection of PtdIns(3,4,5)P3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P3 relative to its precursor, phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P2), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was bench-marked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsPns. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P3 and PtdIns(4,5)P2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.


2006 ◽  
Vol 74 (3) ◽  
pp. 1537-1546 ◽  
Author(s):  
Renata C. P. Baida ◽  
Márcia R. M. Santos ◽  
Mirian S. Carmo ◽  
Nobuko Yoshida ◽  
Danielle Ferreira ◽  
...  

ABSTRACT We previously reported the isolation of a novel protein gene family, termed SAP (serine-, alanine-, and proline-rich protein), from Trypanosoma cruzi. Aided by the availability of the completed genome sequence of T. cruzi, we have now identified 39 full-length sequences of SAP, six pseudogenes and four partial genes. SAPs share a central domain of about 55 amino acids and can be divided into four groups based on their amino (N)- and carboxy (C)-terminal sequences. Some SAPs have conserved N- and C-terminal domains encoding a signal peptide and a glycosylphosphatidylinositol anchor addition site, respectively. Analysis of the expression of SAPs in metacyclic trypomastigotes by two-dimensional electrophoresis and immunoblotting revealed that they are likely to be posttranslationally modified in vivo. We have also demonstrated that some SAPs are shed into the extracellular medium. The recombinant SAP exhibited an adhesive capacity toward mammalian cells, where binding was dose dependent and saturable, indicating a possible ligand-receptor interaction. SAP triggered the host cell Ca2+ response required for parasite internalization. A cell invasion assay performed in the presence of SAP showed inhibition of internalization of the metacyclic forms of the CL strain. Taken together, these results show that SAP is involved in the invasion of mammalian cells by metacyclic trypomastigotes, and they confirm the hypothesis that infective trypomastigotes exploit an arsenal of surface glycoproteins and shed proteins to induce signaling events required for their internalization.


2007 ◽  
Vol 51 (7) ◽  
pp. 2403-2411 ◽  
Author(s):  
Ana M. Sanchez ◽  
Diane Thomas ◽  
Eugene J. Gillespie ◽  
Robert Damoiseaux ◽  
Joseph Rogers ◽  
...  

ABSTRACT Anthrax lethal toxin is one of the fundamental components believed to be responsible for the virulence of Bacillus anthracis. In order to find novel compounds with anti-lethal toxin properties, we used a cell-based assay to screen a collection of approximately 500 small molecules. Nineteen compounds that blocked lethal toxin-mediated killing of RAW 264.7 macrophages were identified, and we report here on the characterization of the two most potent antitoxic compounds, amiodarone and bepridil. These drugs are used to treat cardiac arrhythmia or angina in humans at doses similar to those that provide protection against lethal toxin in vitro. Our results support a model whereby the antitoxic properties of both drugs result from their ability to block endosomal acidification, thereby blocking toxin entry. Amiodarone was tested in vivo and found to significantly increase survival of lethal toxin-challenged Fischer rats.


2018 ◽  
Author(s):  
Anis Senoussi ◽  
Jonathan Lee Tin Wah ◽  
Yoshihiro Shimizu ◽  
Jérôme Robert ◽  
Alfonso Jaramillo ◽  
...  

AbstractRiboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo and they have a fast turnover and a low metabolic burden. However, riboregulators are generally designed in silico and tested in vivo, which only provides a yes/no evaluation of their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable quantitative information about the performances of in silico designed riboregulators. In particular, we use the ribosome as an exquisite molecular machine that detects functional riboregulators, precisely measures their concentration and linearly amplifies the signal by generating a fluorescent protein. We apply this method to characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.


2021 ◽  
Vol 22 (18) ◽  
pp. 9945
Author(s):  
Luisa Galla ◽  
Nicola Vajente ◽  
Diana Pendin ◽  
Paola Pizzo ◽  
Tullio Pozzan ◽  
...  

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.


2000 ◽  
Vol 68 (5) ◽  
pp. 2470-2474 ◽  
Author(s):  
Maen Qa'Dan ◽  
Lea M. Spyres ◽  
Jimmy D. Ballard

ABSTRACT Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.


2001 ◽  
Vol 12 (4) ◽  
pp. 971-980 ◽  
Author(s):  
Nasser M. Rusan ◽  
Carey J. Fagerstrom ◽  
Anne-Marie C. Yvon ◽  
Patricia Wadsworth

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.


1999 ◽  
Vol 340 (2) ◽  
pp. 467-474 ◽  
Author(s):  
Yasunori NAGANAWA ◽  
Kohji ITOH ◽  
Michie SHIMMOTO ◽  
Sachiko KAMEI ◽  
Kyoko TAKIGUCHI ◽  
...  

Fibroblastic cell lines derived from a galactosialidosis patient, stably expressing the chimaeric green fluorescent protein variant (EGFP) gene fused to the wild-type and mutant human lysosomal protective protein/cathepsin A (PPCA) cDNA, were first established as a model system for revealing the sorting and processing of lysosomal enzymes and for investigating the molecular bases of their deficiencies. In the cell line expressing the wild-type PPCA-EGFP chimaera gene (EGFP-PPwild), an 81 kDa form (27 kDa EGFP fused to the C-terminus of the 54 kDa PPCA precursor) was produced, then processed into the mature 32/20 kDa two-chain form free of the EGFP domain. The intracellular cathepsin A, α-N-acetylneuraminidase and β-galactosidase activities, which are deficient in the parent fibroblastic cells, could also be significantly restored in the cells. In contrast with the uniform and strong fluorescence throughout the cytoplasm and nucleus in the mock-cell line expressing only EGFP cDNA, weak reticular and punctate fluorescence was distributed throughout the EGFP-PPwild cell line. Bafilomycin A1, a potent inhibitor of vacuolar ATPase and intracellular acidification, induced the distribution of Golgi-like perinuclear fluorescence throughout the living and fixed cells, in which only the 81 kDa product was detected. After removal of the agent, time-dependent transport of the chimaeric protein from the Golgi apparatus to the prelysosomal structure in living cells was monitored with a confocal laser scanning microscope system. Leupeptin caused the distribution of lysosome-like granular fluorescence throughout the cytoplasm in the fixed cells, although it was hardly observed in living cells. The latter agent also dose-dependently induced an increase in the intracellular amount of the 81 kDa product containing the EGFP domain and inhibited the restoration of cathepsin A activity in the EGFP-PPwild cells after the removal of bafilomycin A1. In parallel, both the mature two-chain form and PPCA function disappeared. These results suggested that the chimaera gene product was transported to acidic compartments (endosomes/lysosomes), where proteolytic processing of the PPCA precursor/zymogen, quenching of the fluorescence, and random degradation of the EGFP portion occurred. A cell line stably expressing a chimaeric gene with a mutant PPCA cDNA containing an A1184 → G (Y395C) mutation, commonly detected in Japanese severe early-infantile type of galactosialidosis patients, showed an endoplasmic reticulum (ER)-like reticular fluorescence pattern. The PPCA-immunoreactive gene product was hardly detected in this cell line. The mutant chimaeric product was suggested to be degraded rapidly in the ER before transport to post-ER compartments. A cell line expressing the chimaeric gene with a T746 → A (Y249N) PPCA mutation exhibited both ER-like reticular and granular fluorescence on the reticular structure that was stronger than that in the EGFP-PPwild cells. Some of them contained large fluorescent inclusion-body-like structures. The ineffectiveness of transport inhibitors in the distribution changes in the two mutant chimaeric proteins suggested that they were not delivered to acidic compartments. Therefore this expression system can possibly be applied to the direct analysis of the sorting defects of mutant gene products in living cells and will be useful for the molecular investigation of lysosomal diseases, including galactosialidosis.


2020 ◽  
Author(s):  
Marcus Y. Chin ◽  
Anand R. Patwardhan ◽  
Kean-Hooi Ang ◽  
Austin L. Wang ◽  
Carolina Alquezar ◽  
...  

ABSTRACTLysosomes are important sites for macromolecular degradation, defined by a profoundly acidic lumenal pH of ~4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein-based pH biosensor called FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes). This sensor was targeted to lysosomes with LAMP1 and reported lumenal pH between 3.0 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content imaging. We expressed FIRE-pHLy in several cellular models as well as the multicellular organism C. elegans and quantified its alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust and versatile lysosomal pH biosensor that has broad applications for investigating pH dynamics in aging and lysosome-related diseases, as well as in lysosome-based drug discovery.


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