Biology subjects

Joliot, A.

Publications and source records attributed to Joliot, A..

7 recordsLinked to original sources

bioRxiv2026

LUCID-EV: a robust and quantitative bioluminescent assay for the detection of EV cytosolic delivery in the absence of VSV-G expression

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

cell biology↗

bioRxiv2026

Measuring capture, internalization and cytosolic delivery of extracellular vesicle-embedded syntenin

Extracellular vesicles (EVs) mediate cell-to-cell communication and are considered potential drug delivery vehicles. Nevertheless, whether EV-embedded cargo can be efficiently delivered into the cytosol of recipient cells remains debated. Here, we investigated the fate of syntenin, a well-established internal cargo of small EVs (sEVs). Using quantitative assays, we show that [~]85% of internalized sEV-embedded syntenin can be delivered to the cytosol of recipient cells within short periods of time. Yet, even at low dose, we find that the internalization of sEVs carrying syntenin is rather inefficient ([~]0.03% of the administered dose). Moreover, we observe that the capture of sEVs by recipient cells is non-saturable over time and largely more efficient than their internalization. Finally, we identify the N-terminal domain of syntenin and the phosphorylation state of a Src-targeted tyrosine residue in this domain, as key determinants for its incorporation into sEVs that support cytosolic delivery. These findings challenge, current views in the field by indicating that sEV internalization may be a marginal process (on the contrary to capture) and that cytosolic delivery can be highly efficient. Moreover, our study identifies molecular determinants governing cytosolic delivery of sEV-embedded syntenin.

cell biology↗

bioRxiv2024

Electrical control of the transduction channels' gating force in sensory hair cells.

The inner ears hair cells rely on mechanosensitive ion channels to convert vibrations of their hair bundles into electrical signals. The mechanical correlates of channel gating--the gating force and the gating swing--are fundamental determinants of hair-cell mechanosensitivity but are still poorly understood. Here we show that varying the electrical potential across the sensory hair-cell epithelium continuously modulates the gating force, by up to {+/-}100%. Our observations also revealed an abrupt transition between states of weak and strong gating force at a threshold potential, so that strong gating forces associated to high mechanosensitivity are observed only when the calcium influx through the channels is large enough, but not too large. Gating-force changes, remarkably enough, were explained by the modulability of the gating swing, ranging from values comparable to the channel pore size to nearly tenfold larger. Gating-swing control is expected to underly the hair cells ability to tune its mechanosensitivity to minute sound stimuli. Table of contentThe inner ears hair cells rely on mechanosensitive ion channels to convert vibrations of their hair-bundle into electrical signals. We show that varying the electrical potential (U) across the sensory epithelium modulates a key determinant of mechanosensitivity--the gating force (FG)--by modulating the gating swing (d), ranging from the size of the channels pore to nearly tenfold larger. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/628311v3_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@18425bborg.highwire.dtl.DTLVardef@c9512forg.highwire.dtl.DTLVardef@f10c0borg.highwire.dtl.DTLVardef@14dc154_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Detection of tumor-derived extracellular vesicles interactions with immune cells is dependent on EV-labelling methods

Cell-cell communication within the complex tumor microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Understanding the interactions between TD-EVs and immune cells is essential for understanding immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs), analyzed by imaging flow cytometry and multicolor spectral flow cytometry. EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labeled either with the lipophilic dye MemGlow-488 (MG-488), with Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), or through expression of a MyrPalm-superFolder GFP (sfGFP) that incorporates into EVs during their biogenesis using a genetically engineered cell line. Our results showed that these different labeling strategies, although analyzed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labeling method. Moreover, MG-488-labeled liposomes behaved similarly to MG-488 EVs, highlighting the predominant role of the labelling strategy on the visualization and analysis of TD-EVs uptake by immune cell types. Consequently, the use of different EV labeling methods has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

cell biology↗

bioRxiv2021

Homosalate boosts the release of tumor-derived Extracellular Vesicles with anti-anoikis properties

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63- or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we discovered that: 1) the two drugs act on EVs generated in distinct subcellular compartments and 2) EVs released upon treatment with Homosalate, but not with Bafilomycin A1, conferred anti-anoikis properties to another recipient tumor cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harboring distinct functional properties.

cell biology↗

bioRxiv2021

Reciprocal regulation of Shh trafficking and H2O2 levels via a noncanonical BOC-Rac1 pathway

Among molecules that bridge environment, cell metabolism, and cell signaling, H2O2 recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery and that physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. A feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

cell biology↗

bioRxiv2020

Specific interaction of Penetratin with cell surface partners measured with biomembrane force probe

Penetratin is a Cell Penetrating Peptide able to cross the cell plasma membrane possibly bound to a cargo molecule to be delivered into the cell. The mechanism of its entry is poorly known. A key to a molecular description of this mechanism is to identify the partners of Penetratin at the cell surface during its adhesion and internalization. We used the Biomembrane Force Probe to identify the partners during the first second of adhesion of Penetratin on the cell plasma membrane. We evidenced that heparan sulfates are the first partners after contact as well as unknown partners hidden by sialic acids. Experiments of binding of Penetratin on vesicles bearing charged or sulfated lipids showed no adhesion pointing that a negatively charged partner is not enough and there is a specificity for certain chemical groups bearing the charges. A model of the measured forces of interaction enabled to determine the adhesion energy of a Penetratin with heparan sulfates on a cell to be in the range 18 to 22 kBT.

biophysics↗