Biology subjects

Mercier, V.

Publications and source records attributed to Mercier, V..

11 recordsLinked to original sources

bioRxiv2026

Dynamic S-acylation controls CMG2 maturation, extracellular matrix regulation, and anthrax toxin entry

CMG2/ANTXR2 functions as a Collagen VI receptor and the primary portal for anthrax toxin entry. We find that CMG2 is regulated by ordered cycles of S-acylation and deacylation throughout its life cycle. Following synthesis in the endoplasmic reticulum, acylation by ZDHHC7 on two juxtamembrane cysteines protects folding intermediates from ER-associated degradation, resulting in a 5-fold increase in CMG2 biogenesis. The cytosolic thioesterase APT2 can remove these acyl chains, thereby controlling CMG2 levels. In the Golgi, CMG2 acylation by ZDHHC3 on a third cysteine to permit Arf6-dependent transport to the plasma membrane. At the cell surface, S-acylated CMG2 recruits APT2 in response to ligand binding, enabling release from the actin cytoskeleton and endocytosis. Accordingly, blocking APT2 suppresses the intracellular delivery of anthrax toxin, and inhibits CMG2-dependent Collagen VI degradation. These results define S-acylation-deacylation cycles as key regulators of CMG2 biogenesis and function, and highlight APT2 inhibition as a strategy to modulate CMG2 levels or prevent anthrax intoxication.

cell biology↗

bioRxiv2024

TORC2-regulated sterol redistribution mediates recovery from membrane perturbation by small amphipathic molecules.

To maintain plasma membrane (PM) integrity, cells need to acutely regulate PM lipid composition. The Target Of Rapamycin (TOR) complex 2 is a protein kinase that acts as a central regulator of PM homeostasis, but the mechanisms by which it monitors and reacts to membrane stresses are poorly understood. To address this knowledge gap, we characterized a family of amphiphilic molecules that physically perturb PM organization and in doing so inhibit TORC2 in yeast and mammalian cells. Using fluorescent lipid associated reporters in budding yeast, we show that these small molecules first cause a transient increase in the amount of biochemically accessible ergosterol at the PM. Contemporaneous TORC2 inhibition stimulates a rapid removal of accessible ergosterol from the PM by the PM-ER sterol transporters Lam2 and Lam4, necessary for TORC2 reactivation. Thus, we show that TORC2 acts in a feedback loop to control active sterol levels at the PM and introduce sterols as possible TORC2 signalling modulators.

cell biology↗

bioRxiv2023

SARS-CoV-2 shifts transcription of host gene to increase Spike acylation and boost infectivity

SARS-CoV-2 infection requires Spike protein mediating fusion between the viral and cellular membranes. The fusogenic activity of Spike requires its post-translational lipid modification by host S-acyltransferases, predominantly ZDHHC20. Previous observations indicate that SARS-CoV-2 infection augments the S-acylation of Spike when compared to transfection. Here, we find that SARS-CoV-2 infection triggers a change in the transcriptional start site of the zddhc20 gene, both in cells and in an in vivo infection model, resulting in a 67-amino-acid-long N-terminally extended protein with 37-times higher Spike acylating activity, leading to enhanced viral infectivity. Furthermore, we observed the same induced transcriptional change in response to other challenges, such as chemically induced colitis, indicating that SARS-CoV-2 hijacks an existing cell damage response pathway to generate more infectious viruses.

cell biology↗

bioRxiv2022

Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes

Measuring forces within living cells remains a technical challenge. We developed hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing and can report on membrane tension. Here, we describe technical optimization of the probe imaging, and diverse characterizations in various biological and in vitro systems. We provide a guideline to measure biophysical parameters of cellular membranes by FLIM microscopy with Flipper probes, providing evidences that flippers can report long range forces in cells, tissues and organsi.

biophysics↗

bioRxiv2022

Retromer oligomerization drives SNX-BAR coat assembly and membrane constriction

The retromer coat mediates protein exit from endosomes and impacts many signaling pathways, lysosomal biogenesis, and diseases such as Parkinsons, Alzheimers and COVID-19. Retromer complexes (CSC in yeast) form coats by interconnecting sorting nexins (SNX). The dynamics of this process is poorly explored. Here, we analyze the oligomerization of CSC/SNX-BAR retromer coats on oriented synthetic lipid tubules. SNX-BARs and CSC assemble a static tubular coat that does not exchange subunits. Coat formation proceeds bidirectionally, adding new subunits at both ends of the coat. High concentrations of SNX-BARs alone suffice to constrict membrane tubes to an invariant radius of 19 nm. At lower concentrations, CSC-complexes must drive constriction, which requires their oligomerization. CSCs populate the SNX-BAR layer at densities that increase with the starting radius of the membrane tube. We hence propose that retromer-mediated crosslinking of SNX-BARs at variable densities tunes the coat according to the energy required to deform the membrane. This model is supported by the effects of mutations interfering with retromer oligomerization, which impair retromer function in yeast and human cells.

cell biology↗

bioRxiv2021

Quantitative coupling of cell volume and membrane tension during osmotic shocks

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as a quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase, quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. During these asymmetric recoveries, tension and volume remained coupled. Pharmacological disruption of the cytoskeleton and functional inhibition of ion channels and mTOR all affected tension and volume responses, proving that a passive mechanism is necessary and critical for the cell to adapt fast. The coupling between them was, nonetheless, maintained for a few exceptions suggesting that volume and tension regulations are independent from the regulation of their coupling.

biophysics↗

bioRxiv2020

ALG-2 interacting protein-X (Alix) is required for activity-dependent bulk endocytosis at brain synapses

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity dependent bulk endocytosis (ADBE). Alix (ALG-2 interacting protein X)/ PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with, and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ABDE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix alters synaptic recovery during physiological or pathological repeated stimulations.

neuroscience↗

bioRxiv2019

Vps4 triggers sequential subunit exchange in ESCRT-III polymers that drives membrane constriction and fission

ESCRT-III is a ubiquitous complex which catalyzes membrane fission from within membrane necks via an as yet unknown mechanism. Here, we reconstituted in vitro the ESCRT-III complex onto membranes. We show that based on variable affinities between ESCRT-III proteins and the ATPase Vps4, subunits are recruited to the membrane in a Vps4-driven sequence that starts with Snf7 and ends with Did2 and Ist1 which, together, form a fission-active subcomplex. Sequential recruitment of ESCRT-III subunits is coupled to membrane remodeling. Binding of Did2 promoted the formation of membrane protrusions which later constricted and underwent fission mediated by the recruitment of Ist1. Overall, our results provide a mechanism to explain how a sequence of ESCRT-III subunits drives membrane deformation and fission.

biochemistry↗

bioRxiv2019

ENDOSOMAL MEMBRANE TENSION CONTROLS ESCRT-III-DEPENDENT INTRA-LUMENAL VESICLE FORMATION

Plasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether membrane tension of organelles regulates their functions, notably intracellular traffic. The ESCRT-III complex is the major membrane remodelling complex that drives Intra-Lumenal Vesicle (ILV) formation on endosomal membranes. Here, we made use of a new fluorescent membrane tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro, and correlates with increased ILVs formation in ESCRT-III decorated endosomes in vivo. Finally, we find that endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and of endosome trafficking, leading us to conclude that membrane tension can control organelle functions. One Sentence SummaryMembrane tension decrease facilitates membrane remodeling by ESCRT-III polymerization during intra-lumenal vesicle formation.

cell biology↗

bioRxiv2018

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells

In specialized cell types, lysosome-related organelles support regulated secretory pathways, while in non-specialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in non-specialized cells, and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel MCOLN1, as well as FYCO1, the AP1 adaptor and its partner Gadkin. We conclude that endolysosomes in non-specialized cells can acquire secretory functions elicited by cyclodextrin, and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

cell biology↗

bioRxiv2018

LimeSeg: A coarsed-grained lipid membrane simulation for 3D image segmentation

Bioimage analysis is an important preliminary step required for data representation and quantitative studies. To carry out these tasks, we developed LimeSeg, an easy-to-use, efficient and modular 3D image segmentation method. Based on the idea of SURFace ELements, LimeSeg resembles a highly coarse-grained simulation of a lipid membrane in which a set of particles, analogous to lipid molecules, are attracted to local image maxima. The particles are self-generating and self-destructing thus providing the ability for the membrane to evolve towards the contour of the object of interest. We characterize the emergent mechanical properties of this system and show how it can be used to segment many 3D objects from numerous types of image of biological samples (brain MRI, cell epithelium, cellular organelles). LimeSeg is available as a Fiji plugin that includes simple commands, a 3D visualizer, and customization options via ImageJ scripting.

bioinformatics↗