Biology subjects

Le Roux, A.-L.

Publications and source records attributed to Le Roux, A.-L..

8 recordsLinked to original sources

bioRxiv2025

Mechanical forces stimulate Golgi export

Cells face diverse mechanical stimuli that vary with cell type, state, and pathological conditions. Mechanobiology investigates how cells sense and respond to these forces. While most work has focused on the cell surface and nucleus as primary mechanosensors, how intracellular organelles adapt to extracellular mechanical forces remains largely unknown. Here, we show that extracellular mechanical signals influence the secretory function of the Golgi apparatus. By subjecting adherent cells to mechanical challenges -cell spreading on different ligands, altered substrate stiffness, or equibiaxial strain-, we reveal that extracellular forces modulate Golgi-to-cell surface carrier biogenesis, thereby regulating exocytosis. Together with changes in Golgi membrane tension, we identify molecular determinants of the mechanotransduction pathway, including microtubule acetylation, diacylglycerol production, and protein kinase D activity. In turn, inhibition of Golgi export suppresses this mechanoresponse and causes impaired cell spreading. These findings uncover a bidirectional mechanotransduction axis in which extracellular mechanics tune Golgi secretory output, providing a framework for investigating organelle-based mechanoadaptation in physiology and disease.

cell biology↗

bioRxiv2024

Parameters for sensing lipid loosening at the inner nuclear membrane by amphipathic helices

Amphipathic helices (AHs) are ubiquitous protein motifs that modulate targeting to organellar membranes by sensing differences in bulk membrane properties. However, the adaptation between membrane-targeting AHs and the nuclear membrane environment that surrounds the genome is poorly understood. Here, we computationally screened for candidate AHs in a curated list of characterized and putative human inner nuclear membrane (INM) proteins. Cell biological and in vitro experimental assays combined with computational calculations demonstrated that AHs detect lipid packing defects over electrostatics to bind to the INM, indicating that the INM is loosely packed under basal conditions. Membrane tension resulting from hypotonic shock further promoted AH binding to the INM, whereas cell-substrate stretch did not enhance recruitment of membrane tension-sensitive AHs. Together, our work demonstrates the rules driving lipid-protein interactions at the INM, and its implications in the response of the nucleus to different stimuli.

cell biology↗

bioRxiv2024

Lipid-driven SRC self-association modulates its transformation capacity

Src tyrosine kinase is anchored to the plasma membrane, however, the contribution of membrane lipids to its regulation remains elusive. Here we report Src self-association through a lysine cluster in the Src SH4 region mediated by lipids in human cells and in vitro. Mutation of the lysine cluster to arginine modulates Src self-association and its transforming function in human cells. This mechanism may also apply to other membrane-associated signaling proteins that have similar lysine clusters in their unstructured regions. Lipid-anchored micron-sized condensates of full length Src are formed in supported homogeneous lipid bilayers (i.e independently of lipid phase separation). Condensates are also formed by the purified Src N-terminal regulatory element, including the myristoylated SH4 domain, the intrinsically disordered Unique domain and the globular SH3 domain. The isolated SH4 domain forms small protein-lipid clusters, but not micron-size condensates. Our findings reveal lipid-mediated kinase self-association as an additional mechanism for Src regulation. Significance StatementCondensate formation, often associated to intrinsically disordered protein regions is emerging as a widespread regulatory mechanism. Protein phase separation has been linked to the regulation of kinases recruited to condensates formed by other proteins. Here we show i) that the N-terminal disordered region of Src kinase drives lipid-mediated self-association to form condensates on the cell surface, ii) a lysine cluster in the N-terminal SH4 domain is essential for this process, iii) mutation of these lysine residues to arginine increases self-association in vitro and in vivo, and iv) self-association of Src results in enhanced transforming capacity in human cells. Similar lysine clusters are present in juxtamembrane regions suggest a general mechanism for modulating protein-protein interactions by lipids. The article shows the formation of condensates by Src on supported lipid membranes mediated by lipids, dependent of a conserved lysine cluster in its SH4 domain, and modulating Srcs transforming capacity. O_LIFull length Src and truncated variants including its myristoylated SH4 domain self-associate on the surface of homogeneous lipid bilayers. C_LIO_LIThe interaction of a lysine cluster in SH4 domain with lipids modulates Src self-association C_LIO_LIAtomic force microscopy shows the formation of micron-size condensates and smaller clusters on the surface of supported lipid bilayers, depending on the lipid composition or the presence of mutations in the lysine cluster C_LIO_LISrc self-association is confirmed in human cells and the Src transforming function of Src in human cells is modulated by lipid-mediated self-association C_LI

molecular biology↗

bioRxiv2022

Lipid-mediated dimerization of membrane-anchored c-Src is driven by a cluster of lysine residues in the N-terminal SH4 domain.

The membrane-anchored c-Src tyrosine kinase mediates signaling from a wide range of cell surface receptors controlling cell growth, adhesion, and survival. c-Src deregulation is associated with cancer. Dimerization appears to be a novel layer of regulation through a yet unclear mechanism. Binding of c-Src tyrosine kinase to the plasma membrane is mediated by the myristoylated and strongly positively charged N-terminal SH4 domain. Although activation of c-Src is known to require phosphorylation by a second c-Src molecule, electrostatic repulsion between the charged residues was considered to prevent dimerization. Here we show that a cluster of positively charged lysine residues in c-Src SH4 domain not only does not prevent dimerization but, in fact, enhances it through a lipid-mediated process. Dimerization not only depends on the number of positive charges but also on their position and the nature of the charged residues. Replacement of lysine by arginine increases dimerization in vitro and in vivo and, in HEK293T cells, causes a two-fold increase in tyrosine phosphorylation. Lipid mediated protein-protein interactions induced by clusters of basic residues may represent a general mechanism for modulating cell signaling, consistent with the abundance of positively charged residues in the juxta membrane region of many signaling proteins.

biophysics↗

bioRxiv2022

Mechanical strain stimulates COPII-dependent trafficking via Rac1

Secretory trafficking from the endoplasmic reticulum (ER) is subject to regulation by extrinsic and intrinsic factors. While much of the focus has been on biochemical triggers, little is known whether and how the ER is subject to regulation by mechanical signals. Here, we show that COPII-dependent ER-export is regulated by mechanical strain. Mechanotransduction to the ER was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulate ER-to-Golgi transport. Altogether, we establish an unprecedented link between mechanical strain and export from the ER.

cell biology↗

bioRxiv2021

A mechanosensing mechanism mediated by IRSp53 controls plasma membrane shape homeostasis at the nanoscale

As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by the I-BAR protein IRSp53, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios. TeaserCell stretch cycles generate PM evaginations of {approx}100 nm which are sensed by IRSp53, triggering a local event of actin polymerization that flattens and recovers PM shape.

biophysics↗

bioRxiv2021

The force loading rate drives cell mechanosensing through both reinforcement and fluidization

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton undergoes fluidization and softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon occurs at the organ level. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.

biophysics↗

bioRxiv2020

Dynamic Mechanochemical feedback between curvedmembranes and BAR protein self-organization

In many physiological situations, BAR proteins interact with, and reshape, pre-existing curved membranes, contributing to essential cellular processes. However, the non-equilibrium and timedependent process of reshaping, and its dependence on initial membrane shape, remains largely unknown. Here we explain, both experimentally and through modelling, how a BAR protein dynamically interacts with mechanically bent lipid membranes. We capture protein binding to curved membranes, and characterize a variety of dynamical reshaping events depending on membrane shape and protein arrangement. The events can be generally understood by an isotropic-to-nematic phase transition, in which low curvature templates with isotropic protein orientation progress towards highly curved lipid tubes with nematic protein arrangement. Our findings also apply in cells, where mechanical stretch triggers BAR-protein-membrane interactions that enable potential mechanotransduction mechanisms. Our results characterize and broaden the reshaping processes of BAR proteins on mechanically constrained membranes, demonstrating the interplay between membrane mechanical stimuli and BAR protein response.

biophysics↗