Biology subjects

Hivroz, C.

Publications and source records attributed to Hivroz, C..

6 recordsLinked to original sources

bioRxiv2025

The MARK2 kinase acts as a gatekeeper of CD28-dependent co-stimulation in T cells

Naive T cell activation requires not only antigen recognition through the TCR but also a co-stimulatory signal, mainly provided by CD28. Here, using a T cell-specific conditional knockout (cKO) model, we identify the microtubule-affinity kinase 2 (MARK2) as a key intracellular checkpoint that limits CD28-mediated co-stimulation. In vivo, MARK2 deficiency promotes the development of central memory T cells, enhances basal glycolysis activity in naive CD8 T cells, and leads to the development of systemic autoimmunity in aged mice. In MARK2-deficient CD8 T cells, TCR engagement alone drives sustained proliferation, cytokine production, and glycolysis, processes that normally require CD28 co-stimulation. Single-cell transcriptomic analysis reveals that MARK2 regulates the expression of genes involved in CD28 signaling and metabolic switch. We show that MARK2 restrains the PI3K-AKT-mTORC1 pathway by limiting CD28-driven transcriptional and metabolic programs. Mechanistically, we demonstrate that MARK2 phosphorylates CREB regulated transcription coactivator 2 (CRTC2) and suppresses CREB-mediated transcription and mTOR activation, whereas CD28 engagement lifts this inhibition. Together, our results redefine the role of CD28 that not only amplifies TCR signaling but also relieves a MARK2-dependent inhibitory signal. This work provides new insights into T cell activation, metabolism and immune tolerance with potential implications for immunotherapeutic strategies in cancer and autoimmunity.

immunology↗

bioRxiv2023

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes

T cell activation induces a metabolic switch generating energy required for proliferation, survival, and fueling their functions. Thus, it is essential to monitor metabolism associated to subcellular functional and structural changes. We used non-invasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NADH co-enzyme during T lymphocyte activation. 2P-FLIM measurements of the protein-bound and free NADH ratios provides a readout of the redox state (NAD+/ NADH) of the cells, and thus of their OXPHOS and glycolysis rates. Using this method, we followed the dynamics of fraction of bound NADH (fb NADH) in live single cells. Comparing fb NADH between resting and activated T cells, we show that T cell activation induces a rapid switch toward glycolysis. The switch takes only 10 minutes and remains stable for at least one hour. Three-dimensional (3D) analysis revealed that the intracellular distribution of fb NADH is symmetrically distributed in resting cells, whereas increases at the contact zone in activated cells. Finally, we show that fb NADH negatively correlates with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study shows that 2P-FLIM measurement of fb NADH is well suited to follow a fast metabolic switch in 3D, in single T lymphocytes with subcellular resolution.

immunology↗

bioRxiv2020

Retrograde And Anterograde Transport Of LAT-Vesicles During The Immunological Synapse Formation: Defining The Finely-Tuned Mechanism

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T-cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of 4 proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute in both pathways, are in our cellular context specifically and respectively involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

cell biology↗

bioRxiv2019

Single-cell immuno-mechanics: rapid viscoelastic changes are a hallmark of early leukocyte activation.

To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. Using an innovative micropipette rheometer, we show in three different cell types that when stimulated by microbeads mimicking target cells, leukocytes become up to ten times stiffer and more viscous. These mechanical changes initiate within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. The current results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new way to investigate cell mechanical processes. Our findings also suggest that dynamic immuno-mechanical measurements provide an identifier of leukocyte type and an indicator of the cells state of activation.

biophysics↗

bioRxiv2019

Dynamic Palmitoylation Events Following T-Cell Receptor Signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi.

biochemistry↗

bioRxiv2019

Mammalian Amoeboid Swimming is propelled by molecular and not protrusion-based paddling in Lymphocytes

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like fibroblasts crawling, relies on maturation of adhesion complexes and actin fiber traction, while the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on 2D and in 3D solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimentally and computationally that leukocytes do swim, and that propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell envelope. We model the propulsion as a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. This mechanism explains that swimming is five times slower than the cortex retrograde flow. Resultantly the ubiquitous ability of mammalian amoeboid cells to migrate in various environments can be explained for lymphocytes by a single machinery of envelope treadmilling.

biophysics↗