Biology subjects

Theodoly, O.

Publications and source records attributed to Theodoly, O..

9 recordsLinked to original sources

bioRxiv2025

Bivalency of natalizumab promotes inhibition of dynamic VLA-4 adhesion beyond affinity gain

Natalizumab, a monoclonal IgG4 antibody used in the treatment of multiple sclerosis (MS), inhibits VLA-4 binding to VCAM-1, thereby reducing leukocyte recruitment to inflamed tissues. The intricate mechanisms underlying these effects remain unclear, particularly concerning the heavy-chain shuffling of IgG4. We conducted an in vitro study to quantify the impact of bivalent IgG and monovalent Fab forms of natalizumab on the capture and migration of human primary memory T lymphocytes under shear stress, using VCAM-1 and SDF-1-coated surfaces. IgG natalizumab at concentrations near its cell surface EC50 showed significant of capture and resistance of adherent cells to shear stress, whereas significantly higher doses of Fab natalizumab, up to 100-fold greater than its cell surface EC50, were needed to achieve similar effects. These findings highlight that receptor occupancy alone may not adequately predict the functional outcomes of inhibitor antibodies. For optimal therapeutic effect, inhibition of cell-surface adhesion may require specific kinetic and geometric binding properties of antibodies.

immunology↗

bioRxiv2024

Microvalve-based devices for flow-free gradient generators

Experiments with gradients of soluble bioactive species have significantly advanced with microfluidic developments that enable cell observation and stringent control of environmental conditions. While some methodologies rely on flow to establish gradients, other opt for flow-free conditions, which is particularly beneficial for studying non-adherent and/or shear-sensitive cells. In flow-free devices, bioactive species diffuse either through resistive microchannels in "microchannel-based" devices, a porous membrane in "membrane-based" devices, or a hydrogel in "gel-based" devices. However, despite significant advancements over traditional methods such as "Boyden chambers", these technologies have not widely disseminated in biological laboratories, arguably due to entrenched practices and the intricate skills required for conducting microfluidic assays. Here, we integrated Quake-type pneumatic microvalves in place of microgrooves, membranes, or gels, and developed devices with precise control over residual flow, establishment initial gradient, and long-term stability of gradients. The "Microvalve-based" approach enables the generation of the automatization of delicate microfluidic manipulations, which paves the way for routine applications of controlled and tunable flow-free gradients in academic laboratories and biomedical units.

biophysics↗

bioRxiv2023

Integrin-like adhesin CglD confers traction and stabilizes bacterial focal adhesions involved in myxobacterial gliding motility

AO_SCPLOWBSTRACTC_SCPLOWIntegrins are crucial for eukaryotic cell attachment and motility within the extracellular matrix (ECM) via focal-adhesion formation, with their evolutionary emergence important for the development of multicellularity. Intriguingly, single gliding cells of the predatory deltaproteobacterium Myxococcus xanthus form bacterial focal-adhesion (bFA) sites; therein, helically-trafficked motors become immobilized at anchored locations through Glt apparatus association with cell-surface integrin I-domain-like adhesin CglB. Using traction-force, bead-force, and total internal reflection-fluorescence microscopies combined with biochemical approaches, we herein identify the von Willebrand A domain-containing cell-surface lipoprotein CglD to be a {beta}-integrin-like outer-membrane lipoprotein that functionally associates with and anchors the trans-envelope Glt-CglB gliding apparatus, stabilizing and efficiently anchoring this assembly at bFAs. Calcium dependence governs CglD importance, consistent with its integrated ECM eukaryotic cartilage oligomeric matrix protein domains. CglD thus confers mechanosensory and mechanotransductory capabilities to the gliding apparatus, helping explain bFA-mediated trans-envelope force transduction, from inner-membrane-embedded motors to the cell surface.

microbiology↗

bioRxiv2023

Naive T lymphocytes chemotax to CCL21 but not to S1P-rich serum

Naive T lymphocytes traffic through the organism in their search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on the chemokine CCL21, while exit into efferent lymphatics relies on the sphingolipid S1P. Surprisingly, while both molecules are claimed chemotactic, a quantitative analysis of naive T lymphocyte migration along defined gradients is missing. Here, we used a reductionist in vitro approach to study the real-time, single-cell response of naive T lymphocytes to CCL21 and S1P-rich serum. Using high-throughput microfluidic and optical micropatterning ad hoc tools, we show that CCL21 triggers long-range chemotaxis whereas S1P-rich serum does not. Instead, S1P-rich serum triggers a transient polarization that may represent a brief transmigration step through exit portals. Our data thus validate naive T lymphocyte chemotaxis towards CCL21 but not S1P, which complements in vivo observations and is of interest for a better tailoring of immunosuppressive drugs.

biophysics↗

bioRxiv2020

Controlling T cells shape, mechanics and activation by micropatterning.

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping. This allowed us to control at the same time the position, shape, mechanics and activation state of T cells. Once adhered on shaped patterns we examined the capacities of T cells to be activated with soluble aCD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 (aCD45) antibody decorated surface is not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. We further demonstrate this by expressing MEGF10 as a non immune adhesion receptor in T cells to obtain the very same spreading area on PLL substrates and Young modulus than immobilized cells on aCD45, while retaining similar activation capabilities using soluble aCD3 or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenised populations in standardized T cell activation assays.

biophysics↗

bioRxiv2019

Chitosan films for microfluidic studies of single bacteria and perspectives for antibiotics susceptibility testing

Single cell microfluidics is powerful to study bacteria and determine their susceptibility to antibiotics treatment. Glass treatment by adhesive molecules is a potential solution to immobilize bacterial cells and perform microscopy but traditional cationic polymers such as poly-lysine deeply affect bacterial physiology. In this work, we chemically characterized a class of chitosan polymers for their biocompatibility when adsorbed to glass. Chitosan chains of known length and composition allowed growth of Escherichia coli cells without any deleterious effects on cell physiology. Combined with a machine-learning approach, this method could measure the antibiotics susceptibility of a diversity of clinical strains in less than 1 hour and with higher accuracy than current methods. Last, chitosan polymers also supported growth of Klebsiella pneumoniae, another bacterial pathogen of clinical significance. The low cost of chitosan slides and their simple implementation makes them highly versatile for research as well as clinical use.

microbiology↗

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↗

bioRxiv2019

Different integrins mediate haptotaxis of T lymphocytes towards either lower or higher adhesion zones

Guidance of cells by molecules anchored on a substrate, known as haptotaxis, is arguably crucial in development, immunology and cancer, however the exact cues and mechanisms driving cell orientation in vivo are hardly identified. Adhesive haptotaxis has been described in the case of mesenchymatous cells that develop strong pulling forces with their substrates and orient via a tug of war mechanism - a competition between cells pulling edges. In the case of amoeboid cells that migrate with minimal interaction with their substrate, existence of adhesive haptotaxis remains unclear. Here, we studied the crawling of human T lymphocytes on substrates with spatially modulated adhesivity, and observed haptotaxis with surface concentrations of integrin ligands found on high endothelial veinules. Overexpression of ICAM-1 and VCAM-1 molecules observed in vivo at transmigration portals can therefore promote leukocyte recruitment. Mechanistically, we show that integrin-mediated haptotaxis of lymphocytes differ both from active chemotaxis, because no mechanotransduction was detected, and from the passive tug of war mechanism of mesenchymatous cells, because different integrins support opposite phenotypes. Cells favored more adherent zones with VLA-4 and, counterintuitively, less adherent zones with LFA-1. These results reveal that integrins control differential adhesive haptotaxis behaviors without mechanotransduction, and this smart capability may support unsuspected ways for cells path selection.

biophysics↗

bioRxiv2018

A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes

The recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with mechanisms only partially deciphered. We studied here the guidance by flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins LFA-1 (L{beta}2) and VLA-4 (4{beta}1), and showed that cells segregated in two populations of opposite orientation for combined adhesion. Sharp decisions of orientation were shown to rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-dominant downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrins affinity, combined with an inhibiting crosstalk of LFA-1 toward VLA-4. At the cellular level, directivity with or against the flow is mechanically mediated by the passive orientation of detached uropod or lamellipod by flow. This complete chain of logical events provides a unique mechanistic picture of a guiding mechanism, from stimuli to cell orientation.\n\nSignificanceCellular guidance is crucial to many biological functions, but the precise mechanisms remain unclear. We have analyzed here an original phenotype of flow-guided cells mimicking leukocytes crawling into the blood vessels and showed that the controlling parameter of cells decision to migrate upstream or downstream was the relative number of two specific adhesion molecules, the integrins LFA-1 and VLA-4. The spatial polarisation of integrins affinity and an intermutually feedback of their activation create a bistable system where cells adhere either by their tip or their tail and orient respectively downstream or upstream. This mechanism therefore proposes a complete chain of event from stimuli to cell orientation and differs strongly from the chemotaxis paradigm because stimuli trigger no signaling.

biophysics↗