TCR and CD28 Concomitant Stimulation Elicits a Distinctive Calcium Response in Naive T Cells

T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

International audienceWe introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells

Guidage optique dans les cristaux plasmoniques 1D et 2D

This thesis is devoted to the study of waveguiding properties of surface plasmons polaritons (group velocity, radiative coupling, absorption) on 1D and 2D plasmonic crystals composed of a metallic film periodically drilled with nanoscaled apertures (slits or holes). We have developed a setup for the measurements of optical transmission and reflexion on large spectral (1-16 μm) and angular (0−60°) ranges. It allows to obtain dispersion diagramms with a high resolution (±0.3°, 0.5 cm−1). In 1D plasmonic crystals, we evidence modulations of radiative losses, due to the coupling between surface plasmons propagating along air/metal and substrate/metal interfaces. It induces two propagation regimes : a radiative regime with negligible absorption, and a low-loss regime nearly uncoupled to the free space. Moreover we show that we can continuously tune the propagation regime with a slight modification of the optical index of the substrate (1%). It opens up new perspectives for the external control of radiative/non-radiative coupling of guiding modes. We also present the dispersion properties of surface plasmons on 2D anisotropic plasmonic crystals with different periods along the two symmetry axes. We reveal a band gap far from the borders of the first Brillouin zone. It is induced by the coupling of three surface plasmons propagating in nearly orthogonal directions. One of these coupled modes presents a strong radiative coupling and a weak group velocity.Ce travail de thèse porte sur l'étude des propriétés de guidage des polaritons-plasmons de surface (vitesse de groupe, couplage radiatif, absorption) dans des cristaux plasmoniques 1D et 2D constitués de films métalliques percés d'ouvertures nanométriques périodiques (fentes ou trous). Nous avons développé un banc de mesures optiques (transmission et réflexion) sur un large domaine angulaire (0 − 60°) et spectral (1-16 μm). Il permet d'obtenir les diagrammes de dispersion des modes de surface avec une haute résolution (±0.3°, 0.5 cm−1). Dans les cristaux plasmoniques 1D, nous montrons une modulation des pertes radiatives due au couplage des plasmons de surface se propageant le long des interfaces air/métal et substrat/métal. Il en résulte deux régimes de propagation : un régime radiatif présentant une absorption négligeable, et un régime faible perte très peu couplé à l'espace libre. De plus nous montrons qu'une faible variation de l'indice du substrat (1%) permet de passer d'un régime de propagation à un autre et ouvre la voie à un contrôle externe des couplages radiatifs et non radiatifs des modes guidés.Nous présentons également les propriétés dispersives des plasmons de surface excités sur les interfaces de cristaux plasmoniques 2D anisotropes ayant des périodes différentes selon ses deux axes de symétrie. Nous montrons l'existence d'une bande interdite loin des bords de la zone de Brillouin, et mettant en jeu un couplage entre trois plasmons de surface se propageant dans des directions quasi-orthogonales. Un des modes couplés présente un fort couplage radiatif et une faible vitesse de groupe

Coupled dipole method to compute optical torque : Application to a micropropeller.

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Guidage optique dans les cristaux plasmoniques 1D et 2D

Ce travail de thèse porte sur l'étude des propriétés de guidage des polaritons-plasmons de surface (vitesse de groupe, couplage radiatif, absorption) dans des cristaux plasmoniques 1D et 2D constitués de films métalliques percés d'ouvertures nanométriques périodiques (fentes ou trous). Nous avons développé un banc de mesures optiques (transmission et réflexion) sur un large domaine angulaire (0-60) et spectral (1-16 m). Il permet d'obtenir les diagrammes de dispersion des modes de surface avec une haute résolution (+-0.3, 0.5 cm-1). Dans les cristaux plasmoniques 1D, nous montrons une modulation des pertes radiatives due au couplage des plasmons de surface se propageant le long des interfaces air/métal et substrat/métal. Il en résulte deux régimes de propagation: un régime radiatif présentant une absorption négligeable, et un régime faible perte très peu couplé à l'espace libre. De plus nous montrons qu'une faible variation de l'indice du substrat (1 %) permet de passer d'un régime de propagation à un autre et ouvre la voie à un contrôle externe des couplages radiatifs et non radiatifs des modes guidés. Nous présentons également les propriétés dispersives des plasmons de surface excités sur les interfaces de cristaux plasmoniques 2D anisotropes ayant des périodes différentes selon ses deux axes de symétrie. Nous montrons l'existence d'une bande interdite loin des bords de la zone de Brillouin, et mettant en jeu un couplage entre trois plasmons de surface se propageant dans des directions quasi-orthogonales. Un des modes couplés présente un fort couplage radiatif et une faible vitesse de groupe.This thesis is devoted to the study of waveguiding properties of surface plasmons polaritons (group velocity, radiative coupling, absorption) on 1D and 2D plasmonic crystals composed of a metallic film periodically drilled with nanoscaled apertures (slits or holes). We have developed a setup for the measurements of optical transmission and reflexion on large spectral (1-16 m) and angular (0-60) ranges. It allows to obtain dispersion diagramms with a high resolution (+-0.3, 0.5 cm-1). In 1D plasmonic crystals, we evidence modulations of radiative losses, due to the coupling between surface plasmons propagating along air/me\-tal and substrate/metal interfaces. It induces two propagation regimes: a radiative regime with negligible absorption, and a low-loss regime nearly uncoupled to the free space. Moreover we show that we can continuously tune the propagation regime with a slight modification of the optical index of the substrate (1 %). It opens up new perspectives for the external control of radiative/non-radiative coupling of guiding modes. We also present the dispersion properties of surface plasmons on 2D anisotropic plasmonic crystals with different periods along the two symmetry axes. We reveal a band gap far from the borders of the first Brillouin zone. It is induced by the coupling of three surface plasmons propagating in nearly orthogonal directions. One of these coupled modes presents a strong radiative coupling and a weak group velocity.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

MreB Forms Subdiffraction Nanofilaments during Active Growth in Bacillus subtilis

The construction of the bacterial cell envelope is a fundamental topic, as it confers its integrity to bacteria and is consequently the target of numerous antibiotics. MreB is an essential protein suspected to regulate the cell wall synthetic machineries. Despite two decades of study, its localization remains the subject of controversies, its description ranging from helical filaments spanning the entire cell to small discrete entities. The true structure of these filaments is important because it impacts the model describing how the machineries building the cell wall are associated, how they are coordinated at the scale of the entire cell, and how MreB mediates this regulation. Our results shed light on this debate, revealing the size of native filaments in B. subtilis during growth. They argue against models where MreB filament size directly affects the speed of synthesis of the cell wall and where MreB would coordinate distant machineries along the side wall.The actin-like MreB protein is a key player of the machinery controlling the elongation and maintenance of the cell shape of most rod-shaped bacteria. This protein is known to be highly dynamic, moving along the short axis of cells, presumably reflecting the movement of cell wall synthetic machineries during the enzymatic assembly of the peptidoglycan mesh. The ability of MreB proteins to form polymers is not debated, but their structure, length, and conditions of establishment have remained unclear and the subject of conflicting reports. Here we analyze various strains of Bacillus subtilis, the model for Gram-positive bacteria, and we show that MreB forms subdiffraction-limited, less than 200 nm-long nanofilaments on average during active growth, while micron-long filaments are a consequence of artificial overaccumulation of the protein. Our results also show the absence of impact of the size of the filaments on their speed, orientation, and other dynamic properties conferring a large tolerance to B. subtilis toward the levels and consequently the lengths of MreB polymers. Our data indicate that the density of mobile filaments remains constant in various strains regardless of their MreB levels, suggesting that another factor determines this constant

3D stochastic process simulation for better interpretation of molecular dynamics related to cell wall biogenesis observed with TIRF microscopy

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3D stochastic process simulation for better interpretation of molecular dynamics related to cell wall biogenesis observed with TIRF microscopy

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Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

International audienceOur goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)(1). Single-molecule videomicroscopy, offering millisecond and nanometric resolution(1-11), allows a detailed representation of membrane organization(12-14) by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis(15,16). Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.The nanometric accuracy(17) obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods(17) such as STORM18, PALM(19,20), RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales