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

RUN and FYVE domain-containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17– positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells

SAPHIR: a Shiny application to analyze tissue section images

International audienceStudy of cell populations in tissues using immunofluorescence is a powerful method for both basic and medical research. Image acquisitions performed by confocal microscopy notably allow excellent lateral resolution and more than 10 parameter measurement when using spectral or multiplex imaging. Analysis of such complex images can be very challenging and easily lead to bias and misinterpretation. Here, we have developed the Shiny Analytical Plot of Histological Image Results (SAPHIR), an R shiny application for histo-cytometry using scatterplot representation of data extracted by segmentation. It offers many features, such as filtering of spurious data points, selection of cell subsets on scatterplot, visualization of scatterplot selections back into the image, statistics of selected data and data annotation. Our application allows to quickly characterize labeled cells, from their phenotype to their number and location in the tissue, as well as their interaction with other cells

Stromal cell networks regulate thymocyte migration and dendritic cell behavior in the thymus.

International audienceAfter entry into thymus, T cell progenitors migrate in the cortex and the medulla while completing their education. Recent reports have documented the dynamic and tortuous behavior of thymocytes. However, other than chemokines and/or segregated thymic substrates, the factors contributing to the dynamic patterns of thymocyte movement are poorly characterized. By combining confocal and dynamic two-photon microscopy, we demonstrate that thymocytes continuously migrate on thymic stromal cell networks. In addition to constituting "roads" for thymocytes, we observed that these networks also provide a scaffold on which dendritic cells attach themselves. These results highlight the central role of stromal microanatomy in orchestrating the multiple cellular interactions necessary for T cell migration/development within the thymus

The <i>Salmonella</i> effector SifA initiates a kinesin-1 and kinesin-3 recruitment process mirroring that mediated by Arl8a and Arl8b

International audienceWhen intracellular, pathogenic Salmonella reside in a membrane compartment composed of interconnected vacuoles and tubules, the formation of which depends on the translocation of bacterial effectors into the host cell. Cytoskeletons and their molecular motors are prime targets for these effectors. In this study, we show that the microtubule molecular motor KIF1Bβ (a splice variant of KIF1B), a member of the kinesin-3 family, is a key element for the establishment of the Salmonella replication niche as its absence is detrimental to the stability of bacterial vacuoles and the formation of associated tubules. Kinesin-3 interacts with the Salmonella effector SifA but also with SKIP (also known as PLEKHM2), a host protein complexed to SifA. The interaction with SifA is essential for the recruitment of kinesin-3 on Salmonella vacuoles whereas that with SKIP is incidental. In the non-infectious context, however, the interaction with SKIP is essential for the recruitment and activity of kinesin-3 only on a fraction of the lysosomes. Finally, our results show that, in infected cells, the presence of SifA establishes a kinesin-1 and kinesin-3 recruitment pathway that is analogous to and functions independently of that mediated by the Arl8a and Arl8b GTPases

Peyer's Patch Dendritic Cells Sample Antigens by Extending Dendrites Through M Cell-Specific Transcellular Pores

International audienceBACKGROUND & AIMS: Peyer's patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo. METHODS: We performed 2-photon microscopy on explants of PPs from mice in which the enhanced green fluorescent protein gene was inserted into the lysozyme M locus (lys-EGFP mice), allowing fluorescence detection of LysoDC. RESULTS: LysoDC extended dendrites through M-cell-specific transcellular pores to the gut lumen. The M-cell adhesion molecules junctional adhesion molecule-A and epithelial cell adhesion molecule were recruited to sites of transcellular migration. Transcellular dendrites scanned the M-cell apical surface and the gut luminal content; they were able to take pathogenic bacteria and inert particles in the lumen before retracting back to the SED. CONCLUSIONS: We describe an antigen sampling mechanism that occurs in PPs and involves cooperation between M cells of the follicle-associated epithelium and DCs of the subepithelial dome. This process might be developed to target vaccines to the mu-cosa

Can one detect atrial fibrillation using a wrist-type photoplethysmographic device?

This study aims at evaluating the potential of a wrist-type photoplethysmographic (PPG) device to discriminate between atrial fibrillation (AF) and other types of rhythm. Data from 17 patients undergoing catheter ablation of various arrhythmias were processed. ECGs were used as ground truth and annotated for the following types of rhythm: sinus rhythm (SR), AF, and ventricular arrhythmias (VA). A total of 381/1370/415 10-s epochs were obtained for the three categories, respectively. After pre-processing and removal of segments corresponding to motion artifacts, two different types of feature were derived from the PPG signals: the interbeat interval-based features and the wave-based features, consisting of complexity/organization measures that were computed either from the PPG waveform itself or from its power spectral density. Decision trees were used to assess the discriminative capacity of the proposed features. Three classification schemes were investigated: AF against SR, AF against VA, and AF against (SR&VA). The best results were achieved by combining all features. Accuracies of 98.1/95.9/95.0 %, specificities of 92.4/88.7/92.8 %, and sensitivities of 99.7/98.1/96.2 % were obtained for the three aforementioned classification schemes, respectively

An Adaptive Organization Index to Characterize Atrial Fibrillation using Wrist-Type Photoplethysmographic Signals

The performance of photoplethysmography (PPG)- based wearable monitors to diagnose atrial fibrillation (AF) remains unknown to date. This study aims at assessing the performance of new indices quantifying the level of organization in PPG signals to diagnose AF. A database made of 18 adult patients undergoing catheter ablation of various cardiac arrhythmias was used. PPG signals were recorded using a wrist-type sensor. A 12-lead ECG was used as gold standard. ECGs were annotated by experts and selected segments were divided into 4 categories: sinus rhythm (SR), regularly paced rhythm (RPR), irregularly paced rhythm (IPR) and AF. The level of organization of the various PPG signals was measured using an adaptive organization index (AOI), defined as the ratio of the power of the fundamental frequency and the first harmonic to the total power of the PPG signal, computed with adaptive band-pass filters. A total of 2806/803/852/287 10-second epochs were considered for AF/SR/RPR/IPR classes. The following mean AOI values were measured: 0.45±0.11 for AF, 0.73±0.19 for SR, 0.78±0.20 for RPR and 0.610.19 for IPR classes. Importantly, the AF AOI was significantly smaller than that of the other categories (p<0.001), indicating a higher degree of disorganization

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power. (C) 2018 Elsevier Inc. All rights reserved