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Endo-lysosomes transport along microtubules and clustering in the perinuclear area are two necessary steps for microbes to activate specialized phagocyte functions. We report that RUN and FYVE domain-containing protein 3 exists as two alternative isoforms distinguishable by the presence of a C-terminal FYVE domain and by their affinity for PtdIns(3)P on endosomal membranes. The FYVE domain-bearing isoform (iRUFY3) is preferentially expressed in immune cells and up-regulated upon activation by microbes and cytokines. iRUFY3 is necessary for ARL8b+/LAMP1+ endo-lysosomes positioning in the pericentriolar organelles cloud of LPS-activated macrophages. We show that iRUFY3 controls macrophages migration, MHC II presentation and responses to Interferon-γ while being required for Intracellular Salmonella replication. Specific inactivation of Rufy3 in phagocytes leads to aggravated pathologies in mouse upon LPS injection or bacterial pneumonia. This study highlights iRUFY3 function, as a novel modulator of immunity, controlling endo-lysosomes dynamics, and ultimately regulating the function of activated phagocytes.

When 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. This article has an associated First Person interview with the first author of the paper.

SummaryLung-resident memory B cells (MBCs) provide localized protection against reinfection in the respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. scRNA-seq analysis and confocal imaging revealed that two main transcriptionally distinct subsets of MBCs colonize the lung peribronchial niche after infection. These subsets arise from different progenitors and are both class-switched, somatically mutated and intrinsically biased in their differentiation fate towards plasma cells. Combined analysis of antigen-specificity and B cell receptor repertoire unveiled a highly permissive selection process that segregates these subsets into “bona fide” virus-specific MBCs and “bystander” MBCs with no apparent specificity for eliciting viruses. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

As a first line of defence against the environment, the epidermis protect animals from infection and physical damage. In C. elegans, wounding the epidermal epithelium triggers both an immune reaction and a repair response. Exactly how these are controlled, and the degree to which they are inter-connected remains unclear. To address these questions, we established a simple system for simultaneously inflicting precise laser wounds and imaging at high spatial and temporal resolution. We show that in C. elegans, wounding provokes a rapid sealing of the plasma membrane, involving reorganisation of phosphatidylinositol 4,5- bisphosphate domains. This is followed by a radial recruitment at the wound site of EBP-2/EB1, a protein that binds the plus ends of microtubules. EB1 recruitment is accompanied by a reorganisation of microtubules, required for the subsequent recruitment of actin and wound closure. It is also required for the directed trafficking towards the site of injury of the key signalling protein SNF-12. In the absence of SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of the innate immune response.

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.

Cancer‐germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer‐germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T‐cell receptor specific for the P1A35–43 peptide associated with H‐2Ld, although able to induce regression of P1A‐expressing P815 mastocytoma cells, were much less effective against P1A‐expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A‐specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H‐2Ld. The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video‐microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+]i) were induced by both types of P1A‐expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB‐Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB‐Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour–CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule‐1, the ligand for LFA‐1, which was not detected on the melanoma cells.