Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

peer reviewedMacrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization

Shaping the cerebral cortex by cellular crosstalk.

peer reviewedThe cerebral cortex is the brain's outermost layer. It is responsible for processing motor and sensory information that support high-level cognitive abilities and shape personality. Its development and functional organization strongly rely on cell communication that is established via an intricate system of diffusible signals and physical contacts during development. Interfering with this cellular crosstalk can cause neurodevelopmental disorders. Here, we review how crosstalk between migrating cells and their environment influences cerebral cortex development, ranging from neurogenesis to synaptogenesis and assembly of cortical circuits

Real-time Recordings of Migrating Cortical Neurons from GFP and Cre Recombinase Expressing Mice.

The cerebral cortex is one of the most intricate regions of the brain that requires elaborate cell migration patterns for its development. Experimental observations show that projection neurons migrate radially within the cortical wall, whereas interneurons migrate along multiple tangential paths to reach the developing cortex. Tight regulation of the cell migration processes ensures proper positioning and functional integration of neurons to specific cerebral cortical circuits. Disruption of neuronal migration often leads to cortical dysfunction and/or malformation associated with neurological disorders. Unveiling the molecular control of neuron migration is thus fundamental to understanding the physiological or pathological development of the cerebral cortex. In this unit, protocols allowing detailed analysis of patterns of migration of both interneurons and projection neurons under different experimental conditions (i.e., loss or gain of function) are presented

Molecular mechanisms of long-distance transport: a new role for local translation?

Axonal microtubules (MT) regulate long-distance transport of cargos to peripheral sub-compartments. Among those cargoes, mRNAs and their associated RNA-binding proteins are transported as membraneless ribonucleoprotein (RNP) granules that, together with ribosomes, can hitchhike on fast-moving membrane-bound organelles for their transport along MTs and subsequent translation. Deep-sequencing studies have characterized the axonal translatome of several cellular subtypes, unravelling developmental translation programs that support compartment-specific biological processes. Importantly, components of the translation machinery including ribosomes, tRNAs and the translation elongation factor 1a (eEF1a) have also been identified at the surface of distinct organelle subtypes suggesting that some organelles may serve as platforms for mRNA translation. Accordingly, axonal late endosomes provide sites for local protein synthesis contributing to the maintenance of the local mitochondria proteome. However, while active protein translation on fast-moving organelles has only been described in the fungi Ustilago maydis, the role for a functional coordination between organelle transport and local translation remains poorly understood in neuronal cells. In order to characterize the population of transcripts actively translated on moving organelles, we use a transgenic mouse model that co-expresses fluorescently-labelled motile organelles and the RiboTag system. By combining sequential centrifugation and fluorescent flow-cytometry, we isolate motile organelles whose associated ribosome-bound mRNAs will be further characterized via translatome studies. We further established an in vitro model of neuron-like cells that undergo axonal transport dynamics and local translation events, and that expresses the SunTag system to probe the translation dynamics of selected candidate mRNAs. This work will therefore combine translatome analysis of sorted motile vesicles with imaging of translation dynamics to elucidate the functional contribution of motile organelles to the axonal proteome, thus revealing new insights into the regulation of local translation in neurons

Molecular mechanisms of long-distance transport: a new role for local translation?

Axonal microtubules (MT) regulate long-distance transport of cargos to peripheral sub-compartments. Among those cargoes, mRNAs and their associated RNA-binding proteins are transported as membraneless ribonucleoprotein (RNP) granules that, together with ribosomes, can hitchhike on fast-moving membrane-bound organelles for their transport along MTs and subsequent translation. Importantly, components of the translation machinery including ribosomes, tRNAs and the translation elongation factor 1a (eEF1a) have also been identified at the surface of distinct organelle subtypes suggesting that some organelles may serve as platforms for mRNA translation. Accordingly, axonal late endosomes provide sites for local protein synthesis contributing to the maintenance of the local mitochondria proteome. However, while active protein translation on fast-moving organelles has only been described in the fungi Ustilago maydis, the role for a functional coordination between organelle transport and local translation remains poorly understood in neuronal cells. In order to characterize the population of transcripts actively translated on moving organelles, we use a transgenic mouse model that co-expresses fluorescently-labelled motile organelles and the RiboTag system. By combining sequential centrifugation and fluorescent flow-cytometry, we isolate motile organelles whose associated ribosome-bound mRNAs will be further characterized via translatome studies. We further established an in vitro model of neuron-like cells that undergo axonal transport dynamics and local translation events, and that expresses the SunTag system to probe the translation dynamics of selected candidate mRNAs. This work will therefore combine translatome analysis of sorted motile vesicles with imaging of translation dynamics to elucidate the functional contribution of motile organelles to the axonal proteome, thus revealing new insights into the regulation of local translation in neurons

NF-κB Signaling in Ex-Vivo Mouse Intestinal Organoids.

peer reviewedWe describe here a protocol to assess NF-κB activation in ex-vivo organoids generated from mouse intestinal crypts. These structures are maintained in culture as crypt-villus forming organoids. These ex-vivo organoids maintain both self-renewal and multilineage differentiation overtime. We also describe the generation of ex-vivo organoids from Apc-mutated mouse intestinal crypts. Both wild-type and Apc-mutated organoids respond very well to NF-κB-activating signals such as TNFα but not to LPS. The kinetic of NF-κB activation in response to these signals in ex-vivo intestinal organoids is very similar to what we see in 2D cell lines. This protocol provides investigators a powerful tool to assess NF-κB activation in both healthy and transformed intestinal epitheliums maintained in culture as 3D structures

From Downscaling to Single-Cell Proteomic: Understanding and minimizing the downscaling effect

peer reviewedINTRODUCTION: Cellular systems consist of a variety of cells with distinct molecular and functional properties based on their location and timing. The characterization of proteome heterogeneity in these systems is a key to enhancing medical research and precision medicine, which requires quantification of proteins at the single-cell level. Liquid chromatography mass spectrometry (LC-MS) is a well-suited technique for proteomics analysis. However, the drop in performance is an inherent effect of decreasing the starting sample material amount. This downscaling effect is strongly related to the sensibility of MS instrument and the peptide loss during sample preparation. A comprehensive study of all contributions to the sample downscaling effect is essential to properly optimize single-cell proteomic methods and thus for minimizing the performance drop. METHODS: Here we present results from downscaled proteomic analysis together with a software-assisted strategy to evaluate and furthering the understanding of the sample downscaling effect on the performance drop for bottom-up proteomic analysis. In this approach, a sample condition (e.g., sample preparation protocol, LC-MS method, MS instruments) is evaluated by monitoring performance when the injected quantity of peptides is reduced. Each peptide signal intensity is monitored in function of the injected quantity in LC-MS. The signal drop for each peptides can then be study independently in function of their intrinsic properties (e.g., mass, charge, hydrophobicity factor, acidic/basis residue ratio) to highlight the causes of peptide loss. PRELIMINARY DATA: This approach was first used to evaluate and compare the performances of LC-MS methods (e.g. sensitivity, feature detection) in a high throughput context on QExactive (Thermo) and timsTOF (Bruker) instruments. This study led to a comprehensive comparison of the performances of these two MS for proteomics of low amount to single-cell level samples. The same workflow was then applied to evaluate specific peptide interactions (binding) with the vial surface. Peptides from a HeLa tryptic digest standard were chosen as peptide mix model for this study to avoid the contribution of sample preparation on the analysis performance. This study was first conducted with Total Recovery glass vials from Waters. The loss of peptide signal was assessed by a downscaling experiment on a set of vials containing peptides at different concentration levels (from 180ng/µL to 10ng/µL). As expected, the total peptide signal decreases as the total peptide concentration is reduced. However, the drop in peptide signal was not homogenous in regards to peptide hydrophobicity factors. This observation has been related to preferential and significant peptide binding on the vial surface. These interactions becoming non-negligible when peptide concentration is downscaled. Based on these results, vials molded in different polymeric materials (e.g., glass-filled polymers, Cyclic olefin polymers, polypropylene, poly(methyl methacrylate, polyether ether ketone) were tested with our downscaling approach. The results helped determine the best candidate polymeric material for vials or other laboratory consumables, minimizing peptide-surface interactions, for single cell proteomic analysis or low starting material experiments LC-MS analyses. As preliminary results, poly(methyl methacrylate), PMMA, vials showed promising behaviors for downscaled proteomics increasing the number of hydrophobic peptides detected by LC-MS compared to glass vial. This improvement leads to a 15% increase in identified proteins. NOVEL ASPECT: Influence of microtube material for single-cell sample preparation and related bioinformatic tools for performance check and protocol optimization.ChipOmics (Win2WAL