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Abstract :
[en] 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.
