SummaryLocal translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

SignificanceDe novo protein synthesis in neuronal axons plays important roles in neural circuit formation, maintenance, and disease. Key to the selectivity of axonal protein synthesis is whether an mRNA is present at the right place to be translated, but the mechanisms behind axonal mRNA localization remain poorly understood. In this work, we quantitatively analyze the link between axonal β-actin mRNA trafficking and its localization patterns. By developing a single-molecule approach to live-image β-actin mRNAs in axons, we explore the biophysical drivers behind β-actin mRNA motion and uncover a mechanism for generating increased density at the axon tip by differences in motor protein-driven transport speeds. These results provide mechanistic insight into the control of local translation through mRNA trafficking.

Kymographs are graphical representations of spatial position over time, which are often used in biology to visualise the motion of fluorescent particles, molecules, vesicles, or organelles moving along a predictable path. Although in kymographs tracks of individual particles are qualitatively easily distinguished, their automated quantitative analysis is much more challenging. Kymographs often exhibit low signal-to-noise-ratios (SNRs), and available tools that automate their analysis usually require manual supervision. Here we developed KymoButler, a Deep Learning-based software to automatically track dynamic processes in kymographs. We demonstrate that KymoButler performs as well as expert manual data analysis on kymographs with complex particle trajectories from a variety of different biological systems. The software was packaged in a web-based ‘one-click’ application for use by the wider scientific community (http://kymobutler.deepmirror.ai). Our approach significantly speeds up data analysis, avoids unconscious bias, and represents another step towards the widespread adaptation of Machine Learning techniques in biological data analysis.

Specifying one oocyte from many In many animals, only one cell from a cyst of germ cells is selected to become the oocyte. Using fruit flies as a model, Nashchekin et al . identified the microtubule minus end-binding protein Patronin/CAMSAP as a key factor for specifying oocyte fate. Patronin amplifies an initial asymmetry provided by the fusome to form a noncentrosomal microtubule network focused on one cell, along which dynein transports oocyte fate determinants. This mechanism for selecting a single oocyte may be shared in other organisms. —BAP

Extrinsic cues trigger the local translation of specific mRNAs in growing axons via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a mechanism linking signals to local translation but it is not known how broadly this mechanism operates, nor whether it can selectively regulate mRNA translation. We report that receptor-ribosome coupling is employed by multiple guidance cue receptors and this interaction is mRNA-dependent. We find that different receptors associate with distinct sets of mRNAs and RNA-binding proteins. Cue stimulation of growing Xenopus retinal ganglion cell axons induces rapid dissociation of ribosomes from receptors and the selective translation of receptor-specific mRNAs. Further, we show that receptor-ribosome dissociation and cue-induced selective translation are inhibited by co-exposure to translation-repressive cues, suggesting a novel mode of signal integration. Our findings reveal receptor-specific interactomes and suggest a generalizable model for cue-selective control of the local proteome.