LAUSR

Significance De 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. During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that β-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that β-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate β-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments.