Abstract
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.
Highlights
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
Tight regulation of mRNA-localization patterns ensures axons are enriched with different sets of mRNAs to their cell body [11,12,13,14]. mRNA-localization patterns vary across the axon itself, where differential enrichment occurs between the sensing growth cone tip and the axon shaft according to mRNA species [12, 15]
molecular beacons (MBs) are composed of a short antisense oligonucleotide loop complementary to the mRNA of interest followed by a GC-rich stem, placing a fluorophore and quencher in close proximity
Summary
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. Localizing mRNA to different subcellular locations is an evolutionarily conserved mechanism to control protein expression It facilitates axis patterning of the Drosophila embryo and mating-type switching in budding yeast and promotes the migration of mammalian fibroblasts 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
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