Cue-Polarized Transport of β-actin mRNA Depends on 3′UTR and Microtubules in Live Growth Cones

Abstract

Guidance cues trigger fast responses in axonal growth cones such as directional turning and collapse that require local protein synthesis. An attractive cue-gradient, such as Netrin-1, triggers de novo synthesis of β-actin localized to the near-side compartment of the growth cone that promotes F-actin assembly and attractive steering. How this precise spatial asymmetry in mRNA translation arises across the small expanse of the growth cone is poorly understood. Pre-localized mRNAs in the vicinity of activated receptors could be selectively translated and/or new mRNAs could be trafficked into the area. Here we have performed live imaging of fluorescent-tagged β-actin mRNA to investigate mRNA trafficking dynamics in Xenopus retinal ganglion cell (RGC) axons and growth cones in response to Netrin-1. A Netrin-1 gradient was found to elicit the transport of β-actin mRNA granules to the near-side of growth cones within a 4–7 min window. This polarized mRNA trafficking depended on the 3′ untranslated region (UTR) since mRNA-Δ3′UTR mutant failed to exhibit cue-induced localization. Global application of Netrin-1 significantly increased the anterograde movement of β-actin mRNA along axons and also promoted microtubule-dependent mRNA excursions from the central domain of the growth cone into the periphery (filopodia and lamellipodia). Dual channel imaging revealed β-actin mRNA riding behind the microtubule plus-end tracking protein, EB1, in movements along dynamic microtubules into filopodia. The mRNA-EB1 movements were unchanged by a Netrin-1 gradient indicating the dynamic microtubules themselves do not underlie the cue-induced polarity of RNA movement. Finally, fast-moving elongated “worm-like” trains of Cy3-RNA, distinct from mitochondria, were seen transporting RNA along axons in vitro and in vivo suggesting the existence of a novel transport organelle. Overall, the results provide evidence that the axonal trafficking of β-actin mRNA can be regulated by the guidance cue Netrin-1 to transduce the polarity of an extracellular stimulus and that the 3′UTR is essential for this cue-induced regulation.

Axonal precursor mi RNA s hitchhike on endosomes and locally regulate the development of neural circuits

“…Many components of pre‐miRNA dynamics that we report here resemble those of mRNA in axons. We document similar bidirectional trafficking (Leung et al , ; Turner‐Bridger et al , ) and faster anterograde than retrograde transport (Turner‐Bridger et al , ). Maximal velocities and average speed of active puncta are also comparable (Leung et al , ; Turner‐Bridger et al , ).…”

“…We document similar bidirectional trafficking (Leung et al , ; Turner‐Bridger et al , ) and faster anterograde than retrograde transport (Turner‐Bridger et al , ). Maximal velocities and average speed of active puncta are also comparable (Leung et al , ; Turner‐Bridger et al , ). In contrast to our study, the dominant mRNA trajectories previously measured (Turner‐Bridger et al , ) were confined and diffusive, with only a small proportion being directed, while the majority of puncta we detected adopted an overall active and directed trajectory.…”

“…Equal frequencies of anterogradely and retrogradely moving puncta were detected ( Fig 2D). Upon subdivision of the puncta population based on speed [stationary (< 0.2 lm/s), moving (0.2-0.5 lm/s), and fast-moving puncta (> 0.5 lm/s) (Maday et al, 2014;Leung et al, 2018)], we observed that the fast anterogradely moving puncta were significantly faster on average than their retrograde counterpart ( Fig 2E). These observations likely reflect the properties of molecular motors: Anterograde kinesins are highly processive motors, while retrograde dyneins make frequent back and side steps (Maday et al, 2014).…”

“…Recent studies have provided great insight into axonal mRNA trafficking (Alami et al , ; Leung et al , ; Turner‐Bridger et al , ). Many components of pre‐miRNA dynamics that we report here resemble those of mRNA in axons.…”

Precursor miRNAs are trafficked along axons associated with vesicles and locally processed to regulate growth cone steering

“…3D), suggesting the existence of a balanced bidirectional transport. We next analyzed stationary (<0.2 μm/s), moving (0.2-0.5 μm/s) and fast moving puncta (>0.5 μm/s) (Leung et al, 2018;Maday et al, 2014) separately. No difference was observed between anterograde and retrograde average velocity for puncta slower than 0.5 μm/s (0.2 μm/s: antero, 0.066 ± 0.007 μm/s; retro, 0.084 ± 0.008 μm/s; 0.2-0.5 μm/s: antero, 0.33 ± 0.02 μm/s; retro, 0.32 ± 0.01 μm/s).…”