Abstract
Neuronal development requires highly coordinated regulation of the cytoskeleton within the developing axon. This dynamic regulation manifests itself in axonal branching, turning and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily functions to repel axons from inappropriate targets, induces cytoskeletal rearrangements that result in growth cone collapse1. These effects require intra-axonal messenger RNA translation. Here we show that transcripts for RhoA, a small guanosine triphosphatase (GTPase) that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3′ untranslated region (UTR). Sema3A induces intra-axonal translation of RhoA mRNA, and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a new mechanism for the regulation of RhoA signalling.
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Acknowledgements
We thank S. Schlesinger for Sindbis plasmids and N. O'Connor for advice on 3D deconvolution and axonal volume calculation. This work is supported by the National Institute of Mental Health (S.R.J.), the National Alliance for Autism Research (S.R.J.), the Charles A. Dana foundation and the Medical Scientist Training Program (E.Z.M.). A.J. is supported by D. Johnston (Baylor Medical College).
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Supplementary information
Supplementary Figure Legends
This file contains legends for Supplementary Figures S1-S9.
Supplementary Table S1
This table contains oligonucleotide sequences used in in situ hybridization and RT-PCR.
Supplementary Figure S1
Effects of protein synthesis inhibitors on growth cone collapse in severed DRG axons.
Supplementary Figure S2
In situ hybridization against RhoA and other transcripts in DRG axons.
Supplementary Figure S3
Isolation of DRG axons using a modified Boyden chamber.
Supplementary Figure S4
Quantification of in situ hybridization signals in axons and cell bodies.
Supplementary Figure S5
Effect of increased NGF concentration on growth cone collapse.
Supplementary Figure S6
Effects of vehicle and anisomycin treatment on the RhoA reporter.
Supplementary Figure S7
Alignment of the 3′UTRs of RhoA mRNA from different species.
Supplementary Figure S8
Inhibition of growth cone collapse by C. botulinum C3 exoenzyme.
Supplementary Figure S9
Infectivity and expression levels of Sindbis-IRES virions.
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Wu, K., Hengst, U., Cox, L. et al. Local translation of RhoA regulates growth cone collapse. Nature 436, 1020–1024 (2005). https://doi.org/10.1038/nature03885
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DOI: https://doi.org/10.1038/nature03885
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