Skip to main content
SpringerLink
Log in

Profiling Locally Translated mRNAs in Regenerating Axons

  • Protocol
  • First Online:
Axon Regeneration

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2636))

Abstract

Spatial and temporal regulation of protein expression plays important roles in many cellular functions, particularly for highly polarized cell types. While the subcellular proteome can be altered by relocalizing proteins from other domains of the cell, transporting mRNAs to subcellular domains provides a means to locally synthesize new proteins in response to different stimuli. Localized protein synthesis is a critical mechanism in neurons that extend dendrites and axons long distances from their cell bodies. Here, we discuss methodologies that have been developed to study localized protein synthesis using axonal protein synthesis as an example. We provide an in-depth method using dual fluorescence recovery after photobleaching to visualize sites of protein synthesis using reporter cDNAs that encode two different localizing mRNAs along with diffusion-limited fluorescent reporter proteins. We show how this method can be used to determine how extracellular stimuli and different physiological states can alter the specificity of local mRNA translation in real time.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Smith TP, Sahoo PK, Kar AN, Twiss JL (2020) Intra-axonal mechanisms driving axon regeneration. Brain Res 1740:146864. https://doi.org/10.1016/j.brainres.2020.146864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sahoo PK, Smith DS, Perrone-Bizzozero N, Twiss JL (2018) Axonal mRNA transport and translation at a glance. J Cell Sci 131(8):jcs196808. https://doi.org/10.1242/jcs.196808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dalla Costa I, Buchanan CN, Zdradzinski MD, Sahoo PK, Smith TP, Thames E, Kar AN, Twiss JL (2021) The functional organization of axonal mRNA transport and translation. Nat Rev Neurosci 22(2):77–91. https://doi.org/10.1038/s41583-020-00407-7

    Article  CAS  PubMed  Google Scholar 

  4. Namjoshi SV, Raab-Graham KF (2017) Screening the molecular framework underlying local dendritic mRNA translation. Front Mol Neurosci 10:45. https://doi.org/10.3389/fnmol.2017.00045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Aakalu G, Smith WB, Nguyen N, Jiang C, Schuman EM (2001) Dynamic visualization of local protein synthesis in hippocampal neurons. Neuron 30(2):489–502. https://doi.org/10.1016/S0896-6273(01)00295-1

    Article  CAS  PubMed  Google Scholar 

  6. Holt CE, Martin KC, Schuman EM (2019) Local translation in neurons: visualization and function. Nat Struct Mol Biol 26(7):557–566. https://doi.org/10.1038/s41594-019-0263-5

    Article  CAS  PubMed  Google Scholar 

  7. Papandreou ME, Palikaras K, Tavernarakis N (2020) Assessment of de novo protein synthesis rates in Caenorhabditis elegans. J Vis Exp 163. https://doi.org/10.3791/61170

  8. Terenzio M, Koley S, Samra N, Rishal I, Zhao Q, Sahoo PK, Urisman A, Marvaldi L, Oses-Prieto JA, Forester C, Gomes C, Kalinski AL, Di Pizio A, Doron-Mandel E, Perry RB, Koppel I, Twiss JL, Burlingame AL, Fainzilber M (2018) Locally translated mTOR controls axonal local translation in nerve injury. Science 359(6382):1416–1421. https://doi.org/10.1126/science.aan1053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Perry RB, Doron-Mandel E, Iavnilovitch E, Rishal I, Dagan SY, Tsoory M, Coppola G, McDonald MK, Gomes C, Geschwind DH, Twiss JL, Yaron A, Fainzilber M (2012) Subcellular knockout of importin beta1 perturbs axonal retrograde signaling. Neuron 75(2):294–305. https://doi.org/10.1016/j.neuron.2012.05.033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Perry RB, Rishal I, Doron-Mandel E, Kalinski AL, Medzihradszky KF, Terenzio M, Alber S, Koley S, Lin A, Rozenbaum M, Yudin D, Sahoo PK, Gomes C, Shinder V, Geraisy W, Huebner EA, Woolf CJ, Yaron A, Burlingame AL, Twiss JL, Fainzilber M (2016) Nucleolin-mediated RNA localization regulates neuron growth and cycling cell size. Cell Rep 16(6):1664–1676. https://doi.org/10.1016/j.celrep.2016.07.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sahoo PK, Kar AN, Samra N, Terenzio M, Patel P, Lee SJ, Miller S, Thames E, Jones B, Kawaguchi R, Coppola G, Fainzilber M, Twiss JL (2020) A Ca2+-dependent switch activates axonal casein kinase 2alpha translation and drives G3BP1 granule disassembly for axon regeneration. Curr Biol 30(24):4882–4895 e4886. https://doi.org/10.1016/j.cub.2020.09.043

    Article  CAS  Google Scholar 

  12. Sahoo PK, Lee SJ, Jaiswal PB, Alber S, Kar AN, Miller-Randolph S, Thames E, Taylor EE, Smith T, Singh B, Ho TS-Y, Urisman A, Chand S, Pena EA, Burlingame AL, Woolf CJ, Fainzilber M, English AW, Twiss JL (2018) Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration. Nat Commun 9:3358. https://doi.org/10.1038/s41467-018-05647-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yudin D, Hanz S, Yoo S, Iavnilovitch E, Willis D, Gradus T, Vuppalanchi D, Segal-Ruder Y, Ben-Yaakov K, Hieda M, Yoneda Y, Twiss JL, Fainzilber M (2008) Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve. Neuron 59(2):241–252. https://doi.org/10.1016/j.neuron.2008.05.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zheng JQ, Kelly TK, Chang B, Ryazantsev S, Rajasekaran AK, Martin KC, Twiss JL (2001) A functional role for intra-axonal protein synthesis during axonal regeneration from adult sensory neurons. J Neurosci 21(23):9291–9303. https://doi.org/10.1523/jneurosci.21-23-09291.2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16(9):1055–1069. https://doi.org/10.1016/S0006-3495(76)85755-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gomes C, Merianda TT, Lee SJ, Yoo S, Twiss JL (2014) Molecular determinants of the axonal mRNA transcriptome. Dev Neurobiol 74(3):218–232. https://doi.org/10.1002/dneu.22123

    Article  CAS  PubMed  Google Scholar 

  17. Willis DE, Twiss JL (2011) Profiling axonal mRNA transport. Methods Mol Biol 714:335–352. https://doi.org/10.1007/978-1-61779-005-8_21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Koppel I, Fainzilber M (2018) Omics approaches for subcellular translation studies. Mol Omics 14(6):380–388. https://doi.org/10.1039/c8mo00172c

    Article  CAS  PubMed  Google Scholar 

  19. Dorrbaum AR, Alvarez-Castelao B, Nassim-Assir B, Langer JD, Schuman EM (2020) Proteome dynamics during homeostatic scaling in cultured neurons. elife 9. https://doi.org/10.7554/eLife.52939

  20. Kar AN, Macgibeny MA, Gervasi NM, Gioio AE, Kaplan BB (2013) Intra-axonal synthesis of eukaryotic translation initiation factors regulates local protein synthesis and axon growth in rat sympathetic neurons. J Neurosci 33(17):7165–7174. https://doi.org/10.1523/JNEUROSCI.2040-12.2013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Enam SU, Zinshteyn B, Goldman DH, Cassani M, Livingston NM, Seydoux G, Green R (2020) Puromycin reactivity does not accurately localize translation at the subcellular level. elife 9. https://doi.org/10.7554/eLife.60303

  22. Weiler IJ, Spangler CC, Klintsova AY, Grossman AW, Kim SH, Bertaina-Anglade V, Khaliq H, de Vries FE, Lambers FA, Hatia F, Base CK, Greenough WT (2004) Fragile X mental retardation protein is necessary for neurotransmitter-activated protein translation at synapses. Proc Natl Acad Sci U S A 101(50):17504–17509. https://doi.org/10.1073/pnas.0407533101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sanz E, Yang L, Su T, Morris DR, McKnight GS, Amieux PS (2009) Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci U S A 106(33):13939–13944. https://doi.org/10.1073/pnas.0907143106

    Article  PubMed  PubMed Central  Google Scholar 

  24. Rozenbaum M, Rajman M, Rishal I, Koppel I, Koley S, Medzihradszky KF, Oses-Prieto JA, Kawaguchi R, Amieux PS, Burlingame AL, Coppola G, Fainzilber M (2018) Translatome regulation in neuronal injury and axon regrowth. eNeuro 5(2). https://doi.org/10.1523/ENEURO.0276-17.2018

  25. Doyle JP, Dougherty JD, Heiman M, Schmidt EF, Stevens TR, Ma G, Bupp S, Shrestha P, Shah RD, Doughty ML, Gong S, Greengard P, Heintz N (2008) Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135(4):749–762. https://doi.org/10.1016/j.cell.2008.10.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ostroff LE, Santini E, Sears R, Deane Z, Kanadia RN, LeDoux JE, Lhakhang T, Tsirigos A, Heguy A, Klann E (2019) Axon TRAP reveals learning-associated alterations in cortical axonal mRNAs in the lateral amgydala. elife 8. https://doi.org/10.7554/eLife.51607

  27. Fusco CM, Desch K, Dörrbaum AR, Wang M, Staab A, Chan ICW, Vail E, Villeri V, Langer JD, Schuman EM (2021) Neuronal ribosomes dynamically exchange ribosomal proteins in a context-dependent manner. bioRxiv:2021.2003.2025.437026. https://doi.org/10.1101/2021.03.25.437026

Download references

Acknowledgments

This work was supported by grants from the National Institutes of Health (R01NS089633 and R01NS117821 to JLT), National Science Foundation (MCB-1020970 to JLT), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to JLT), and South Carolina Spinal Cord Injury Research Fund (2019-PD-02 to PKS). JLT is the incumbent SmartState Chair in Childhood Neurotherapeutics at the University of South Carolina.

Author information

Authors and Affiliations

  1. Department of Biological Sciences, University of South Carolina, Columbia, SC, USA

    Pabitra K. Sahoo & Jeffery L. Twiss

Authors
  1. Pabitra K. Sahoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeffery L. Twiss
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pabitra K. Sahoo .

Editor information

Editors and Affiliations

  1. Department of Biology, Appalachian State University, Boone, NC, USA

    Ava J. Udvadia

  2. Office of Technology Development, Medical College of Wisconsin, Milwaukee, WI, USA

    James B. Antczak

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sahoo, P.K., Twiss, J.L. (2023). Profiling Locally Translated mRNAs in Regenerating Axons. In: Udvadia, A.J., Antczak, J.B. (eds) Axon Regeneration. Methods in Molecular Biology, vol 2636. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3012-9_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3012-9_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3011-2

  • Online ISBN: 978-1-0716-3012-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation