Skip to main content
SpringerLink
Log in

A Protocol for Single-Molecule Translation Imaging in Xenopus Retinal Ganglion Cells

  • Protocol
  • First Online:
Single Molecule Microscopy in Neurobiology

Part of the book series: Neuromethods ((NM,volume 154))

  • 642 Accesses

Abstract

Single-molecule translation imaging (SMTI) is a straightforward technique for the direct quantification of local protein synthesis. The protein of interest is fused to a fast-folding and fast-bleaching fluorescent protein, allowing one to monitor the appearance of individual fluorescence events after photobleaching of pre-existing proteins in the cell under investigation. The translation of individual molecules is then indicated by photon bursts of sub-second length that appear over a dark background. The method thus shares attributes with fluorescence recovery after photobleaching (FRAP) microscopy. Resulting datasets are similar to those generated by localization-based super-resolution microscopy techniques and can be used both to generate density maps of local protein production and to quantify the kinetics of local synthesis. The detailed protocol described in this chapter uses a Venus-β-actin fusion construct to visualize and measure the β-actin mRNA translational activity in Xenopus retinal ganglion cell growth cones upon Netrin-1 stimulation, which can be readily adapted for detecting translation events of other mRNAs in various cell types.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Ströhl F, Lin JQ, Laine RF et al (2017) Single molecule translation imaging visualizes the dynamics of local β-actin synthesis in retinal axons. Sci Rep 7:709. https://doi.org/10.1038/s41598-017-00695-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ifrim MF, Williams KR, Bassell GJ (2015) Single-molecule imaging of PSD-95 mRNA translation in dendrites and its dysregulation in a mouse model of fragile X syndrome. J Neurosci 35:7116–7130. https://doi.org/10.1523/JNEUROSCI.2802-14.2015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dickson BJ (2002) Molecular mechanisms of axon guidance. Science 298:1959–1964. https://doi.org/10.1126/science.1072165

    Article  CAS  PubMed  Google Scholar 

  4. O’Donnell M, Chance RK, Bashaw GJ (2009) Axon growth and guidance: receptor regulation and signal transduction. Annu Rev Neurosci 32:383–412. https://doi.org/10.1146/annurev.neuro.051508.135614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kolodkin AL, Tessier-Lavigne M (2011) Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb Perspect Biol 3:1–14. https://doi.org/10.1101/cshperspect.a001727

    Article  CAS  Google Scholar 

  6. Harris WA, Holt CE, Bonhoeffer F (1987) Retinal axons with and without their somata, growing to and arborizing in the tectum of Xenopus embryos: a time-lapse video study of single fibres in vivo. Development 101:123–133

    CAS  PubMed  Google Scholar 

  7. Campbell DS, Holt CE (2001) Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 32:1013–1026. https://doi.org/10.1016/S0896-6273(01)00551-7

    Article  CAS  PubMed  Google Scholar 

  8. Leung K-M, van Horck FPG, Lin AC et al (2006) Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1. Nat Neurosci 9:1247–1256. https://doi.org/10.1038/nn1775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Piper M, Anderson R, Dwivedy A et al (2006) Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones. Neuron 49:215–228. https://doi.org/10.1016/j.neuron.2005.12.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Welshhans K, Bassell GJ (2011) Netrin-1-induced local beta-actin synthesis and growth cone guidance requires zipcode binding protein 1. J Neurosci 31:9800–9813. https://doi.org/10.1523/JNEUROSCI.0166-11.2011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wu KY, Hengst U, Cox LJ et al (2005) Local translation of RhoA regulates growth cone collapse. Nature 436:1020–1024. https://doi.org/10.1038/nature03885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yao J, Sasaki Y, Wen Z et al (2006) An essential role for beta-actin mRNA localization and translation in Ca2+-dependent growth cone guidance. Nat Neurosci 9:1265–1273. https://doi.org/10.1038/nn1773

    Article  CAS  PubMed  Google Scholar 

  13. Bassell GJ, Zhang H, Byrd AL et al (1998) Sorting of beta-actin mRNA and protein to neurites and growth cones in culture. J Neurosci 18:251–265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang C, Han B, Zhou R, Zhuang X (2016) Real-time imaging of translation on single mRNA transcripts in live cells. Cell 165:990–1001. https://doi.org/10.1016/j.cell.2016.04.040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yan X, Hoek TA, Vale RD, Tanenbaum ME (2016) Dynamics of translation of single mRNA molecules in vivo. Cell 165:976–989. https://doi.org/10.1016/j.cell.2016.04.034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wu B, Eliscovich C, Yoon YJ, Singer RH (2016) Translation dynamics of single mRNAs in live cells and neurons. Science 352:1430–1435. https://doi.org/10.1126/science.aaf1084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Morisaki T, Lyon K, DeLuca KF et al (2016) Real-time quantification of single RNA translation dynamics in living cells. Science 352:1425–1429. https://doi.org/10.1126/science.aaf0899

    Article  CAS  PubMed  Google Scholar 

  18. Tatavarty V, Ifrim MF, Levin M et al (2012) Single-molecule imaging of translational output from individual RNA granules in neurons. Mol Biol Cell 23:918–929. https://doi.org/10.1091/mbc.E11-07-0622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nagai T, Ibata K, Park ES et al (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90. https://doi.org/10.1038/nbt0102-87

    Article  CAS  PubMed  Google Scholar 

  20. Nieuwkoop PD, Faber J (1994) Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. Garland Publishing, New York

    Google Scholar 

  21. Falk J, Drinjakovic J, Leung KM et al (2007) Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus. BMC Dev Biol 7:107. https://doi.org/10.1186/1471-213X-7-107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wong HH-W, Holt CE (2018) Targeted electroporation in the CNS in Xenopus embryos. Methods Mol Biol 1865:119–131. https://doi.org/10.1007/978-1-4939-8784-9_9

    Article  CAS  PubMed  Google Scholar 

  23. Cagnetta R, Frese CK, Shigeoka T et al (2018) Rapid cue-specific remodeling of the nascent axonal proteome. Neuron 99:29–46.e4. https://doi.org/10.1016/j.neuron.2018.06.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783. https://doi.org/10.1016/S0006-3495(02)75618-X

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wong HH-W, Lin JQ, Ströhl F et al (2017) RNA docking and local translation regulate site-specific axon remodeling in vivo. Neuron 95:852–868.e8. https://doi.org/10.1016/j.neuron.2017.07.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wolter S, Löschberger A, Holm T et al (2012) rapidSTORM: accurate, fast open-source software for localization microscopy. Nat Methods 9:1040–1041. https://doi.org/10.1038/nmeth.2224

    Article  CAS  PubMed  Google Scholar 

  27. Sholl DA (1953) Dendritic organization in the neurons of the visual and motor cortices of the cat. J Anat 87:387–406

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Xing L, Bassell GJ (2013) MRNA localization: an orchestration of assembly, traffic and synthesis. Traffic 14:2–14. https://doi.org/10.1111/tra.12004

    Article  CAS  PubMed  Google Scholar 

  29. Chabanon H, Mickleburgh I, Hesketh J (2004) Zipcodes and postage stamps: mRNA localisation signals and their trans-acting binding proteins. Brief Funct Genomic Proteomic 3:240–256. https://doi.org/10.1093/bfgp/3.3.240

    Article  CAS  PubMed  Google Scholar 

  30. Piper M, Salih S, Weinl C et al (2005) Endocytosis-dependent desensitization and protein synthesis-dependent resensitization in retinal growth cone adaptation. Nat Neurosci 8:179–186. https://doi.org/10.1038/nn1380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Turner-Bridger B, Jakobs M, Muresan L et al (2018) Single-molecule analysis of endogenous β-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons. Proc Natl Acad Sci U S A 115:E9697–E9706. https://doi.org/10.1073/pnas.1806189115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Leung K-M, Lu B, Wong HH-W et al (2018) Cue-polarized transport of β-actin mRNA depends on 3′UTR and microtubules in live growth cones. Front Cell Neurosci 12:1–19. https://doi.org/10.3389/fncel.2018.00300

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Florian Ströhl and Julie Qiaojin Lin contributed equally to this work.

Authors and Affiliations

  1. Department of Physics and Technology, UiT The Arctic University of Norway, Tromsø, Norway

    Florian Ströhl

  2. Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK

    Florian Ströhl, Julie Qiaojin Lin, Francesca W. van Tartwijk & Clemens F. Kaminski

  3. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

    Julie Qiaojin Lin, Hovy Ho-Wai Wong & Christine E. Holt

  4. Department of Clinical Neurosciences, UK Dementia Research Institute at the University of Cambridge, Cambridge, UK

    Julie Qiaojin Lin & Clemens F. Kaminski

  5. Centre of Research in Neuroscience, Brain Repair and Integrative Neuroscience Programme, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montréal, QC, Canada

    Hovy Ho-Wai Wong

Authors
  1. Florian Ströhl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julie Qiaojin Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francesca W. van Tartwijk
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hovy Ho-Wai Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christine E. Holt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Clemens F. Kaminski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Clemens F. Kaminski .

Editor information

Editors and Affiliations

  1. Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

    Nobuhiko Yamamoto

  2. Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan

    Yasushi Okada

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ströhl, F., Lin, J.Q., van Tartwijk, F.W., Wong, H.HW., Holt, C.E., Kaminski, C.F. (2020). A Protocol for Single-Molecule Translation Imaging in Xenopus Retinal Ganglion Cells. In: Yamamoto, N., Okada, Y. (eds) Single Molecule Microscopy in Neurobiology . Neuromethods, vol 154. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0532-5_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0532-5_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0531-8

  • Online ISBN: 978-1-0716-0532-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation