Skip to main content

Imaging the Cytoskeleton in Live Xenopus laevis Embryos

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 586))

Summary

Historically, much of our understanding of actin filaments, microtubules and intermediate filaments has come from the study of fixed cells and tissues. But the cytoskeleton is inherently dynamic, and so developing the means to image it in living cells has proved crucial. Advances in confocal microscopy and fluorescent protein technologies have allowed us to dynamically image the cytoskeleton at high resolution and so learn much more about its cellular functions. However, most of this work has been performed in cultured cells, and a critical next step is to understand how the cytoskeleton functions in the context of an intact organism. We, and others, have developed methods to image the cytoskeleton in living vertebrate embryos. Here, we describe an approach to image the cytoskeleton in embryos of the frog, Xenopus laevis, using mRNA to express fluorescently tagged cytoskeletal probes and confocal microscopy to visualize their dynamic behavior.

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 1567–1572

    Article  CAS  PubMed  Google Scholar 

  2. Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., and Lukyanov, S.A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17, 969–973

    Article  CAS  PubMed  Google Scholar 

  3. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233

    Article  CAS  PubMed  Google Scholar 

  4. Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., and Tsien, R.Y. (2002). A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99, 7877–7882

    Article  CAS  PubMed  Google Scholar 

  5. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802–805

    Article  CAS  PubMed  Google Scholar 

  6. Heim, R. and Tsien, R.Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6, 178–182

    Article  CAS  PubMed  Google Scholar 

  7. Megason, S.G. and Fraser, S.E. (2003). Digitizing life at the level of the cell: high-performance laser-scanning microscopy and image analysis for in toto imaging of development. Mech Dev 120, 1407–1420

    Article  CAS  PubMed  Google Scholar 

  8. Kieserman, E.K., Glotzer, M., and Wallingford, J.B. (2008). Developmental regulation of central spindle assembly and cytokinesis during vertebrate embryogenesis. Curr Biol 18, 116–123

    Article  CAS  PubMed  Google Scholar 

  9. Woolner, S., O’Brien, L.L., Wiese, C., and Bement, W.M. (2008). Myosin-10 and actin filaments are essential for mitotic spindle function. J Cell Biol 182, 77–88

    Article  CAS  PubMed  Google Scholar 

  10. Miller, A.L., and Bement, W.M. (2009). Regulation of cytokinesis by Rho GTPase flux. Nature Cell Biology 11, 71–7

    Google Scholar 

  11. Mikhailov, A. and Gundersen, G.G. (1998). Relationship between microtubule dynamics and lamellipodium formation revealed by direct imaging of microtubules in cells treated with nocodazole or taxol. Cell Motil Cytoskeleton 41, 325–340

    Article  CAS  PubMed  Google Scholar 

  12. Charras, G.T., Hu, C.K., Coughlin, M., and Mitchison, T.J. (2006). Reassembly of contractile actin cortex in cell blebs. J Cell Biol 175, 477–490

    Article  CAS  PubMed  Google Scholar 

  13. Peng, H.B. and Kay, B.K. (1991). Xenopus laevis: practical uses in cell and molecular biology. Methods Cell Biol 36, 679–681

    Article  PubMed  Google Scholar 

  14. Burkel, B.M., von Dassow, G., and Bement, W.M. (2007). Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell Motil Cytoskeleton 64, 822–832

    Article  CAS  PubMed  Google Scholar 

  15. Faire, K., Waterman-Storer, C.M., Gruber, D., Masson, D., Salmon, E.D., and Bulinski, J.C. (1999). E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics. J Cell Sci 112(Pt 23), 4243–4255

    CAS  PubMed  Google Scholar 

  16. Pertz, O. and Hahn, K.M. (2004). Designing biosensors for Rho family proteins – deciphering the dynamics of Rho family GTPase activation in living cells. J Cell Sci 117, 1313–1318

    Article  CAS  PubMed  Google Scholar 

  17. Kim, S.H., Li, Z., and Sacks, D.B. (2000). E-cadherin-mediated cell-cell attachment activates Cdc42. J Biol Chem 275, 36999–37005

    Article  CAS  PubMed  Google Scholar 

  18. Benink, H.A. and Bement, W.M. (2005). Concentric zones of active RhoA and Cdc42 around single cell wounds. J Cell Biol 168, 429–439

    Article  CAS  PubMed  Google Scholar 

  19. Yonemura, S., Hirao-Minakuchi, K., and Nishimura, Y. (2004). Rho localization in cells and tissues. Exp Cell Res 295, 300–314

    Article  CAS  PubMed  Google Scholar 

  20. Sokac, A.M., Co, C., Taunton, J., and Bement, W. (2003). Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs. Nat Cell Biol 5, 727–732

    Article  CAS  PubMed  Google Scholar 

  21. Ma, C., Benink, H.A., Cheng, D., Montplaisir, V., Wang, L., Xi, Y., Zheng, P.P., Bement, W.M., and Liu, X.J. (2006). Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation. Curr Biol 16, 214–220

    Article  CAS  PubMed  Google Scholar 

  22. Bement, W.M., Benink, H.A., and von Dassow, G. (2005). A microtubule-dependent zone of active RhoA during cleavage plane specification. J Cell Biol 170, 91–101

    Article  CAS  PubMed  Google Scholar 

  23. Sive, H.L., Grainger, R.M., and Harland, R.M. (1998). Early Development of Xenopus laevis: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York

    Google Scholar 

  24. Heasman, J. (2002). Morpholino oligos: making sense of antisense? Dev Biol 243, 209–214

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Woolner, S., Miller, A.L., Bement, W.M. (2009). Imaging the Cytoskeleton in Live Xenopus laevis Embryos. In: Gavin, R. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology, vol 586. Humana Press. https://doi.org/10.1007/978-1-60761-376-3_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-376-3_2

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-375-6

  • Online ISBN: 978-1-60761-376-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics