Skip to main content
SpringerLink
Log in
Book cover

Cytoskeleton Methods and Protocols pp 151–163Cite as

Studying Cytoskeletal Dynamics in Living Cells Using Green Fluorescent Protein

  • Protocol

  • 526 Accesses

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 161))

Abstract

The cytoskeleton is a dynamic structure comprised of at least three distinct cellular filament systems: microfilaments, intermediate filaments, and microtubules. Microfilaments are composed of assembled globular actin monomers that form a filamentous system involved in the maintenance of cell shape and polarity. Intermediate filaments are made of fibrous proteins including vimentin, cytokeratin, nuclear lamins, and neurofilament proteins that assemble into fibers providing mechanical stability to animal cells. Microtubules are formed by the assembly of tubulin (α and β subunits) producing long, rigid polymers which govern the location of membrane-bounded organelles and other cellular components. While each filament system has specific cellular functions, an emerging view of the cytoskeleton is that actin, intermediate filaments and microtubule networks function in concert to provide the cell with stability, polarity and organization.

This is a preview of subscription content, 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   74.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

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. Tsien R. Y. (1998) The Green Fluorescent Protein.Annu. Rev. Biochem. 67, 509–544.

    Article  PubMed  CAS  Google Scholar 

  2. Haseloff J. (1999) GFP variants for multispectral imaging of living cells. Methods Cell Biol. 58, 139–151.

    Article  PubMed  CAS  Google Scholar 

  3. Pepperkok R., Squire A., Geley S., and Bastiaens P. I. (1999) Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence life time imaging microscopy. Curr. Biol. 9, 269–272.

    Article  PubMed  CAS  Google Scholar 

  4. Mitra R. D., Silva C. M., and Youvan D. C. (1996) Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. Gene 173, 13–17.

    Article  PubMed  CAS  Google Scholar 

  5. Day R. N. (1998) Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy.Mol. Endocrin. 12, 1410–1419.

    Article  CAS  Google Scholar 

  6. Mahajan N. P., Linder K., Berry G., Gordon G. W., Heim R., and Herman B. (1998) Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat. Biotech. 16, 547–552.

    Article  CAS  Google Scholar 

  7. DeAngelis D. A., Miesenbock G., Zemelman B. V., and Rothman J. E. (1998) PRIM: proximity imaging of green fluorescent protein-tagged polypeptides. Proc. Nat. Acad. Sci. 95, 12,312–12,316.

    Article  CAS  Google Scholar 

  8. Westphal M., Jungbluth A., Heidecker M., Muhlbauer B., Heizer C., Schwartz J., et al. (1997) Microfilament dynamics during cell movement and chemotaxis moni tored using a GFP-actin fusion protein. Curr. Biol. 7, 176–183.

    Article  PubMed  CAS  Google Scholar 

  9. Choidas A., Jungbluth A., Sechi A., Murphy J., Ullrich A., and Marriott G. (1998) The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells.Eur.J. Cell Biol. 77, 81–90.

    PubMed  CAS  Google Scholar 

  10. Doyle T. and Botstein D. (1996) Movement of yeast cortical actin cytoskeleton visualized in vivo. Proc. Natl. Acad. Sci. USA 93, 3886–3891.

    Article  PubMed  CAS  Google Scholar 

  11. Verkhusha V., Tsukita S., and Oda H. (1999) Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP-actin fusion. FEBS Letters. 445, 395–401.

    Article  PubMed  CAS  Google Scholar 

  12. Fischer M., Kaech S., Knutti D., and Matus A. (1998) Rapid actin-based plasticity in dendritic spines. Neuron 20, 847–854.

    Article  PubMed  CAS  Google Scholar 

  13. Ballestrem C., Wehrle-Haller B., and Imhof B. (1998) Actin dynamics in living mammalian cells. J. Cell Sci. 111, 1649–1658.

    PubMed  CAS  Google Scholar 

  14. Heidemann S., Kaech S., Buxbaum R., and Matus A. (1999) Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts. J. Cell Biol. 145, 109–122.

    Article  PubMed  CAS  Google Scholar 

  15. Yoon M., Moir R., Prahlad V., and Goldman R. (1998) Motile properties of vimentin intermediate filament networks in living cells. J. Cell Biol. 143, 147–157.

    Article  PubMed  CAS  Google Scholar 

  16. Prahlad V., Yoon M., Moir R., Vale R., and Goldman R. (1998) Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks. J. Cell Biol. 143, 159–170.

    Article  PubMed  CAS  Google Scholar 

  17. Carminati J. and Stearns T. (1997) Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex.J. Cell Biol. 138, 629–641.

    Article  PubMed  CAS  Google Scholar 

  18. Straight A., Marshall W., Sedat J., and Murray A. (1997) Mitosis in living budding yeast: anaphase A but no metaphase plate. Science 277, 574–578.

    Article  PubMed  CAS  Google Scholar 

  19. Cleveland D. (1988) Autoregulated instability of tubulin mRNAs: a novel eukaryotic regulatory mechanism. Trends. Biochem. Sci. 13, 339–343.

    Article  PubMed  CAS  Google Scholar 

  20. Ludin B. and Matus A. (1998) GFP illuminates the cytoskeleton. Trends Cell Biol. 8, 72–77.

    Article  PubMed  CAS  Google Scholar 

  21. Mermall V., Post P., and Mooseker M. (1998) Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science 279, 527–533.

    Article  PubMed  CAS  Google Scholar 

  22. Moores S., Sabry J., and Spudich J. (1996) Myosin dynamics in live Dictyostelium cells. Proc. Natl. Acad. Sci. USA 93, 443–446.

    Article  PubMed  CAS  Google Scholar 

  23. Moss J., Price A., Raz E., Driever W., and Rosenthal N. (1996) Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish. Gene 173, 89–98.

    Article  PubMed  CAS  Google Scholar 

  24. Hirokawa N. (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519–526.

    Article  PubMed  CAS  Google Scholar 

  25. Hirokawa N., Noda Y., and Okda Y. (1998) Kinesin and dynein superfamily proteins in organelle transport and cell division. Curr. Opin. Cell Biol. 10, 60–73.

    Article  PubMed  CAS  Google Scholar 

  26. Huyett A., Kahana J., Silver P., Zeng X., and Saunders W. (1998) The Kar3p and Kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers. J. Cell Sci. 111, 295–301.

    PubMed  CAS  Google Scholar 

  27. Miller R., Heller K., Frisen L., Wallack D., Loayza D., Gammie A., and Rose M. (1998) The kinesin-related proteins, Kip2p and Kip3p, function differently innuclear migration in yeast. Mol. Biol. Cell. 9, 2051–2068.

    PubMed  CAS  Google Scholar 

  28. Endow S. and Komma D. (1996) Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd-GFP fusionproteins. J. Cell Sci. 109, 2429–2442.

    PubMed  CAS  Google Scholar 

  29. Gibbons I. (1981) Cilia and flagella of eukaryotes. J. Cell Biol. 91, 107s–124s.

    Article  PubMed  CAS  Google Scholar 

  30. Vallee R., Wall J., Paschal B., and Shpetner H. (1988) Microtubule-associatedprotein 1C from brain is a two-headed cytosolic dynein. Nature 5332, 561–563.

    Article  Google Scholar 

  31. Holzbaur E. and Vallee R. (1994) DYNEINS: molecular structure and cellular function. Ann. Rev. Cell Biol. 510, 339–372.

    Article  Google Scholar 

  32. Criswell P., Ostrowski L., and Asai D. (1996) A novel cytoplasmic dynein heavy chain: expression of DHC1b in mammalian ciliated epithelial cells. J. Cell Sci. 109, 1891–1898.

    PubMed  CAS  Google Scholar 

  33. Vaisberg E., Grisson P., and McIntosh J. (1996) Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles.J. Cell Biol. 143, 831–842.

    Article  Google Scholar 

  34. Shaw S., Yeh E., Salmon E., and Bloom K. (1996) Digital time-lapsed DIC/Fluorescence imaging of dynein-GFP reveals dynamics of astral microtubules in Saccharomyces cerevisiae throughout the cell cycle. Mol. Biol. Cell. 7s, 398a.

    Google Scholar 

  35. Shaw S., Yeh E., Maddox P., Salmon E., and Bloom K. (1997) Astral microtubule dynamics in yeast: a microtubule-based searching mechanism for spindleorientation and nuclear migration into the bud. J. Cell Biol. 139, 985–994.

    Article  PubMed  CAS  Google Scholar 

  36. McNiven M. (1998) Dynamin: a molecular motor with pinchase action. Cell 94, 151–154.

    Article  PubMed  CAS  Google Scholar 

  37. Cao H., Garcia F., and McNiven M. (1998) Differential distribution of dynamin isoforms in mammalian cells. Mol. Biol. Cell. 9, 2595–2609.

    PubMed  CAS  Google Scholar 

  38. Cook T. A., Mesa K., and Urrutia R. (1996) Three dynamin-encoding genes are differentially expressed in developing rat brain.J. Neurochem. 67, 927–931.

    Article  PubMed  CAS  Google Scholar 

  39. Cook T. A., Urrutia R., and McNiven M. A. (1994) Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues. Proc. Natl. Acad. Sci. USA 91, 644–648.

    Article  PubMed  CAS  Google Scholar 

  40. Nakata T., Takemura R., and Hirokawa N. (1993) A novel member of the dynamin family of GTP-binding proteins is expressed specifically in the testis. J. Cell Sci. 105, 1–5.

    PubMed  CAS  Google Scholar 

  41. Obar R. A., Collins C. A., Hammarback J. A., Shpetner H. S., and Vallee R. B. (1990) Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins.Nature 347, 256–261.

    Article  PubMed  CAS  Google Scholar 

  42. Shpetner H. and Vallee R. (1989) Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell 59, 421–432.

    Article  PubMed  CAS  Google Scholar 

  43. Sontag J.-M., Fykse E. M., Ushkaryov Y., Liu J.-P., Robinson P. J., and Südhof T.C. (1994) Differential expression and regulation of multiple dynamins. J. Biol. Chem. 269, 4547–4554.

    PubMed  CAS  Google Scholar 

  44. Presley J., Cole N., Schroer T., Hirschberg K., Zaal K., and Lippincott-Schwartz J. (1997) ER-to-Golgi transport visualized in living cells. Nature 389, 81–85.

    Article  PubMed  CAS  Google Scholar 

  45. Suzuki Y., Yasunaga T., Ohkura R., Wakabayashi T., and Sutoh K. (1998) Swing of the lever arm of a myosin motor at the isomerization and phosphaterelease steps. Nature 396, 380–383.

    Article  PubMed  CAS  Google Scholar 

  46. Iwane A., Funatsu T., Harada Y., Tokunaga M., Ohara O., Morimoto S., and Yanagida T. (1997) Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro. FEBS Letters 407, 235–238.

    Article  PubMed  CAS  Google Scholar 

  47. Romberg L., Pierce D., and Vale R. (1998) Role of the kinesin neck region in processive microtubule-based motility. J. Cell Biol. 140, 1407–1416.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Center for Basic Research in Digestive Diseases, Department of Gastroenterology and Hepatology, Mayo Clinic and Foundation, Rochester, MN

    Yisang Yoon & Mark A. McNiven

  2. Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic and Foundation, Rochester, MN

    Kelly R. Pitts

Authors
  1. Yisang Yoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kelly R. Pitts
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark A. McNiven
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Biology, Brooklyn College, The City University of New York, NY

    Ray H. Gavin

Rights and permissions

Reprints and permissions

Copyright information

© 2001 Humana Press Inc.

About this protocol

Cite this protocol

Yoon, Y., Pitts, K.R., McNiven, M.A. (2001). Studying Cytoskeletal Dynamics in Living Cells Using Green Fluorescent Protein. In: Gavin, R.H. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology™, vol 161. Humana Press. https://doi.org/10.1385/1-59259-051-9:151

Download citation

  • DOI: https://doi.org/10.1385/1-59259-051-9:151

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-771-7

  • Online ISBN: 978-1-59259-051-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation