Skip to main content
SpringerLink
Log in
Book cover

Dictyostelium discoideum Protocols pp 261–279Cite as

Application of 2D and 3D DIAS to Motion Analysis of Live Cells in Transmission and Confocal Microscopy Imaging

  • Protocol

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 346))

Abstract

The chemotactic signal in Dictyostelium is a cAMP wave that is relayed over relatively large distances through a cell population during aggregation. Cells exhibit unique behaviors in response to the different spatial, temporal, and concentration components of the cAMP wave, suggesting that distinct signal transduction pathways are evoked in each of the various phases of the wave. For this reason, we designed a set of experimental protocols to test responses of normal and mutant Dictyostelium amoebae to the different components of a wave of chemoattractant. We then used computer-assisted two- (2D) and three-dimensional (3D) technologies (2D and 3D Dynamic Image Analysis System [DIAS]) for analysis of cells in the absence of a chemotactic signal (basic motile behavior) and in response to the temporal, spatial, and concentration components of the wave. As a result, we have elucidated parallel and independent pathways activated by specific phases of the cAMP wave. Likewise, human polymorphonuclear neutrophils (PMNs) respond to experimentally applied waves of the chemotactic peptide fMLP, and also exhibit discrete behavioral responses to the different phases. Using Dictyostelium as a paradigm, we applied our protocols to normal and diseased human PMNs and precisely defined a chemotactic defect. In this chapter, we describe methods for quantifying behaviors in Dictyostelium amoebae, PMNs, and other amoeboid cells using 2D and 3D DIAS. These methods are useful in the reconstruction and motion analysis of most migrating cells with transmitted and/or confocal microscopy.

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   169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
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. Murray, J., Vawter-Hugart, H., Voss, E., and Soll, D. R. (1992) A three-dimensional motility cycle in leukocytes. Cell Motil. Cytoskel. 22, 211–223.

    Article  CAS  Google Scholar 

  2. Wessels, D., Vawter-Hugart, H., Murray, J., and Soll, D. R. (1994) Three dimensional dynamics of pseudopod formation and turning during the motility cycle of Dictyostelium. Cell Motil. Cytoskel. 27, 1–12.

    Article  CAS  Google Scholar 

  3. Kirfel, G., Rigort, A., Borm, B., and Herzog, V. (2004) Cell migration: mechanisms of rear detachment and the formation of migration tracks. Eur. J. Cell Biol. 83, 717–724.

    Article  PubMed  Google Scholar 

  4. Uchida, K., Kitanishi-Yumura, T., and Yumura, S. (2002) Myosin II contributes to the posterior contraction and the anterior extension during the retraction phase in migrating Dictyostelium cells. J. Cell Sci. 116, 51–60.

    Article  Google Scholar 

  5. Geiger, J., Wessels, D., and Soll, D. R. (2003) Human polymorphonuclear leukocytes respond to waves of chemoattractant, like Dictyostelium. Cell Motil. Cytoskel. 56, 27–44.

    Article  Google Scholar 

  6. Firtel, R. and Chung, C. (2000) The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients. Bioessays 22, 603–615.

    Article  PubMed  CAS  Google Scholar 

  7. Devreotes, P. and Janetopoulos, C. (2003) Eukaryotic chemotaxis: distinctions between directional sensing and polarization. J. Biol. Chem. 278, 20,445–20,448.

    Article  PubMed  CAS  Google Scholar 

  8. Parent, C. (2004) Making all the right moves: chemotaxis in neutrophils and Dictyostelium. Curr. Opin. Cell Biol. 16, 14–23.

    Article  Google Scholar 

  9. Soll, D.R., Wessels, D., Heid, P., and Zhang, H. (2003) A contextual framework for characterizing motility and chemotaxis mutants in Dictyostelium discoideum. J. Muscle Res. Cell Motil. 23, 659–672.

    Article  Google Scholar 

  10. Alcantara, E. and Monk, M. (1974) Signal propagation in the cellular slime mould Dictyostelium discoideum. J. Gen. Microbiol. 84, 321–334.

    Google Scholar 

  11. Wyckoff, J., Wang, W., Lin, E., et al. (2004). A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res. 64, 7022–7029.

    Article  PubMed  CAS  Google Scholar 

  12. Wessels, D., Murray, J., and Soll, D. R. (1992) Behavior of Dictyostelium amoebae is regulated primarily by the temporal dynamic of the natural cAMP wave. Cell Motil. Cytoskel. 41, 225–246.

    Article  Google Scholar 

  13. Stepanovic, V., Wessels, D., Daniels, K. Loomis, W. F., and Soll, D. R. (2005) Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis. Euk. Cell 4, 775–786.

    Article  CAS  Google Scholar 

  14. Wessels, D., Brincks, R., Kuhl, S., et al. (2004) RasC plays a selective role in the transduction of temporal gradient information in the cAMP wave of Dictyostelium. Euk. Cell 3, 646–662.

    Article  CAS  Google Scholar 

  15. Kumar, A., Wessels, D., Daniels, K., Alexander, H., Alexander, S., and Soll, D. R. (2004) Sphingosine-1-phosphate plays a role in the suppression of lateral pseudopod formation during Dictyostelium discoideum cell migration and chemotaxis. Cell Motil. Cytoskel. 59, 227–241.

    Article  CAS  Google Scholar 

  16. Zhang, H., Heid, P., Wessels, D., et al. (2003) Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium. Euk. Cell 2, 62–75.

    Article  CAS  Google Scholar 

  17. Falk, D., Wessels, D., Jenkins, L., et al. (2003) Shared, unique and redundant functions of three members of the class I myosins (MyoA, MyoB and MyoF) in motility and chemotaxis in Dictyostelium. J. Cell Sci. 116, 3985–3999.

    Article  PubMed  CAS  Google Scholar 

  18. Zhang, H., Wessels, D., Fey, P., Daniels, K., Chisholm, R., and Soll, D. R. (2002) Phosphorylation of the myosin regulatory light chain plays a role in cell motility and polarity in Dictyostelium chemotaxis. J. Cell Sci. 115, 1733–1747.

    Article  PubMed  CAS  Google Scholar 

  19. Wessels, D., Zhang, H., Reynolds, J., et al. (2000) The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis. Mol. Biol. Cell 11, 2803–2820.

    PubMed  CAS  Google Scholar 

  20. Wessels, D. and Soll, D. R. (1998) Computer-assisted characterization of the behavioral defects of cytoskeletal mutants of Dictyostelium discoideum, in Motion Analysis of Living Cells (Soll, D. R. and Wessels, D., eds.), John Wiley, Inc., New York: pp. 101–140.

    Google Scholar 

  21. Stepanovic, V., Wessels, D., Goldman, G., Geiger, J., and Soll, D. R. (2004) The chemotaxis defect of Shwachman-Diamond syndrome leukocytes. Cell Motil. Cytoskel. 57, 158–174.

    Article  CAS  Google Scholar 

  22. Soll, D. R. and Voss, E. (1998) Two and three-dimensional computer systems for analyzing how cells crawl, in Motion Analysis of Living Cells (Soll, D. R. and Wessels, D., eds.), John Wiley, Inc., New York: pp. 25–52.

    Google Scholar 

  23. Soll, D. R. (1999) Computer-assisted three-dimensional reconstruction and motion analysis of living, crawling cells. Comp. Med. Imag. Graph. 23, 3–14.

    Article  CAS  Google Scholar 

  24. Soll, D. R., Voss, E., Johnson, O., and Wessels, D. (2000) Three dimensional reconstruction and motion analysis of living crawling cells. Scanning 22, 249–257.

    Article  PubMed  CAS  Google Scholar 

  25. Soll, D. R., Wessels, D., Voss, E., and Johnson, O. (2000) Computer-assisted systems for the analysis of amoeboid cell motility, in Methods in Molecular Biology: Cytoskeleton Methods and Protocols (Gavin, R. H., ed.), Humana, Totowa, NJ: pp. 45–58.

    Chapter  Google Scholar 

  26. Heid, P., Geiger, J., Wessels, D., Voss, E., and Soll, D. R. (2005) Computer assisted analysis of filopod formation and the role of myosin II heavy chain phosphorylation in Dictyostelium. J. Cell Sci. 118, 2225–2237.

    Article  PubMed  CAS  Google Scholar 

  27. Varnum, B. and Soll, D.R. (1984). Effect of cAMP on single cell motility in Dictyostelium. J. Cell Biol. 99, 1151–1155.

    Article  PubMed  CAS  Google Scholar 

  28. Varnum-Finney, B., Voss, E., and Soll, D. R. (1987) Frequency and orientation of pseudopod formation of Dictyostelium discoideum amoebae chemotaxing in a spatial gradient: further evidence for a temporal mechanism. Cell Motil. Cytoskel. 8, 18–26.

    Article  CAS  Google Scholar 

  29. Varnum, B., Edwards, K., and Soll, D. R. (1985) Dictyostelium amoebae alter motility differently in response to increasing versus decreasing temporal gradients of cAMP. J. Cell Biol. 101, 1–5.

    Article  PubMed  CAS  Google Scholar 

  30. Zigmond, S. (1978) A new visual assay of leukocyte chemotaxis, in Leukocyte Chemotaxis: Methods, Physiology and Clinical Implication (Gallin, J. I. and Quie, P. G., eds.), Raven, New York: pp. 57–66.

    Google Scholar 

  31. Shutt, D., Jenkins, L., Carolan, E., et al. (1998) T cell syncytia induced by HIV release T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber. J. Cell Sci. 111, 99–109.

    PubMed  CAS  Google Scholar 

  32. Escalante, R., Wessels, D., Soll, D. R., and Loomis, W. F. (1997) Chemotaxis to cAMP and slug migration in Dictyostelium both depend on MigA, a BTB protein. Mol. Biol. Cell 8, 1763–1775.

    PubMed  CAS  Google Scholar 

  33. Geiger, J., Wessels, D., Lockhart, S., and Soll, D. R. (2004) Release of a potent polymorphonuclear leukocyte chemoattractant is regulated by white-opaque switching in Candida albicans. Infect. Immunl. 72, 667–671.

    Article  CAS  Google Scholar 

  34. Wessels, D., Schroeder, N., Voss, E., Hall, A., Condeelis, J., and Soll, D. R. (1989) cAMP mediated inhibition of intracellular particle movement and actin reorganization in Dictyostelium. J. Cell Biol. 109, 2841–2851.

    Article  PubMed  CAS  Google Scholar 

  35. Wessels, D., Voss, E., von Bergen, N., Burns, R., Stites, J., and Soll, D. R. (1998) A computer-assisted system for reconstructing and interpreting the dynamic three-dimensional relationships of the outer surface, nucleus, and pseudopodia of crawling cells. Cell Motil. Cytoskel. 41, 225–246.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. W. M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, The University of Iowa, Iowa City, IA, USA

    Deborah Wessels, Spencer Kuhl & David R. Soll

Authors
  1. Deborah Wessels
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Spencer Kuhl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David R. Soll
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Center for Biochemistry and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany

    Ludwig Eichinger  & Francisco Rivero  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Wessels, D., Kuhl, S., Soll, D.R. (2006). Application of 2D and 3D DIAS to Motion Analysis of Live Cells in Transmission and Confocal Microscopy Imaging. In: Eichinger, L., Rivero, F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology™, vol 346. Humana Press. https://doi.org/10.1385/1-59745-144-4:261

Download citation

  • DOI: https://doi.org/10.1385/1-59745-144-4:261

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-623-8

  • Online ISBN: 978-1-59745-144-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation