Summary
For decades, Dictyostelium discoideum has been an efficacious and attractive model system for the study of cell motility, primarily because cells become highly motile during the transition from growth phase to aggregation competence and because the haploid genome is readily amenable to mutation. These crawling amoebae, as well as other motile cells such as polymorphonuclear neutrophils (PMNs), extend pseudopodia, retract pseudopodia, and translocate across a substratum even in the absence of chemoattractant. This phenomenon, referred to as basic motile behavior, has been investigated in Dictyostelium through analysis of cytoskeletal mutants. Likewise, many chemotactic signal transduction pathways and networks have been inferred from studies of Dictyostelium mutants. However, before concluding from mutational analyses that a particular molecule or protein plays a role in chemotaxis, it is imperative to first precisely define its contribution, if any, to basic motile behavior. Here, we describe two-dimensional and three-dimensional technologies that can be coupled with 2D and 3D Dynamic Image Analysis System (2D and 3D-DIAS) software for the analysis of cell motility, shape changes, pseudopod formation, and localization of tagged molecules during basic motile behavior. In addition, we describe a method to analyze the 3D trajectories of microspheres attached to the surface of crawling Dictyostelium cells. We include information on microscopy, image acquisition techniques, and computer hardware that could be reproduced in a typical laboratory setting for motion analysis using 2D and 3D-DIAS software. Finally, we highlight features available in DIAS that have proven insightful in identifying defects in basic motile behavior exhibited by various cytoskeletal and putative signal transduction mutants.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Soll, D. R., Wessels, D., Heid, P., and Zhang, H. (2002) A contextual framework for characterizing motility and chemotaxis in Dictyostelium discoideum. J. Muscle Res. Cell Motil. 23, 659–672
Soll, D. R. (1988) “DMS”, a computer-assisted system for quantitating motility, the dynamics of cytoplasmic flow and pseudopod formation: its application to Dictyostelium chemotaxis. Cell Motil. Cytoskeleton 10, 91–106
Soll, D. R. (1999) Computer-assisted three-dimensional reconstruction and motion analysis of living, crawling cells. Comput. Med. Imaging Graph. 23, 3–14
Soll, D. R. (1995) The use of computers in understanding how animal cells crawl. Int. Rev. Cytol. 163, 43–104
Soll, D. R. and Voss, E. (1998) Two and three-dimensional computer systems for analyzing how cells crawl. In: Soll, D. R. and Wessels, D. (eds.), Motion Analysis of Living Cells. Wiley, New York, pp. 25–52
Soll, D. R., Voss, E., Johnson, O., and Wessels, D. J. (2000) Three-dimensional reconstruction and motion analysis of living crawling cells. Scanning 22, 249–257
Soll, D. R., Wessels, D., Voss, E., and Johnson, O. (2001) Computer-assisted systems for the analysis of amoeboid cell motility. In: Gavin, R. H. (ed.), Methods in Molecular Biology: Cytoskeleton Methods and Protocols, Vol. 161. Humana Press, Totowa, NJ, pp. 45–58
Soll, D. R., Wessels, D., Heid, P. J., and Voss, E. (2003) Computer-assisted reconstruction and motion analysis of the three-dimensional cell. ScientificWorldJournal 3, 827–841
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. and Rivero, F. (eds.), Methods in Molecular Biology: Dictyostelium discoideum Protocols, Vol. 346. Humana Press, Totowa, NJ, pp. 261–279
Soll, D. R., Voss, E., Wessels, D., and Kuhl, S. (2007) Computer-assisted systems for dynamic 3D reconstruction and motion analysis of living cells. In: Shorte, S. (ed.), Cell Motility. Springer, Germany, pp. 365–384
Cocucci, S. and Sussman, M. (1970) RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J. Cell Biol. 4, 399–407
Varnum, B. and Soll, D. R. (1984) Effect of cAMP on single cell motility in Dictyostelium. J. Cell Biol. 99, 1151–1155
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
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. Cytoskeleton 8, 18–26
Geiger, J., Wessels, D., and Soll, D. R. (2003) Human polymorphonuclear leukocytes respond to waves of chemoattractant, like Dictyostelium. Cell Motil. Cytoskeleton 56, 27–44
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
Soll, D. R. (1979) Timers in developing systems. Science 203, 841–849
Wessels, D., Brincks, R., Kuhl, S., Stepanovic, V., Daniels, K. J., Weeks, G., et al. (2004) RasC plays a role in transduction of temporal gradient information in the cyclic-AMP wave of Dictyostelium discoideum. Eukaryot. Cell 3, 646–662
Wessels, D., Soll, D. R., Knecht, D., Loomis, W. F., DeLozanne, A., and Spudich, J. (1988) Cell motility and chemotaxis in Dictyostelium amoebae lacking myosin heavy chain. Dev. Biol. 128, 164–177
Wessels, D., Murray, J., Jung, G., Hammer, J., and Soll, D. R. (1991) Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility. Cell Motil. Cytoskeleton 20, 301–315
Cox, D., Condeelis, J., Wessels, D., Soll, D., Kern, H., and Knecht, D. (1992) Targeted disruption of the ABP-120 gene leads to cells with altered motility. J. Cell Biol. 116, 943–955
Titus, M., Wessels, D., Spudich, J., and Soll, D. R. (1992) The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility. Mol. Biol. Cell 4, 233–246
Wessels, D. and Soll, D. R. (1998) Computer-assisted characterization of the behavioral defects of cytoskeletal mutants of Dictyostelium discoideum. In: Soll, D.R. and Wessels, D. (eds), Motion Analysis of Living Cells. Wiley, New York, pp. 101–140
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
Heid, P., Voss, E., and Soll, D. R. (2002) 3D-DIASemb: a computer-assisted system for reconstructing and motion analyzing in 4D every cell and nucleus in a developing embryo. Dev. Biol. 245, 329–347
Pang, K., Lee, E., and Knecht, D. (1998) Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous actin structures. Curr. Biol. 8, 405–408
Acknowledgments
The development of 2D-DIAS and 3D-DIAS was supported in part by grants HD18577 and AI40040 from NIH and a generous facility grant from the W.M. Keck Foundation. We would like to thank Daniel Lusche for careful reading of the manuscript and Katie Ekvall for help in its preparation.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press
About this protocol
Cite this protocol
Wessels, D.J., Kuhl, S., Soll, D.R. (2009). Light Microscopy to Image and Quantify Cell Movement. In: Jin, T., Hereld, D. (eds) Chemotaxis. Methods in Molecular Biology™, vol 571. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-198-1_30
Download citation
DOI: https://doi.org/10.1007/978-1-60761-198-1_30
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-197-4
Online ISBN: 978-1-60761-198-1
eBook Packages: Springer Protocols