Abstract
The unicellular eukaryotic amoeba, Dictyostelium discoideum, represents a superb model for examining the molecular mechanism of chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folic acid. Under starved conditions, they lose their sensitivity to pterins and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including ease of growth, genetic tractability, and the conservation of mammalian signaling pathways. In this chapter, we describe the use of the under-agarose chemotaxis assay to understand the signaling pathways controlling directional sensing and motility in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect conserved pathways involved in eukaryotic chemotaxis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Diez S, Gerisch G, Anderson K, Müller-Taubenberger A, Bretschneider T (2005) Subsecond reorganization of the actin network in cell motility and chemotaxis. Proc Natl Acad Sci U S A 102:7601–7606
van Haastert PJM, Devreotes P (2004) Chemotaxis: signaling the way forward. Nat Rev Mol Cell Biol 5:626–634
Nichols JM, Veltman D, Kay RR (2015) Chemotaxis of a model organism: progress with Dictyostelium. Curr Opin Cell Biol 36:7–12
King JS, Insall RH (2009) Chemotaxis: finding the way forward with Dictyostelium. Trends Cell Biol 19:523–530
Artemenko Y, Lampert TJ, Devreotes PN (2014) Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes. Cell Mol Life Sci 71:3711–3747
Jin T, Xu X, Fang J, Isik N, Yan J, Brzostowski JA, Hereld D (2009) How human leukocytes track down and destroy pathogens: lessons learned from the model organism Dictyostelium discoideum. Immunol Res 43:118–127
Pan P, Hall EM, Bonner JT (1975) Determination of the active portion of the folic acid molecule in cellular slime mold chemotaxis. J Bacteriol 122:185–191
Van Haastert PJ, De Wit RJ, Grijpma Y, Konijn TM (1982) Identification of a pterin as the acrasin of the cellular slime mold Dictyostelium lacteum. Proc Natl Acad Sci U S A 79:6270–6274
Pan M, Xu X, Chen Y, Jin T (2016) Identification of a chemoattractant G-protein-coupled receptor for folic acid that controls both chemotaxis and phagocytosis. Dev Cell 36:428–439
Mahadeo DC, Parent CA (2006) Signal relay during the life cycle of Dictyostelium. Curr Top Dev Biol 73:115–140
Varnum B, Soll DR (1981) Chemoresponsiveness to cAMP and folic acid during growth, development, and dedifferentiation in Dictyostelium discoideum. Differentiation 18:151–160
Parent CA, Devreotes PN (1996) Molecular genetics of signal transduction in Dictyostelium. Annu Rev Biochem 65:411–440
Gerisch G, Keller HU (1981) Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J Cell Sci 52:1–10
Zicha D, Dunn GA, Brown AF (1991) A new direct-viewing chemotaxis chamber. J Cell Sci 99:769–775
Muinonen-Martin AJ, Veltman DM, Kalna G, Insall RH (2010) An improved chamber for direct visualisation of chemotaxis. PLoS One 5:e15309
Cutler JE, Munoz JJ (1974) A simple in vitro method for studies on chemotaxis. Proc Soc Exp Biol Med 147:471–474
Lauffenburger D, Rothman C, Zigmond SH (1983) Measurement of leukocyte motility and chemotaxis parameters with a linear under-agarose migration assay. J Immunol 131:940–947
Laevsky G, Knecht DA (2001) Under-agarose folate chemotaxis of Dictyostelium discoideum amoebae in permissive and mechanically inhibited conditions. BioTechniques 31:1140–1149
Laevsky G, Knecht DA (2003) Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments. J Cell Sci 116:3761–3770
Xu XS, Lee E, Chen T, Kuczmarski E, Chisholm RL, Knecht DA (2001) During multicellular migration, myosin ii serves a structural role independent of its motor function. Dev Biol 232:255–264
Devreotes PN, Zigmond SH (1988) Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium. Annu Rev Cell Biol 4:649–686
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N (2010) Computer control of microscopes using μManager. Curr Protoc Mol Biol Chapter 14: Unit14.20
Edelstein AD, Tsuchida MA, Amodaj N, Pinkard H, Vale RD, Stuurman N (2014) Advanced methods of microscope control using μManager software. J Biol Methods 1
Lemieux MG, Janzen D, Hwang R, Roldan J, Jarchum I, Knecht DA (2014) Visualization of the actin cytoskeleton: different F-actin-binding probes tell different stories. Cytoskeleton (Hoboken) 71:157–169
Lauffenburger DA, Tranquillo RT, Zigmond SH (1988) Concentration gradients of chemotactic factors in chemotaxis assays. Methods Enzymol 162:85–101
Pan P, Wurster B (1978) Inactivation of the chemoattractant folic acid by cellular slime molds and identification of the reaction product. J Bacteriol 136:955–959
Aubry L, Firtel R (1999) Integration of signaling networks that regulate Dictyostelium differentiation. Annu Rev Cell Dev Biol 15:469–517
Tweedy L, Knecht DA, Mackay GM, Insall RH (2016) Self-generated chemoattractant gradients: attractant depletion extends the range and robustness of chemotaxis. PLoS Biol 14:e1002404
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Brazill, D., Knecht, D.A. (2022). Chemotaxis: Under Agarose Assay. In: Gavin, R.H. (eds) Cytoskeleton . Methods in Molecular Biology, vol 2364. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1661-1_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1661-1_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1660-4
Online ISBN: 978-1-0716-1661-1
eBook Packages: Springer Protocols