Dictyostelium Cell Dynamics

Carole A. Parent1

1 National Cancer Institute, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.5
DOI:  10.1002/0471143030.cb1205s09
Online Posting Date:  May, 2001
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Abstract

This unit describes culturing and imaging of D. discoideum amoebae to study fundamental cellular responses, such as motility and directed migration. The system displays powerful molecular genetics that can be used to link structural determinants of proteins with in vivo cellular functions.

     
 
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Table of Contents

  • Basic Protocol 1: Imaging GFP‐Labeled Protiens in Live Single Cells
  • Alternate Protocol 1: Imaging GFP‐Labeled Proteins Following a Uniform Increase in Chemoattractant
  • Alternate Protocol 2: Imaging GFP‐Labeled Proteins in a Chemoattractant Gradient
  • Basic Protocol 2: Imaging GFP‐Labeled Proteins in Aggregation Stream and Mound Preparations
  • Basic Protocol 3: GFP‐Labeled Proteins in Slug Preparations
  • Support Protocol 1: Growing D. discoideum Axenically
  • Support Protocol 2: Growing D. discoideum on Bacterial Laws
  • Support Protocol 3: Plasmid Construction and Transformation
  • Support Protocol 4: Phenotypic Screening by Immunoblotting
  • Support Protocol 5: Phenotypic Screening by Development on Non‐Nutrient Agar
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Imaging GFP‐Labeled Protiens in Live Single Cells

  Materials
  • Transformed D. discoideum cells (see protocol 8) expressing desired green fluorescent protein (GFP) fusion protein
  • Phosphate buffer (PB; see recipe)
  • Developmental buffer (DB; see recipe)
  • Stock solution of adenosine 3′,5′‐cyclic monophosphate (cAMP, sodium salt) to give appropriate final concentration, for developed cells only
  • Inverted microscope equipped with a charge‐coupled device (CCD) camera, mercury light source, and appropriate filter sets for GFP imaging
  • Computer with either IPLab‐Spectrum (Scanalytics) or Openlab (Improvision) acquisition software
  • Chambered coverglass (e.g., Lab‐Tek II, Nalge Nunc International)
  • Brass holder designed to fit in a standard mechanical stage holder
  • Peristaltic pump connected to timer (for developed cells only)
  • Additional materials and reagents for growing D. discoideum cells (see Support Protocols protocol 61 and protocol 72) and for counting cells (unit 1.1)

Alternate Protocol 1: Imaging GFP‐Labeled Proteins Following a Uniform Increase in Chemoattractant

  Materials
  • Transformed D. discoideum cells (see protocol 8) expressing desired green fluorescent protein (GFP) fusion protein
  • Developmental buffer (DB; see recipe)
  • Adenosine 3′,5′‐cyclic monophosphate (cAMP, sodium salt) to give appropriate final concentration
  • Phosphate buffer (PB; see recipe)
  • Inverted microscope equipped with a charge‐coupled device (CCD) camera, mercury light source, and appropriate filter sets for GFP imaging
  • Computer with either IPLab‐Spectrum (Scanalytics) or Openlab (Improvision) acquisition software
  • Eight‐chambered coverglass (Lab‐Tek II, Nalge Nunc International)
  • Peristaltic pump connected to timer
  • Additional materials and reagents for growing D. discoideum cells to log phase in liquid medium (see protocol 6) and for counting cells (unit 1.1)

Alternate Protocol 2: Imaging GFP‐Labeled Proteins in a Chemoattractant Gradient

  • 10‐5 to 10‐6 M adenosine 3′,5′‐cyclic monophosphate (cAMP, sodium salt)
  • One‐chambered coverglass (Lab‐Tek II, Nalge Nunc International) or Attofluor cell chamber (Molecular Probes) fitted with a 35‐mm coverslip
  • Micropipet consisting of pulled glass capillary with an opening of ∼0.5 µm (e.g., Femtotips, Eppendorf Scientific)
  • Microinjector (e.g., Eppendorf Scientific)
  • Additional reagents and equipment for imaging GFP‐labeled proteins following a uniform increase in chemoattractant (see protocol 2)
NOTE: A Microloader (Eppendorf Scientific) can be used to load the micropipet.

Basic Protocol 2: Imaging GFP‐Labeled Proteins in Aggregation Stream and Mound Preparations

  Materials
  • Transformed D. discoideum cells (see protocol 8) grown on non‐nutrient agar (see protocol 10)
  • Silicone oil (e.g., Dow Corning 200/20cs, BDH Chemicals)
  • Incubator, 22°C
  • Attofluor cell chamber (Molecular Probes) fitted with a 35‐mm coverslip (optional)
  • Additional reagents and equipment for imaging GFP‐labeled proteins (see protocol 1)

Basic Protocol 3: GFP‐Labeled Proteins in Slug Preparations

  Materials
  • Transformed D. discoideum cells (see protocol 8) grown to log phase in liquid medium (see protocol 6)
  • 35‐mm petri dish containing 0.5 ml of 1% (w/v) aqueous Bacto Agar (Difco)
  • Silicone oil (e.g., Dow Corning 200/20cs, BDH Chemicals)
  • Incubator, 22°C
  • Small rubber ring (1 mm high and 5 mm in diameter)
  • Additional reagents and equipment for imaging GFP‐labeled proteins (see protocol 1)

Support Protocol 1: Growing D. discoideum Axenically

  Materials
  • D. discoideum cells (ATCC)
  • HL5 medium (see recipe)
  • Phosphate buffer (PB, see recipe)
  • 100‐mm tissue culture plate
  • 250‐ml tissue culture flask
  • Incubator, 22°C, with shaker

Support Protocol 2: Growing D. discoideum on Bacterial Laws

  Materials
  • Klebsiella aerogenes (ATCC)
  • HL5 medium (see recipe) without dihydrostreptomycin
  • D. discoideum amoebae (ATCC)
  • 100‐mm SM plates (see recipe)
  • Phosphate buffer (PB; see recipe)
  • Incubator, 22°C

Support Protocol 3: Plasmid Construction and Transformation

  Materials
  • D. discoideum cells grown to log phase in liquid medium (see protocol 6)
  • Electroporation buffer (EB; see recipe), ice cold
  • Supercoiled DNA vector encoding a GFP fusion protein
  • 100‐mm SM plate (see recipe)
  • Healing solution (see recipe)
  • HL5 medium (see recipe)
  • 20 mg/ml (w/v) G418 solution (see recipe)
  • Klebsiella aerogenes prepared for bacterial lawns (see protocol 7)
  • Electroporation cuvettes (0.2‐cm electrode gap; e.g., Bio‐Rad), ice cold
  • Electroporator (e.g., Gene Pulser II, Bio‐Rad)
  • Incubator, 22°C, with shaker
  • 24‐well plates
  • Additional reagents and equipment for counting cells (unit 1.1), immunoblotting (see protocol 9 and unit 6.2), and microscopic visualization (see Basic Protocols protocol 11, protocol 42, and protocol 53; see Alternate Protocols protocol 21 and protocol 32)
NOTE: Unless otherwise mentioned, all manipulations are performed aseptically on ice.

Support Protocol 4: Phenotypic Screening by Immunoblotting

  Materials
  • Transformed D. discoideum cells (see protocol 8) grown to log phase in liquid medium (see protocol 6)
  • Developmental buffer (DB; see recipe)
  • 4× SDS sample buffer ( appendix 2A)
  • Anti‐GFP antibody (e.g., Clontech Laboratories)
  • Antibody directed against protein of interest (optional)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)

Support Protocol 5: Phenotypic Screening by Development on Non‐Nutrient Agar

  Materials
  • Transformed D. discoideum cells (see protocol 8) grown to log phase in liquid medium (see protocol 6)
  • Developmental buffer (DB; see recipe)
  • Developmental buffer agar (DB agar; see recipe)
  • 35‐mm petri dish
  • Incubator, 22°C
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Figures

Videos

Literature Cited

Literature Cited
   Aubry, L. and Firtel, R.A. 1999. Integration of signaling networks that regulate Dictyostelium differentiation. Annu. Rev.Cell Dev. Biol. 15:469‐517
   Bonner, J.T. 1982. Comparative biology of cellular slime molds. In The Development of Dictyostelium discoideum (W.F. Loomis, ed.) pp. 1‐33. Academic Press, New York.
   Brown, J.M. and Firtel, R.A. 1998. Phosphorelay signalling: New tricks for an ancient pathway. Curr. Biol. 8:R662‐R665.
   Daunderer, C., Schliwa, M., and Graf, R. 1999. Dictyostelium discoideum: A promising centrosome model system. Biol. Cell 91:313‐320.
   De Lozanne, A. and Spudich, J.A. 1987. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236:1086‐1091.
   Dormann, D., Siegert, F., and Weijer, C.J. 1996. Analysis of cell movement during the culmination phase of Dictyostelium development. Development 122:761‐769.
   Egelhoff, T.T., Titus, M.A., Manstein, D.J., Ruppel, K.M., and Spudich, J.A. 1991. Molecular genetic tools for study of the cytoskeleton in Dictyostelium. Methods Enzymol. 196:319‐334.
   Eichinger, L., Lee, S.S., and Schleicher, M. 1999. Dictyostelium as a model system for studies of the actin cytoskeleton by molecular genetics. Micros. Res. Tech. 47:124‐134.
   Harwood, A.J., Plyte, S.E., Woodgett, J., Strutt, H., and Kay, R.R. 1995. Glycogen synthase kinase 3 regulates cell fate in Dictyostelium. Cell 80:139‐148.
   Howard, P.K., Ahern, K.G., and Firtel, R.A. 1988. Establishment of a transient expression system for Dictyostelium discoideum. Nucl. Acids Res. 16:2613‐2623.
   Hughes, J.E., Kiyosawa, H., and Welker, D.L. 1994. Plasmid maintenance functions encoded on Dictyostelium discoideum nuclear plasmid Ddp1. Mol. Cell. Biol. 14:6117‐6124.
   Kawata, T., Shevchenko, A., Fukuzawa, M., Jermyn, K.A., Totty, N.F., Zhukovskaya, N.V., Sterling, A.E., Mann, M., and Williams, J.G. 1997. SH2 signaling in a lower eukaryote: A STAT protein that regulates stalk cell differentiation in Dictyostelium. Cell 89:909‐916.
   Kay, R.R. and Williams, J.G. 1999. The Dictyostelium genome project: An invitation to species hopping. Trends Genet. 15:294‐297.
   Kessin, R.H. 2000. Dictyostelium: Evolution, Cell Biology, and the Development of Multicellularity. Developmental and Cell Biology Series. Cambridge University Press, Cambridge.
   Kuspa, A. and Loomis, W.F. 1992. Tagging developmental genes in Dictyostelium by restriction enzyme‐mediated integration of plasmid DNA. Proc. Natl. Acad. Sci. U.S.A. 89:8803‐8807.
   Loomis, W.F. 1998. Role of PKA in the timing of developmental events in Dictyostelium cells. Microbiol. Mol. Biol. Rev. 6:684‐694.
   Loomis, W.F., Kuspa, A., and Shaulsky, G. 1998. Two‐component signal transduction systems in eukaryotic microorganisms. Curr. Opin. Microbiol. 1:643‐648.
   Maeda, Y., Inouye, K., and Takeuchi, I. 1997. Dictyostelium, A Model System for Cell and Developmental Biology. Universal Academy Press, Tokyo.
   Manstein, D.J., Schuster, H.P., Morandini, P., and Hunt, D.M. 1995. Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 162:129‐134.
   Nellen, W. and Firtel, R.A. 1985. High‐copy‐number transformants and co‐transformants in Dictyostelium. Gene 39:155‐163.
   Pang, K.M., Lynes, M.A., and Knecht, D.A. 1999. Variables controlling the expression level of exogenous genes in Dictyostelium. Plasmid 41:187‐197.
   Parent, C.A. and Devreotes, P.N. 1996a. Molecular dissection of G protein–mediated signal transduction using random mutagenesis in Dictyostelium. Microb. Genome Methods 1:1‐13.
   Parent, C.A. and Devreotes, P.N. 1996b. Molecular genetics of signal transduction in Dictyostelium. Annu. Rev. Biochem. 65:411‐440.
   Parent, C.A. and Devreotes, P.N. 1999. A cell's sense of direction. Science 284:765‐770.
   Rietdorf, J., Siegert, F., and Weijer, C.J. 1996. Analysis of optical density wave propagation and cell movement during mound formation in Dictyostelium discoideum. Dev. Biol. 177:427‐438.
   Slade, M.B., Emslie, K.R., and Williams, K.L. 1997. Expression of recombinant glycoproteins in the simple eukaryote Dictyostelium discoideum. Biotechnol. Genet. Eng. Rev. 14:1‐35.
   Sussman, M. 1987. Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol. 28:9‐29.
   Thomason, P.A., Traynor, D., and Kay, R.R. 1999. Taking the plunge: Terminal differentiation in Dictyostelium. Trends Genet. 15:15‐19.
   Williams, J.G. 1999. Serpentine receptors and STAT activation: More than one way to twin a STAT. Trends Biochem. Sci. 24:333‐334.
Key References
   Kessin 2000. See above
  This is the most recent book on Dictyostelium cell biology to date. It covers a wide variety of valuable information on the development of Dictyostelium.
   Ludin, B. and Matus, A., in association with Trends Cell Biol. 1999. “GFP in motion”.
  This CD contains beautiful movies highlighting the use of GFP in live cells. Several sections describe results obtained using D. discoideum. Contact Trends in Cell Biology (www.elsevier.com) for more details.
   Spudich, J.A. (ed.) 1987. Dictyostelium discoideum: Molecular Approaches to Cell Biology. Methods Cell Biol. Vol. 28.
  This volume contains details of a variety of molecular and cell biological tools used to study D. discoideum.
   Experientia Vol. 51. 1995.
  This volume contains a series of reviews pertaining to genomics, cell biology, and biochemistry of D. discoideum.
Internet Resources
   http://dicty.cmb.nwu.edu/dicty/dicty.html
  This site serves as a single entry point for the online resources for the D. discoideum community. It provides access to an online database of investigators, current and past editions of the newsletter “Dicty News”, links to cDNA and genome sequencing projects, and a wide variety of useful resources.
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