Skip to main content
SpringerLink
Log in

Dynamic organization of the actin system in the motile cells of Dictyostelium

  • Published:
Journal of Muscle Research & Cell Motility Aims and scope Submit manuscript

Abstract

The actin system forms a supramolecular, membrane-associated network that serves multiple functions in Dictyostelium cells, including cell motility controlled by chemoattractant, phagocytosis, macropinocytosis, and cytokinesis. In executing these functions the monomeric G-actin polymerizes reversibly, and the actin filaments are assembled into membrane-anchored networks together with other proteins involved in shaping the networks and controlling their dynamics. Most impressive is the speed at which actin-based structures are built, reorganized, or disassembled. We used GFP-tagged coronin and Arp3, an intrinsic constituent of the Arp2/3 complex, as examples of proteins that are recruited to highly dynamic actin-filament networks. By fluorescence recovery after photobleaching (FRAP), average exchange rates of cell-cortex bound coronin were estimated. A nominal value of 5 s for half-maximal incorporation of coronin into the cortex, and a value of 7 s for half-maximal dissociation from cortical binding sites has been obtained. Actin dynamics implies also flow of F-actin from sites of polymerization to sites of depolymerization, i.e. to the tail of a migrating cell, the base of a phagocytic cup, and the cleavage furrow in a mitotic cell. To monitor this flow, we expressed in Dictyostelium cells a GFP-tagged actin-binding fragment of talin. This fragment (GFP-TalC63) translocates from the front to the tail during cell migration and from the polar regions to the cleavage furrow during mitotic cell division. The intrinsic dynamics of the actin system can be manipulated in vivo by drugs or other probes that act either as inhibitors of actin polymerization or as stabilizers of filamentous actin. In order to investigate structure–function relationships in the actin system, a technique of reliably arresting transient network structures is in demand. We discuss the potential of electron tomography of vitrified cells to visualize actin networks in their native association with membranes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bray D and White JG (1988) Cortical flow in animal cells. Science 239: 883–888.

    Google Scholar 

  • Claviez M, BrinkMand Gerisch G (1986) Cytoskeleton from a mutant of Dictyostelium discoideum with flattened cells. J Cell Sci 86: 69–82.

    Google Scholar 

  • DeHostos EL, Bradtke B, Lottspeich F, Guggenheim R and Gerisch G (1991) Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein ß subunits. EMBO J 10: 4097–4104.

    Google Scholar 

  • DeHostos EL, Rehfueß C, Bradtke B, Waddell DR, Albrecht R, Murphy J and Gerisch G (1993) Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility. J Cell Biol 120: 163–173.

    Google Scholar 

  • Dierksen K, Typke D, Hegerl R and Baumeister W (1993) Ultramicroscopy 49: 109–120.

    Google Scholar 

  • Dierksen K, Typke D, Hegerl R, Koster AJ and Baumeister W (1992) Towards automatic electron tomography. Ultramicroscopy 40: 71–87.

    Google Scholar 

  • Dormann D, Libotte T, Weijer CJ and Bretschneider T (2002) Simultaneous quanti.cation of cell motility and protein-membrane-association using active contours. Cell Motil Cytoskel 52: 221–230.

    Google Scholar 

  • Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW and Schultz P (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21: 129–228.

    Google Scholar 

  • Eichinger L, Lee SS and Schleicher M (1999) Dictyostelium as model system for studies of the actin cytoskeleton by molecular genetics. Microscopy Res Tech 47: 124–134.

    Google Scholar 

  • Felsenfeld DP, Choquet D and Sheetz MP (1996) Ligand binding regulates the directed movement of β1 integrins on fibroblasts. Nature 383: 438–440.

    Google Scholar 

  • Fukui Y, Engler S, Inoué S and deHostos EL (1999a) Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis. Cell Motil Cytoskel 42: 204–217.

    Google Scholar 

  • Fukui Y, Kitanishi-Yumura T and Yumura S (1999b) Myosin II-independent F-actin flow contributes to cell locomotion in Dictyostelium. J Cell Sci 112: 877–886.

    Google Scholar 

  • Fukui Y, Yumura S and Yumura TK (1987) Agar-overlay immuno-fluorescence: high-resolution studies of cytoskeletal components and their changes during chemotaxis. In: Spudich JA (ed.) Methods Cell Biology (Vol. 28, pp. 347–356). Academic Press, Orlando, FL.

    Google Scholar 

  • Gerisch G and Müller-Taubenberger A (2003) GFP-fusion proteins as fluorescent reporters to study organelle and cytoskeleton dynamics in chemotaxis and phagocytosis. In: Marriott G and Parker I (eds) Methods Enzymology (Vol. 361, Biophotonics) Academic Press, New York.

    Google Scholar 

  • Gerisch G, Albrecht R, Heizer C, Hodgkinson S and Maniak M (1995) Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using green fluorescent protein-coronin fusion protein. Curr Biol 5: 1280–1285.

    Google Scholar 

  • Hall AL, Schlein A and Condeelis J (1988) Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells. J Cell Biochem 37: 285–299.

    Google Scholar 

  • Hegerl R (1996) The EM program package: a platform for image processing in biological electron microscopy. J Struct Biol 116: 30–34.

    Google Scholar 

  • Higgs HN and Pollard TD (2001) Regulation of actin filament network formation through Arp2/3 complex: activation by a diverse array of proteins. Annu Rev Biochem 70: 649–676.

    Google Scholar 

  • Insall R, Müller-Taubenberger A, Machesky L, Köhler J, Simmeth E, Atkinson SJ, Weber I and Gerisch G (2001) Dynamics of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis. Cell Mot Cytoskel 50: 115–128.

    Google Scholar 

  • Koster AJ, Grimm R, Typke D, Hegerl R, Stoschek A, Walz J and Baumeister W (1997) Perspectives of molecular and cellular electron tomography. J Struct Biol 120: 276–308.

    Google Scholar 

  • Lee E, Pang K and Knecht DA (2001) The regulation of actin polymerization and cross-linking in Dictyostelium. Biochim Biophys Acta 1525: 217–227.

    Google Scholar 

  • Lee E, Shelden EA and Knecht DA (1998) Formation of F-actin aggregates in cells treated with actin stabilizing drugs. Cell Motil Cytoskel 39: 122–133.

    Google Scholar 

  • Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J and Pollard TD (1994) Puri.cation of a cortical complex containing two unconventional actins from Acanthamoeba by afinity chromatography on pro.lin-agarose. J Cell Biol 127: 107–115.

    Google Scholar 

  • Maniak M, Rauchenberger R, Albrecht R, Murphy J and Gerisch G (1995) Coronin involved in phagocytosis: dynamics of particleinduced relocalization visualized by a green fluorescent protein tag. Cell 83: 915–924.

    Google Scholar 

  • Manstein DJ, Titus MA, DeLozanne A and Spudich JA (1989) Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J 8: 923–932.

    Google Scholar 

  • Medalia O, Weber I, Frangakis AS, Nicastro D, Gerisch G and Baumeister W (2002) Macromolecular architecture visualized in eukaryotic cells by cryo-electron tomography Science 298: 1209–1213.

    Google Scholar 

  • Neujahr R, Albrecht R, Köhler J, Matzner M, Schwartz J-M, Westphal M and Gerisch G (1998) Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells. J Cell Sci 111: 1227–1240.

    Google Scholar 

  • Neujahr R, Heizer C, Albrecht R, Ecke M, Schwartz J-M, Weber I and Gerisch G (1997) Three-dimensional patterns and redistribution of myosin II and actin in mitotic Dictyostelium cells. J Cell Biol 139: 1793–1804.

    Google Scholar 

  • Pang KM, Lee E and Knecht DA (1998) Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures. Curr Biol 8: 405–408.

    Google Scholar 

  • Peracino B, Borleis J, Jin T, Westphal M, Schwartz J-M, Wu L, Bracco E, Gerisch G, Devreotes P and Bozzaro S (1998) G protein β subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton. J Cell Biol 141: 1529–1537.

    Google Scholar 

  • Podolski JL and Steck TL (1990) Length distribution of F-actin in Dictyostelium discoideum. J Biol Chem 265: 1312–1318.

    Google Scholar 

  • Potma EO, de Boeij WP, Bosgraaf L, Roelofs J, van Haastert PJM and Wiersma DA (2001) Reduced protein di.usion rate by cytoskeleton in vegetative and polarized Dictyostelium cells. Biophys J 81: 2010–2019.

    Google Scholar 

  • Rivero F, Dislich H, Glöckner G and Noegel AA (2001) The Dictyostelium discoideum family of Rho-related proteins. Nucl Acids Rese 29: 1068–1079.

    Google Scholar 

  • Salmon NJ, Jonkman JEN and Stelzer EHK (2001) The compact confocal camera: instrument control software built around databases, for applications in molecular biology. In: Proc of the 12th IEEE Internatl Congress on Real Time for Nuclear and Plasma Science.

  • Schafer DA, Welch MD, Machesky LM, Bridgman PC, Meyer SM and Cooper JA (1998) Visualization and molecular analysis of actin assembly in living cells. J Cell Biol 143: 1919–1930.

    Google Scholar 

  • Schindl M, Wallraff E, Deubzer B, Witke W, Gerisch G and Sackmann E (1995) Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy. Biophys J 68: 1177–1190.

    Google Scholar 

  • Spector I, Shochet NR, Blasberg D and Kashman Y (1989) Latrunculins: novel marine macrolides that disrupt micro.lament organization and a.ect cell growth. I. Comparison with cytochalasin D. Cell Motil Cytoskel 13: 127–144.

    Google Scholar 

  • Stossel TP, Condeelis J, Cooley L, Hartwig JH, Noegel A, Schleicher M and Shapiro SS (2001) Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol 2: 138–145.

    Google Scholar 

  • Vicker MG (2002a) F-actin assembly in Dictyostelium cell locomotion and shape oscillations propagates as a self-organized reaction-diffusion wave. FEBS Lett 510: 5–9.

    Google Scholar 

  • Vicker MG (2002b) Eukaryotic cell locomotion depends on the propagation of self-organized reaction-di.usion waves and oscillations of actin filament assembly. Exp Cell Res 275: 54–66.

    Google Scholar 

  • Vicker MG, Xiang W, Plath PJ and Wosniok W (1997) Pseudopodium extension and amoeboid locomotion in Dictyostelium discoideum: possible autowave behaviour of F-actin. Physica D 101: 317–332.

    Google Scholar 

  • Watts DJ and Ashworth JM (1970) Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem J 119: 171–174.

    Google Scholar 

  • Weber I, Gerisch G, Heizer C, Murphy J, Badelt K, Stock A, Schwartz J-M and Faix J (1999) Cytokinesis mediated through the recruitment of cortexillins into the cleavage furrow. EMBO J 18: 586–594.

    Google Scholar 

  • Weber I, Niewöhner J, Du A, Röhrig U and Gerisch G (2002) A talin fragment as an actin trap visualizing actin flow in chemotaxis, endocytosis, and cytokinesis. Cell Motil Cytoskel 53: 136–149.

    Google Scholar 

  • Weber I, Wallra. E, Albrecht R and Gerisch G (1995) Motility and substrate adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis. J Cell Sci 108: 1519–1530.

    Google Scholar 

  • Westphal M, Jungbluth A, Heidecker M, Mühlbauer B, Heizer C, Schwartz J-M, Marriott G and Gerisch G (1997) Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein. Curr Biol 7: 176–183.

    Google Scholar 

  • White JP and Stelzer EHK (1999) Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol 9: 61–65.

    Google Scholar 

  • Wu L, Valkema R, Van Haastert PJM and Devreotes PN (1995) The G protein β subunit is essential for multiple responses to chemoattractants in Dictyostelium. J Cell Biol 129: 1667–1675.

    Google Scholar 

  • Yumura S (2001) Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells. J Cell Biol 154: 137–145.

    Google Scholar 

Download references

Author information

Author notes

  1. Karmela Barisic

    Present address: Department of Medical Biochemistry and Hematology, University of Zagreb, Domagojeva 2, 10000, Zagreb, Croatia

Authors and Affiliations

  1. Max-Planck-Institut für Biochemie, D-82152, Martinsried, Germany

    Till Bretschneider, Jana Köhler, Ohad Medalia, Karmela Barisic, Wolfgang Baumeister & Günther Gerisch

  2. European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117, Heidelberg, Germany

    James Jonkman & Ernst H.K. Stelzer

  3. Rudjer Boskovic Institut, Bijenicka cesta 54, 10000, Zagreb, Croatia

    Igor Weber

Authors
  1. Till Bretschneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James Jonkman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jana Köhler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ohad Medalia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karmela Barisic
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Igor Weber
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ernst H.K. Stelzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wolfgang Baumeister
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Günther Gerisch
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bretschneider, T., Jonkman, J., Köhler, J. et al. Dynamic organization of the actin system in the motile cells of Dictyostelium . J Muscle Res Cell Motil 23, 639–649 (2002). https://doi.org/10.1023/A:1024455023518

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1024455023518

Keywords

Search

Navigation