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The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development
F. Rivero, B. Koppel, B. Peracino, S. Bozzaro, F. Siegert, C.J. Weijer, M. Schleicher, R. Albrecht, A.A. Noegel
Journal of Cell Science 1996 109: 2679-2691;

Summary

We generated Dictyostelium double mutants lacking the two F-actin crosslinking proteins alpha-actinin and gelation factor by inactivating the corresponding genes via homologous recombination. Here we investigated the consequences of these deficiencies both at the single cell level and at the multicellular stage. We found that loss of both proteins severely affected growth of the mutant cells in shaking suspension, and led to a reduction of cell size from 12 microns in wild-type cells to 9 microns in mutant cells. Moreover the cells did not exhibit the typical polarized morphology of aggregating Dictyostelium cells but had a more rounded cell shape, and also exhibited an increased sensitivity towards osmotic shock and a reduced rate of phagocytosis. Development was heavily impaired and never resulted in the formation of fruiting bodies. Expression of developmentally regulated genes and the final developmental stages that were reached varied, however, with the substrata on which the cells were deposited. On phosphate buffered agar plates the cells were able to form tight aggregates and mounds and to express prespore and prestalk cell specific genes. Under these conditions the cells could perform chemotactic signalling and cell behavior was normal at the onset of multicellular development as revealed by time-lapse video microscopy. Double mutant cells were motile but speed was reduced by approximately 30% as compared to wild type. These changes were reversed by expressing the gelation factor in the mutant cells. We conclude that the actin assemblies that are formed and/or stabilized by both F-actin crosslinking proteins have a protective function during osmotic stress and are essential for proper cell shape and motility.

REFERENCES

    1. Andre E.,
    2. Brink M.,
    3. Gerisch G.,
    4. Isenberg G.,
    5. Noegel A. A.,
    6. Schleicher M.,
    7. Segall J. E. and
    8. Wallraff E.
    (1989). A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein shows normal motility and chemotaxis. J. Cell Biol 108, 985–995
    OpenUrlAbstract/FREE Full Text
    1. Berthold J.,
    2. Stadler J.,
    3. Bozzaro S.,
    4. Fichtner B. and
    5. Gerisch G.
    (1985). Carbohydrate and other epitopes of the contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies. Cell Differ 16, 187–202
    OpenUrlCrossRefPubMedWeb of Science
    1. Bichler G. and
    2. Weijer C. J.
    (1994). A Dictyostelium anterior-like cell mutant reveals sequential steps in the prespore prestalk differentiation pathway. Development 120, 2857–2868
    OpenUrlAbstract
    1. Bozzaro S.,
    2. Merkl R. and
    3. Gerisch G.
    (1987). Cell adhesion: its quantification, assay of the molecules involved and selection of defective mutants in Dictyostelium and Polysphondylium. Meth. Cell Biol 28, 359–385
    OpenUrlCrossRefPubMedWeb of Science
    1. Brink M.,
    2. Gerisch G.,
    3. Isenberg G.,
    4. Noegel A. A.,
    5. Segall J. E.,
    6. Wallraff E. and
    7. Schleicher M.
    (1990). A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor. J. Cell Biol 111, 1477–1489
    OpenUrlAbstract/FREE Full Text
    1. Cantiello H. F.,
    2. Prat A. G.,
    3. Bonventre J. V.,
    4. Cunningham C. C.,
    5. Hartwig J. H. and
    6. Ausiello D. A.
    (1993). Actin-binding protein contributes to cell volume regulatory ion channel activation in melanoma cells. J. Biol. Chem 268, 4596–4599
    OpenUrlAbstract/FREE Full Text
    1. Chowdbury S.,
    2. Smith K. W. and
    3. Gustin M. C.
    (1992). Osmotic stress and the yeast cytoskeleton: phenotype specific suppression of an actin mutation. J. Cell Biol 118, 561–571
    OpenUrlAbstract/FREE Full Text
    1. Claviez M.,
    2. Pagh K.,
    3. Maruta H.,
    4. Baltes W.,
    5. Fisher P. and
    6. Gerisch G.
    (1982). Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin. EMBO J 1, 1017–1022
    OpenUrlPubMedWeb of Science
    1. Condeelis J.,
    2. Hall A.,
    3. Bresnick A.,
    4. Warren V.,
    5. Hock R.,
    6. Bennett H. and
    7. Ogihara S.
    (1988). Actin polymerization and pseudopod extension during amoeboid chemotaxis. Cell Motil. Cytoskel 10, 77–90
    OpenUrlCrossRefPubMedWeb of Science
    1. Cox D.,
    2. Condeelis J.,
    3. Wessels D.,
    4. Soll D.,
    5. Kern H. and
    6. Knecht D. A.
    (1992). Targeted disruption of the ABP-120 gene leads to cells with altered motility. J. Cell Biol 116, 943–955
    OpenUrlAbstract/FREE Full Text
    1. Cox D.,
    2. Wessels D.,
    3. Soll D. R.,
    4. Hartwig J. and
    5. Condeelis J.
    (1996). Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120 Dictyostelium mutants. Mol. Biol. Cell 7, 803–823
    OpenUrlAbstract/FREE Full Text
    1. Cunningham C. C.,
    2. Gorlin J. D.,
    3. Kwiatkowski D. J.,
    4. Hartwig J. H.,
    5. Janmey P. A.,
    6. Byers H. R. and
    7. Stossel T. P.
    (1992). Actin-binding protein requirement for cortical stability and efficient locomotion. Science 255, 325–327
    OpenUrlAbstract/FREE Full Text
    1. Cunningham C. C.
    (1995). Actin polymerization and intracellular solvent flow in cell surface blebbing. J. Cell Biol 129, 1589–1599
    OpenUrlAbstract/FREE Full Text
    1. De Hostos E. L.,
    2. Bradtke B.,
    3. Lottspeich F.,
    4. Guggenheim R. and
    5. Gerisch G.
    (1991). Coronin, an actin-binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G proteinsubunits. EMBO J 10, 4097–4104
    OpenUrlPubMedWeb of Science
    1. De Hostos E. L.,
    2. Rehfueβ C.,
    3. Bradtke B.,
    4. Waddell D. R.,
    5. Albrecht R.,
    6. Murphy J. and
    7. Gerisch G.
    (1993). Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility. J. Cell Biol 120, 163–173
    OpenUrlAbstract/FREE Full Text
    1. De Lozanne A. and
    2. Spudich J. A.
    (1987). Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236, 1086–1091
    OpenUrlAbstract/FREE Full Text
    1. Dingermann T.,
    2. Reindl N.,
    3. Werner H.,
    4. Hildebrandt M.,
    5. Nellen W.,
    6. Harwood A.,
    7. Williams J. and
    8. Nerke K.
    (1989). Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum. Gene 85, 353–362
    OpenUrlCrossRefPubMedWeb of Science
    1. Doolittle K. W.,
    2. Reddy I. and
    3. McNally J. G.
    (1995). 3D analysis of cell movement during normal and myosin-II-null cell morphogenesis in Dictyostelium. Dev. Biol 167, 118–129
    OpenUrlCrossRefPubMedWeb of Science
    1. Early A. E.,
    2. Williams J. G.,
    3. Meyer H. E.,
    4. Por S. B.,
    5. Smith E.,
    6. Williams K. L. and
    7. Gooley A. A.
    (1988). Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA. Mol. Cell. Biol 8, 3458–3466
    OpenUrlAbstract/FREE Full Text
    1. Eichinger L.,
    2. Köppel B.,
    3. Noegel A. A.,
    4. Schleicher M.,
    5. Schliwa M.,
    6. Weijer C. J.,
    7. Witke W. and
    8. Janmey P. A.
    (1996). Mechanical perturbation elicits a phenotypic difference between Dictyostelium wild-type cells and cytoskeletal mutants. Biophys.J 70, 1054–1060
    OpenUrlPubMedWeb of Science
    1. Eliott S.,
    2. Joss G. H.,
    3. Spudich A. and
    4. Williams K. L.
    (1993). Patterns in Dictyostelium discoideum: the role of myosin II in the transition from the unicellular to the multicellular phase. J. Cell Sci 104, 457–466
    OpenUrlAbstract/FREE Full Text
    1. Fisher P. R.,
    2. Merkl R. and
    3. Gerisch G.
    (1989). Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients. J. Cell Biol 108, 973–984
    OpenUrlAbstract/FREE Full Text
    1. Fukui Y.,
    2. Lynch T. J.,
    3. Brzeska H. and
    4. Korn E. D.
    (1989). Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature 341, 328–331
    OpenUrlCrossRefPubMed
    1. Furukawa R. and
    2. Fechheimer M.
    (1994). Differential localization of-actinin and the 30 kD actin-bundling protein in the cleavage furrrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum. Cell Motil. Cytoskel 29, 46–56
    OpenUrlCrossRefPubMed
    1. Gerisch G. and
    2. Keller H. U.
    (1981). Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J. Cell Sci 52, 1–10
    OpenUrlAbstract/FREE Full Text
    1. Hartmann H.,
    2. Noegel A. A.,
    3. Eckerskorn C.,
    4. Rapp S. and
    5. Schleicher M.
    (1989). Ca2+ independent F-actin capping proteins: Cap32/34, a capping protein from Dictyostelium discoideum, does not share sequence homologies with known capping proteins. J. Biol. Chem 264, 12639–12647
    OpenUrlAbstract/FREE Full Text
    1. Haugwitz M.,
    2. Noegel A. A.,
    3. Rieger D.,
    4. Lottspeich F. and
    5. Schleicher M.
    (1991). Dictyostelium discoideum contains two profilin isoforms that differ in structure and function. J. Cell Sci 100, 481–489
    OpenUrlAbstract/FREE Full Text
    1. Haugwitz M.,
    2. Noegel A. A.,
    3. Karakesisoglou J. and
    4. Schleicher M.
    (1994). Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development. Cell 79, 303–314
    OpenUrlCrossRefPubMedWeb of Science
    1. Janssen K.-P.,
    2. Eichinger L.,
    3. Janmey P.,
    4. Noegel A. A.,
    5. Schliwa M.,
    6. Witke W. and
    7. Schleicher M.
    (1996). Viscoelastic properties of F-actin solutions in the presence of normal and mutated actin-binding proteins. Arch. Biochem. Biophys 325, 183–189
    OpenUrlCrossRefPubMedWeb of Science
    1. Jung G. and
    2. Hammer J. A.
    (1990). Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform. J. Cell Biol 110, 1955–1964
    OpenUrlAbstract/FREE Full Text
    1. Knecht D. A.,
    2. Cohen S. M.,
    3. Loomis W. F. and
    4. Lodish H. F.
    (1986). Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors. Mol. Cell. Biol 6, 3973–3983
    OpenUrlAbstract/FREE Full Text
    1. Kuwayama H.,
    2. Ecke M.,
    3. Gerisch G. and
    4. Van Haastert P.
    (1996). Protection against osmotic stress by cGMP mediated myosin phosphorylation. Science 271, 207–209
    OpenUrlAbstract
    1. Kwon H. M. and
    2. Handler J. S.
    (1995). Cell volume regulated transporters of compatible osmolytes. Curr. Opin. Cell Biol 7, 465–471
    OpenUrlCrossRefPubMedWeb of Science
    1. Laemmli U. K.
    (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
    OpenUrlCrossRefPubMedWeb of Science
    1. Luby-Phelps K.
    (1994). Physical properties of cytoplasm. Curr. Opin. Cell Biol 6, 3–9
    OpenUrlCrossRefPubMedWeb of Science
    1. Maniak M.,
    2. Rauchenberger R.,
    3. Albrecht R.,
    4. Murphy J. and
    5. Gerisch G.
    (1995). Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein tag. Cell 83, 915–924
    OpenUrlCrossRefPubMedWeb of Science
    1. Manstein D. J.,
    2. Titus M. A.,
    3. De Lozanne A. and
    4. Spudich J. A.
    (1989). Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J 8, 923–932
    OpenUrlPubMedWeb of Science
    1. Matsudaira P.
    (1991). Modular organization of actin crosslinking proteins. Trends Biochem. Sci 16, 87–92
    OpenUrlCrossRefPubMedWeb of Science
    1. Noegel A. A.,
    2. Harloff C.,
    3. Hirth P.,
    4. Merkl R.,
    5. Modersitzki M.,
    6. Stadler J.,
    7. Weinhart U.,
    8. Westphal M. and
    9. Gerisch G.
    (1985). Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein. EMBO J 4, 3805–3810
    OpenUrlPubMedWeb of Science
    1. Noegel A. A.,
    2. Gerisch G.,
    3. Stadler J. and
    4. Westphal M.
    (1986). Complete sequence and transcript regulation of a cell adhesion protein from aggregating Dictyostelium cells. EMBO J 5, 1473–1476
    OpenUrlPubMedWeb of Science
    1. Noegel A. A.,
    2. Rapp S.,
    3. Lottspeich F.,
    4. Schleicher M. and
    5. Stewart M.
    (1989). The Dictyostelium gelation factor shares a putative actin binding site with-actinins and dystrophin and also has a rod domain containing six 100-residue motifs that appear to have a cross-beta conformation. J. Cell Biol 109, 607–618
    OpenUrlAbstract/FREE Full Text
    1. Pollenz R. S.,
    2. Chen T.-L. L.,
    3. Trivinos-Lagos L. and
    4. Chisholm R. L.
    (1992). The Dictyostelium essential light chain is required for myosin function. Cell 69, 951–962
    OpenUrlCrossRefPubMed
    1. Schleicher M.,
    2. Noegel A. A.,
    3. Schwarz T.,
    4. Wallraff E.,
    5. Brink M.,
    6. Faix J.,
    7. Gerisch G. and
    8. Isenberg G.
    (1988). A Dictyostelium mutant with severe defects in-actinin: Its characterization by cDNA probes and monoclonal antibodies. J. Cell Sci 90, 59–71
    OpenUrlAbstract/FREE Full Text
    1. Schleicher M.,
    2. Andre B.,
    3. Andreoli C.,
    4. Eichinger L.,
    5. Haugwitz M.,
    6. Hofmann A.,
    7. Karakesisoglou J.,
    8. Stöckelhuber M. and
    9. Noegel A. A.
    (1995). Structure/function studies on cytoskeletal proteins in Dictyostelium amoebae as a paradigm. FEBS Lett 369, 38–42
    OpenUrlCrossRefPubMedWeb of Science
    1. Segall J. E.,
    2. Fisher P. R. and
    3. Gerisch G.
    (1987). Selection of chemotaxis mutants of Dictyostelium discoideum. J. Cell Biol 10, 151–161
    OpenUrl
    1. Shelden E. and
    2. Knecht D. A.
    (1995). Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis. J. Cell Sci 108, 1105–1115
    OpenUrlAbstract/FREE Full Text
    1. Siegert F. and
    2. Weijer C.
    (1989). Digital image processing of optical density wave propagation in Dictyostelium discoideum and analysis of the effects of caffeine and ammonia. J. Cell Sci 93, 325–335
    OpenUrlAbstract/FREE Full Text
    1. Siegert F. and
    2. Weijer C. J.
    (1992). Three-dimensional scroll waves organize Dictyostelium slugs. Proc. Nat. Acad. Sci. USA 89, 6433–6437
    OpenUrlAbstract/FREE Full Text
    1. Siegert F. and
    2. Weijer C.
    (1995). Spiral and concentric waves organize multicellular Dictyostelium mounds. Curr. Biol 5, 937–943
    OpenUrlCrossRefPubMedWeb of Science
    1. Simpson P. A.,
    2. Spudich J. A. and
    3. Parham P.
    (1984). Monoclonal antibodies prepared against Dictyostelium actin: characterization and interaction with actin. J. Cell Biol 99, 287–295
    OpenUrlAbstract/FREE Full Text
    1. Springer M. L.,
    2. Patterson B. and
    3. Spudich J. A.
    (1994). Stage specific requirement for myosin II during Dictyostelium development. Development 120, 2651–2660
    OpenUrlAbstract/FREE Full Text
    1. Stossel T. P.
    (1993). On the crawling of animal cells. Science 260, 1086–1094
    OpenUrlAbstract/FREE Full Text
    1. Traynor D.,
    2. Tasaka M.,
    3. Takeuchi I. and
    4. Williams J.
    (1994). Aberrant pattern formation in myosin heavy chain mutants of Dictyostelium. Development 120, 591–601
    OpenUrlAbstract
    1. Vogel G.
    (1987). Endocytosis and recognition mechanisms in Dictyostelium discoideum. Meth. Cell Biol 28, 129–138
    OpenUrlCrossRefPubMed
    1. Wachsstock D. H.,
    2. Schwarz W. H. and
    3. Pollard T. D.
    (1994). Cross-linker dynamics determine the mechanical properties of actin gels. Biophys. J 66, 801–809
    OpenUrlCrossRefPubMedWeb of Science
    1. Wallraff E.,
    2. Schleicher M.,
    3. Modersitzki M.,
    4. Rieger D.,
    5. Isenberg G. and
    6. Gerisch G.
    (1986). Selection of Dictyostelium mutants defective in cytoskeletal proteins: Use of an antibody that binds to the ends of-actinin rods. EMBO J 5, 61–67
    OpenUrlPubMed
    1. Weiner O. H.,
    2. Murphy J.,
    3. Griffiths G.,
    4. Schleicher M. and
    5. Noegel A. A.
    (1993). The actin-binding protein comitin (p24) is a component of the Golgi apparatus. J. CellBiol 123, 23–34
    OpenUrlAbstract/FREE Full Text
    1. Wessels D.,
    2. Soll D. R.,
    3. Knecht D. A.,
    4. Loomis W. F.,
    5. De Lozanne A. and
    6. Spudich J. A.
    (1988). Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain. Dev. Biol 128, 164–177
    OpenUrlCrossRefPubMedWeb of Science
    1. Williams K. L. and
    2. Newell P. C.
    (1976). A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis. Genetics 82, 287–307
    OpenUrlAbstract/FREE Full Text
    1. Witke W.,
    2. Nellen W. and
    3. Noegel A. A.
    (1987). Homologous recombination in the Dictyostelium -actinin gene leads to an altered mRNA and lack of the protein. EMBO J 6, 4143–4148
    OpenUrlPubMedWeb of Science
    1. Witke W.,
    2. Schleicher M. and
    3. Noegel A. A.
    (1992). Redundancy in the microfilament system: Abnormal development of Dictyostelium cells lacking two F-actin crosslinking proteins. Cell 68, 53–62
    OpenUrlCrossRefPubMedWeb of Science
    1. Yumura S.,
    2. Mori H. and
    3. Fukui Y.
    (1984). Localization of actin and myosin for the study of amoeboid movement in Dictyostelium using improved immunofluorescence. J. Cell Biol 99, 894–899
    OpenUrlAbstract/FREE Full Text
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Journal Articles
The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development
F. Rivero, B. Koppel, B. Peracino, S. Bozzaro, F. Siegert, C.J. Weijer, M. Schleicher, R. Albrecht, A.A. Noegel
Journal of Cell Science 1996 109: 2679-2691;
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