Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1996 May;143(1):249–258. doi: 10.1093/genetics/143.1.249

Single Amino Acid Mutations in Drosophila Fascin Disrupt Actin Bundling Function in Vivo

K Cant 1, L Cooley 1
PMCID: PMC1207258  PMID: 8722779

Abstract

Fascins bundle actin filaments into large, tightly packed hexagonal arrays that support diverse cellular processes including microvillar projections and filopodial extensions. In Drosophila, fascin is encoded by the singed locus. Severe singed mutants have gnarled bristles and are female sterile due to a defect in rapid cytoplasm transport during oogenesis. In this paper, we report the results of a large EMS mutagenesis screen to generate new singed alleles. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of fascin in vivo, singed(G409E) mutants have kinked bristles and are fertile with a mild nurse cell cytoplasm transport defect. This mutation is in a small conserved domain near the C-terminus of fascin. A mutation that changes serine 289 to asparagine almost completely inactivates fascin in vivo, singed(S289N) mutants have gnarled bristles and are sterile due to a severe defect in nurse cell cytoplasm transport caused by the absence of nurse cell cytoplasmic actin bundles. A subsequent EMS mutagenesis screen for dominant suppressors of singed(S289N) sterility revealed an intragenic suppressor mutation that changes serine 251 to phenylalanine and restores much of fascin's function. These two mutations, S289N and S251F, draw attention to a central domain in fascin.

Full Text

The Full Text of this article is available as a PDF (5.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams A. E., Botstein D. Dominant suppressors of yeast actin mutations that are reciprocally suppressed. Genetics. 1989 Apr;121(4):675–683. doi: 10.1093/genetics/121.4.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams A. E., Botstein D., Drubin D. G. A yeast actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation. Science. 1989 Jan 13;243(4888):231–233. doi: 10.1126/science.2643162. [DOI] [PubMed] [Google Scholar]
  3. Adams A. E., Botstein D., Drubin D. G. Requirement of yeast fimbrin for actin organization and morphogenesis in vivo. Nature. 1991 Dec 5;354(6352):404–408. doi: 10.1038/354404a0. [DOI] [PubMed] [Google Scholar]
  4. Adams J. C. Formation of stable microspikes containing actin and the 55 kDa actin bundling protein, fascin, is a consequence of cell adhesion to thrombospondin-1: implications for the anti-adhesive activities of thrombospondin-1. J Cell Sci. 1995 May;108(Pt 5):1977–1990. doi: 10.1242/jcs.108.5.1977. [DOI] [PubMed] [Google Scholar]
  5. Appel L. F., Prout M., Abu-Shumays R., Hammonds A., Garbe J. C., Fristrom D., Fristrom J. The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4937–4941. doi: 10.1073/pnas.90.11.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bender H A. Studies on the Expression of Various Singed Alleles in Drosophila Melanogaster. Genetics. 1960 Jul;45(7):867–883. doi: 10.1093/genetics/45.7.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bryan J., Edwards R., Matsudaira P., Otto J., Wulfkuhle J. Fascin, an echinoid actin-bundling protein, is a homolog of the Drosophila singed gene product. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9115–9119. doi: 10.1073/pnas.90.19.9115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bryan J., Kane R. E. Separation and interaction of the major components of sea urchin actin gel. J Mol Biol. 1978 Oct 25;125(2):207–224. doi: 10.1016/0022-2836(78)90345-5. [DOI] [PubMed] [Google Scholar]
  9. Cant K., Knowles B. A., Mooseker M. S., Cooley L. Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension. J Cell Biol. 1994 Apr;125(2):369–380. doi: 10.1083/jcb.125.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooley L., Verheyen E., Ayers K. chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis. Cell. 1992 Apr 3;69(1):173–184. doi: 10.1016/0092-8674(92)90128-y. [DOI] [PubMed] [Google Scholar]
  12. DeRosier D. J., Censullo R. Structure of F-actin needles from extracts of sea urchin oocytes. J Mol Biol. 1981 Feb 15;146(1):77–99. doi: 10.1016/0022-2836(81)90367-3. [DOI] [PubMed] [Google Scholar]
  13. Drubin D. G., Miller K. G., Botstein D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J Cell Biol. 1988 Dec;107(6 Pt 2):2551–2561. doi: 10.1083/jcb.107.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edwards R. A., Bryan J. Fascins, a family of actin bundling proteins. Cell Motil Cytoskeleton. 1995;32(1):1–9. doi: 10.1002/cm.970320102. [DOI] [PubMed] [Google Scholar]
  15. Edwards R. A., Herrera-Sosa H., Otto J., Bryan J. Cloning and expression of a murine fascin homolog from mouse brain. J Biol Chem. 1995 May 5;270(18):10764–10770. doi: 10.1074/jbc.270.18.10764. [DOI] [PubMed] [Google Scholar]
  16. Gans M., Audit C., Masson M. Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster. Genetics. 1975 Dec;81(4):683–704. doi: 10.1093/genetics/81.4.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gutzeit H. O. The role of microfilaments in cytoplasmic streaming in Drosophila follicles. J Cell Sci. 1986 Feb;80:159–169. doi: 10.1242/jcs.80.1.159. [DOI] [PubMed] [Google Scholar]
  18. Heberlein U., Penton A., Falsafi S., Hackett D., Rubin G. M. The C-terminus of the homeodomain is required for functional specificity of the Drosophila rough gene. Mech Dev. 1994 Oct;48(1):35–49. doi: 10.1016/0925-4773(94)90004-3. [DOI] [PubMed] [Google Scholar]
  19. Holthuis J. C., Schoonderwoert V. T., Martens G. J. A vertebrate homolog of the actin-bundling protein fascin. Biochim Biophys Acta. 1994 Sep 13;1219(1):184–188. doi: 10.1016/0167-4781(94)90267-4. [DOI] [PubMed] [Google Scholar]
  20. Honts J. E., Sandrock T. S., Brower S. M., O'Dell J. L., Adams A. E. Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin. J Cell Biol. 1994 Jul;126(2):413–422. doi: 10.1083/jcb.126.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hoover K. K., Chien A. J., Corces V. G. Effects of transposable elements on the expression of the forked gene of Drosophila melanogaster. Genetics. 1993 Oct;135(2):507–526. doi: 10.1093/genetics/135.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kane R. E. Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts. J Cell Biol. 1976 Dec;71(3):704–714. doi: 10.1083/jcb.71.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Komitopoulou K., Gans M., Margaritis L. H., Kafatos F. C., Masson M. Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER with Special Attention to Eggshell Mutants. Genetics. 1983 Dec;105(4):897–920. doi: 10.1093/genetics/105.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mahajan-Miklos S., Cooley L. The villin-like protein encoded by the Drosophila quail gene is required for actin bundle assembly during oogenesis. Cell. 1994 Jul 29;78(2):291–301. doi: 10.1016/0092-8674(94)90298-4. [DOI] [PubMed] [Google Scholar]
  25. Mosialos G., Yamashiro S., Baughman R. W., Matsudaira P., Vara L., Matsumura F., Kieff E., Birkenbach M. Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein. J Virol. 1994 Nov;68(11):7320–7328. doi: 10.1128/jvi.68.11.7320-7328.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Otto J. J., Kane R. E., Bryan J. Formation of filopodia in coelomocytes: localization of fascin, a 58,000 dalton actin cross-linking protein. Cell. 1979 Jun;17(2):285–293. doi: 10.1016/0092-8674(79)90154-5. [DOI] [PubMed] [Google Scholar]
  27. Otto J. J., Kane R. E., Bryan J. Redistribution of actin and fascin in sea urchin eggs after fertilization. Cell Motil. 1980;1(1):31–40. doi: 10.1002/cm.970010104. [DOI] [PubMed] [Google Scholar]
  28. Overton J. The fine structure of developing bristles in wild type and mutant Drosophila melanogaster. J Morphol. 1967 Aug;122(4):367–379. doi: 10.1002/jmor.1051220406. [DOI] [PubMed] [Google Scholar]
  29. Paterson J., O'Hare K. Structure and transcription of the singed locus of Drosophila melanogaster. Genetics. 1991 Dec;129(4):1073–1084. doi: 10.1093/genetics/129.4.1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Petersen N. S., Lankenau D. H., Mitchell H. K., Young P., Corces V. G. forked proteins are components of fiber bundles present in developing bristles of Drosophila melanogaster. Genetics. 1994 Jan;136(1):173–182. doi: 10.1093/genetics/136.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Qian S., Zhang J. Y., Kay M. A., Jacobs-Lorena M. Structural analysis of the Drosophila rpA1 gene, a member of the eucaryotic 'A' type ribosomal protein family. Nucleic Acids Res. 1987 Feb 11;15(3):987–1003. doi: 10.1093/nar/15.3.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spudich J. A., Amos L. A. Structure of actin filament bundles from microvilli of sea urchin eggs. J Mol Biol. 1979 Apr 5;129(2):319–331. doi: 10.1016/0022-2836(79)90285-7. [DOI] [PubMed] [Google Scholar]
  33. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wheatley S., Kulkarni S., Karess R. Drosophila nonmuscle myosin II is required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos. Development. 1995 Jun;121(6):1937–1946. doi: 10.1242/dev.121.6.1937. [DOI] [PubMed] [Google Scholar]
  35. Xue F., Cooley L. kelch encodes a component of intercellular bridges in Drosophila egg chambers. Cell. 1993 Mar 12;72(5):681–693. doi: 10.1016/0092-8674(93)90397-9. [DOI] [PubMed] [Google Scholar]
  36. Yamashiro-Matsumura S., Matsumura F. Intracellular localization of the 55-kD actin-bundling protein in cultured cells: spatial relationships with actin, alpha-actinin, tropomyosin, and fimbrin. J Cell Biol. 1986 Aug;103(2):631–640. doi: 10.1083/jcb.103.2.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yamashiro-Matsumura S., Matsumura F. Purification and characterization of an F-actin-bundling 55-kilodalton protein from HeLa cells. J Biol Chem. 1985 Apr 25;260(8):5087–5097. [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES