Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 May 1;108(5):1717–1726. doi: 10.1083/jcb.108.5.1717

Identification of critical functional and regulatory domains in gelsolin

PMCID: PMC2115573  PMID: 2541138

Abstract

Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11- amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin- severing proteins.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Ampe C., Vandekerckhove J. The F-actin capping proteins of Physarum polycephalum: cap42(a) is very similar, if not identical, to fragmin and is structurally and functionally very homologous to gelsolin; cap42(b) is Physarum actin. EMBO J. 1987 Dec 20;6(13):4149–4157. doi: 10.1002/j.1460-2075.1987.tb02761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. André E., Lottspeich F., Schleicher M., Noegel A. Severin, gelsolin, and villin share a homologous sequence in regions presumed to contain F-actin severing domains. J Biol Chem. 1988 Jan 15;263(2):722–727. [PubMed] [Google Scholar]
  3. Ankenbauer T., Kleinschmidt J. A., Vandekerckhove J., Franke W. W. Proteins regulating actin assembly in oogenesis and early embryogenesis of Xenopus laevis: gelsolin is the major cytoplasmic actin-binding protein. J Cell Biol. 1988 Oct;107(4):1489–1498. doi: 10.1083/jcb.107.4.1489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arpin M., Pringault E., Finidori J., Garcia A., Jeltsch J. M., Vandekerckhove J., Louvard D. Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity. J Cell Biol. 1988 Nov;107(5):1759–1766. doi: 10.1083/jcb.107.5.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bazari W. L., Matsudaira P., Wallek M., Smeal T., Jakes R., Ahmed Y. Villin sequence and peptide map identify six homologous domains. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4986–4990. doi: 10.1073/pnas.85.14.4986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bryan J. Gelsolin has three actin-binding sites. J Cell Biol. 1988 May;106(5):1553–1562. doi: 10.1083/jcb.106.5.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bryan J., Hwo S. Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin. J Cell Biol. 1986 Apr;102(4):1439–1446. doi: 10.1083/jcb.102.4.1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chaponnier C., Janmey P. A., Yin H. L. The actin filament-severing domain of plasma gelsolin. J Cell Biol. 1986 Oct;103(4):1473–1481. doi: 10.1083/jcb.103.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chaponnier C., Yin H. L., Stossel T. P. Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways. J Exp Med. 1987 Jan 1;165(1):97–106. doi: 10.1084/jem.165.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glenney J. R., Jr, Bretscher A., Weber K. Calcium control of the intestinal microvillus cytoskeleton: its implications for the regulation of microfilament organizations. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6458–6462. doi: 10.1073/pnas.77.11.6458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  12. Hasegawa T., Takahashi S., Hayashi H., Hatano S. Fragmin: a calcium ion sensitive regulatory factor on the formation of actin filaments. Biochemistry. 1980 Jun 10;19(12):2677–2683. doi: 10.1021/bi00553a021. [DOI] [PubMed] [Google Scholar]
  13. Hinssen H., Vandekerckhove J., Lazarides E. Gelsolin is expressed in early erythroid progenitor cells and negatively regulated during erythropoiesis. J Cell Biol. 1987 Sep;105(3):1425–1433. doi: 10.1083/jcb.105.3.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hwo S., Bryan J. Immuno-identification of Ca2+-induced conformational changes in human gelsolin and brevin. J Cell Biol. 1986 Jan;102(1):227–236. doi: 10.1083/jcb.102.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Janmey P. A., Chaponnier C., Lind S. E., Zaner K. S., Stossel T. P., Yin H. L. Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking. Biochemistry. 1985 Jul 2;24(14):3714–3723. doi: 10.1021/bi00335a046. [DOI] [PubMed] [Google Scholar]
  16. Janmey P. A., Hvidt S., Peetermans J., Lamb J., Ferry J. D., Stossel T. P. Viscoelasticity of F-actin and F-actin/gelsolin complexes. Biochemistry. 1988 Oct 18;27(21):8218–8227. doi: 10.1021/bi00421a035. [DOI] [PubMed] [Google Scholar]
  17. Janmey P. A., Iida K., Yin H. L., Stossel T. P. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem. 1987 Sep 5;262(25):12228–12236. [PubMed] [Google Scholar]
  18. Janmey P. A., Matsudaira P. T. Functional comparison of villin and gelsolin. Effects of Ca2+, KCl, and polyphosphoinositides. J Biol Chem. 1988 Nov 15;263(32):16738–16743. [PubMed] [Google Scholar]
  19. Janmey P. A., Stossel T. P. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature. 1987 Jan 22;325(6102):362–364. doi: 10.1038/325362a0. [DOI] [PubMed] [Google Scholar]
  20. Kilhoffer M. C., Gérard D. Fluorescence study of brevin, the Mr 92 000 actin-capping and -fragmenting protein isolated from serum. Effect of Ca2+ on protein conformation. Biochemistry. 1985 Sep 24;24(20):5653–5660. doi: 10.1021/bi00341a055. [DOI] [PubMed] [Google Scholar]
  21. Kouyama T., Mihashi K. Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem. 1981;114(1):33–38. [PubMed] [Google Scholar]
  22. Kwiatkowski D. J., Janmey P. A., Mole J. E., Yin H. L. Isolation and properties of two actin-binding domains in gelsolin. J Biol Chem. 1985 Dec 5;260(28):15232–15238. [PubMed] [Google Scholar]
  23. Kwiatkowski D. J., Mehl R., Yin H. L. Genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin. J Cell Biol. 1988 Feb;106(2):375–384. doi: 10.1083/jcb.106.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kwiatkowski D. J., Stossel T. P., Orkin S. H., Mole J. E., Colten H. R., Yin H. L. Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature. 1986 Oct 2;323(6087):455–458. doi: 10.1038/323455a0. [DOI] [PubMed] [Google Scholar]
  25. Lind S. E., Janmey P. A., Chaponnier C., Herbert T. J., Stossel T. P. Reversible binding of actin to gelsolin and profilin in human platelet extracts. J Cell Biol. 1987 Aug;105(2):833–842. doi: 10.1083/jcb.105.2.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lodish H. F. Transport of secretory and membrane glycoproteins from the rough endoplasmic reticulum to the Golgi. A rate-limiting step in protein maturation and secretion. J Biol Chem. 1988 Feb 15;263(5):2107–2110. [PubMed] [Google Scholar]
  27. Matsudaira P., Janmey P. Pieces in the actin-severing protein puzzle. Cell. 1988 Jul 15;54(2):139–140. doi: 10.1016/0092-8674(88)90542-9. [DOI] [PubMed] [Google Scholar]
  28. Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. 1987 Oct 29-Nov 4Nature. 329(6142):840–842. doi: 10.1038/329840a0. [DOI] [PubMed] [Google Scholar]
  29. Stossel T. P., Chaponnier C., Ezzell R. M., Hartwig J. H., Janmey P. A., Kwiatkowski D. J., Lind S. E., Smith D. B., Southwick F. S., Yin H. L. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Vandekerckhove J., Weber K. The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation. Differentiation. 1979;14(3):123–133. doi: 10.1111/j.1432-0436.1979.tb01021.x. [DOI] [PubMed] [Google Scholar]
  32. Way M., Weeds A. Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats. J Mol Biol. 1988 Oct 20;203(4):1127–1133. doi: 10.1016/0022-2836(88)90132-5. [DOI] [PubMed] [Google Scholar]
  33. Yamamoto K., Pardee J. D., Reidler J., Stryer L., Spudich J. A. Mechanism of interaction of Dictyostelium severin with actin filaments. J Cell Biol. 1982 Dec;95(3):711–719. doi: 10.1083/jcb.95.3.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yin H. L. Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein. Bioessays. 1987 Oct;7(4):176–179. doi: 10.1002/bies.950070409. [DOI] [PubMed] [Google Scholar]
  35. Yin H. L., Iida K., Janmey P. A. Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments. J Cell Biol. 1988 Mar;106(3):805–812. doi: 10.1083/jcb.106.3.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yin H. L., Kwiatkowski D. J., Mole J. E., Cole F. S. Structure and biosynthesis of cytoplasmic and secreted variants of gelsolin. J Biol Chem. 1984 Apr 25;259(8):5271–5276. [PubMed] [Google Scholar]
  37. Yin H. L., Stossel T. P. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature. 1979 Oct 18;281(5732):583–586. doi: 10.1038/281583a0. [DOI] [PubMed] [Google Scholar]
  38. Yin H. L., Stossel T. P. Purification and structural properties of gelsolin, a Ca2+-activated regulatory protein of macrophages. J Biol Chem. 1980 Oct 10;255(19):9490–9493. [PubMed] [Google Scholar]
  39. Zaner K. S., Hartwig J. H. The effect of filament shortening on the mechanical properties of gel-filtered actin. J Biol Chem. 1988 Apr 5;263(10):4532–4536. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES