Skip to main content
SpringerLink
Log in

Molecular cloning and functional expression of chromaffin cell scinderin indicates that it belongs to the family of Ca2+-dependent F-actin severing proteins

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Scienderin is a Ca+-dependent actin filament severing protein present in chromaffin cells, platelets and a variety of secretory cells. It has been suggested that scinderin is involved in chromaffin cell F-actin dynamics and that this actin network controls the delivery of secretory vesicles to plasma membrane exocytotic sites. Moreover, scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated cells. Here we describe the molecular cloning, the nucleotide sequence and the expression of bovine chromaffin cell scinderin cDNA. The fusion protein obtained cross-reacts with native scinderin antibodies and binds phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PIP2) and actin in a Ca+-dependent manner. Antibodies raised against the fusion protein produced the same cellular staining patterns for scinderin as anti-native scinderin. Nucleotide and amino acid sequence analysis indicate that scinderin has six domains each containing three internal sequence motifs, two actin and two PIP2 binding sites and has 63 and 53% homology with gelsolin and villin. These data indicate that scinderin is a novel member of the family of Ca2+-dependent F-actin severing proteins which includes gelsolin and villin.

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

Abbreviations

PIP2 :

phosphatidylinositol 4,5 bisphosphate

PKC:

protein kinase C

Sc:

scinderin

PS:

phosphatidyl serine

F-Sc:

scinderin fusion protein

PCR:

polymerase chain reaction

References

  1. Lee RWH, Trifaró J-M: Characterization of anti-actin antibodies and their use in immunocytochemical studies on the localisation of actin in adrenal chromaffin cells in culture. Neuroscience 6: 2087–2108, 1981

    Google Scholar 

  2. Trifaró J-M, Kenigsberg RL, Coté A, Lee RWH, Hikita T: Adrenal paraneuron contractile proteins and stimulus-secretion coupling. Can J Physiol Pharmacol 62: 493–501, 1984

    Google Scholar 

  3. Cheek TR, Burgoyne RD: Nicotine-evoked disassembly of cortical actin filaments in adrenal chromaffin cells. FEBS Lett 207: 110–114, 1986

    Google Scholar 

  4. Trifaró J-M, Lee RWH, Kenigsberg RL, Coté A: Contractile proteins and chromaffin cell function. In: Synthesis Storage and Secretion of Adrenal Catacholamines. Adv Biosci 36: 151–158, 1982

  5. Trifaró J-M, Novas ML, Fournier S, Rodríguez Del Castillo A: Cellular and molecular mechanisms in hormone and neurotransmitter secretion. In: M. Velasco, A. Isreal, E. Romero and H. Silva (eds). Recent Advances in Pharmacology and Therapeutics. Elsevier, Amsterdam, 1989, pp 15–20

    Google Scholar 

  6. Burgoyne RD, Cheek TR: Reorganisation of peripheral actin filaments as a prelude of exocytosis. Biosci Rep 7: 281–288, 1987

    Google Scholar 

  7. Burgoyne RD: Control of exocytosis in adrenal chromaffin cells. Biochim Biophys Acta 1071: 174–202, 1991

    Google Scholar 

  8. Friedman JE, Lelkes PI, Rosenheck K, Oplatka A: The possible implication of membrane associated actin in stimulus secretion in adrenal chromaffin cells. Biochem Biophys Res Commun 96: 1717–1723, 1980

    Google Scholar 

  9. Lelkes PI, Friedman JE, Rosenheck K, Oplatka A: Destabilization of actin filaments as a requirement for the secretion of catecholamines from permeabillized chromaffin cells. FEBS Lett 208: 357–363, 1986

    Google Scholar 

  10. Sontag J-M, Aunis D, Bader M-F: Peripheral actin filaments control calcium-mediated catecholamine release from streptolysis O-permeabilized chromaffin cell. Eur J Cell Biol 46: 316–326, 1988

    Google Scholar 

  11. Vitale ML, Rodríguez Del Castillo A, Trifaró J-M: Protein kinase C activation by phorbol esters induces chromaffin cell cortical filamentous actin disassembly and increases the intitial rate of exocytosis in response to nicotinic receptor stimulation. Neuroscience 51: 463–474, 1992

    Google Scholar 

  12. Burgoyne RD, Morgan A, O'Sullivan AJ: The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C. Cell Signalling 1: 323–334, 1989

    Google Scholar 

  13. Marxen P, Bigalke H: Tetanus and botulinum A toxins inhibit stimulated F-actin rearrangement in chromaffin cells. NeuroReport 2: 33–36, 1991

    Google Scholar 

  14. Vitale ML, Rodríguez Del castillo A, Tchakarov L, Trifaró J-M: Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin. J Cell Biol 113: 1057–1067. 1991

    Google Scholar 

  15. Trifaró J-M, Bader M-F, Doucet J-P: Chromaffin cell cytoskeleton: its possible role in secretion. Can J Biochem Cell Biol 63: 661–669, 1985

    Google Scholar 

  16. Bader M-F, Trifaró J-M, Langley OK, Thiersé D, Aunis D: Secretory cell actin-binding proteins identification of gelsolin-like protein in chromaffin cells. J Cell Biol 102: 636–646, 1986

    Google Scholar 

  17. Rodríguez Del Castillo A, Lemaire S, Tchakarov L, Jeyapragasan M, Doucet JP, Vitale ML, Trifaró J-M: Chromaffin cell scinderin: a novel calcium-dependent actin filament severing protein. EMBO J 9: 43–52, 1990

    Google Scholar 

  18. Yin H, Stossel TP: Control of cytomplasmic actin gel-sol transformation by gelsolin, a calcium dependent regulatory protein. Nature 281: 583–586, 1979

    Google Scholar 

  19. Yin H, Albrecht JH, Fattoum A: Identification of gelsolin, a calcium-dependent regulatory protein of actin gel-sol transformation and its intracellular distribution in a variety of cells and tissues. J Cell Biol 91: 901–906, 1981

    Google Scholar 

  20. Stossel TP, Chaponnier C, Ezzel R, Hartwig JH, Janmey PA, Kwiatowski DJ, Lind SE, Smith DB, Southwick FS, Yin HL: Non-muscle actin binding proteins. Ann Rev Cell Biol 1: 353–402, 1985

    Google Scholar 

  21. Tchakarov L, Vitale ML, Jeyapragasan M, Rodríguez Del Castillo A, Trifaró J-M: Expression of scinderin, an actin filament-severing protein, in different tissues. FEBS Lett 268: 209–212, 1990

    Google Scholar 

  22. Rodríguez Dell Castillo A, Vitale ML, Tchakarov L, Trifaró J-M: Human platelets contain scinderin, a calcium dependent actin filamentsevering protein. Thromb Haemost 67: 248–251, 1992

    Google Scholar 

  23. Rodríguez Del Castillo A, Vitale ML, Trifaró J-M: Ca2+ and pH determine the interation of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation. J Cell Biol 119: 797–810, 1992

    Google Scholar 

  24. Yin HL, Iida K, Janmey PA: Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments. J Cell Biol 106: 805–812, 1988

    Google Scholar 

  25. Maekawa S, Sakai H: Inhibition of actin regulatory activity of the 74-kDa protein from bovine adrenal medulla (Adseverin) by some phospholipds. J Biol Chem 265: 10940–10942, 1990

    Google Scholar 

  26. Trifaró J-M, Rodríguez Del Castillo A, Vitale ML: Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis. Mol Neurobiol 6: 339–358, 1992

    Google Scholar 

  27. Marcu MG, Rodríguez Del Castillo A, Trifaró J-M: Molecular cloning of bovine chromaffin cell scinderin (Sc) cDNA reveals acting and polyphosphoinositide (PPI) binding domains. XII Int. Cong. Pharmacol: Can J Physiol Pharmacol 72: 246, 1994

    Google Scholar 

  28. Trifaró J-M, Lee RWH: Morphological characteristics and stimultion-secretion coupling in bovine adrenal chromaffin cell cultures. Neuroscience 5: 1533–1546, 1980

    Google Scholar 

  29. Birnboim HC: Rapid extractionof high molecular weight RNA from cultured cells and granulocytes for Northern analysis. Nucleic Acid Res 16: 1487–1497, 1988

    Google Scholar 

  30. Aviv H, Leder P: Purification of biologically active globin messenger RNA by chromatography on oligothmidylic acid-cellulose. Proc Natl Acad Sci 69: 1408–1412, 1972

    Google Scholar 

  31. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York, 1989

    Google Scholar 

  32. Sanger F, Nicklen S, Coulson A: A DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467, 1977

    Google Scholar 

  33. Frohman M, Dush M, Martin G: Rapid production of full length cDNAs from rare transcripts: amplification using a single gene specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002, 1988

    Google Scholar 

  34. Doucet J-P, Trifaró J-M: A discontinuous and highly porous sodium dodecyl sulphate-polyacrylamide slab gel system of high resolution. Anal Biochem 168: 265–271, 1988

    Google Scholar 

  35. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from acrylamide gels to nitrocellulose sheets procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979

    Google Scholar 

  36. Henvick RM, Hunkapiller MW, Hood LE, Dreyere WJ: A gas-liquid solid phase peptide and protein sequenator. J Biol Chem 256: 7990–7997, 1981

    Google Scholar 

  37. Smith DB, Johnson KS: Single-step purification of polypeptides expressed inE. coli as fusions with glutathione S-transferase. Gene 67: 31–40, 1988

    Google Scholar 

  38. Caldwell PC: Calcium chelation and buffers. In: A. W. Cuthbert (ed). Calcium and cellular function. McMillan and Company Limited, London, 1970, pp 10–16

    Google Scholar 

  39. McLaughlin PJ, Gooch JT, Mannherz H-G, Weeds AG: Structure of gelsolin segement I-actin complex and the mechanism of filament severing. Nature 364: 685–692, 1993

    Google Scholar 

  40. Rhee SG, Suh PG, Ryu SH, Sang YL: Studies of inositol phospholipid-specific Phospholipase C. Science 244: 546–550, 1989

    Google Scholar 

  41. Yu F-X, Sun H-Q, Janmey PA, Yin HL: Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin. J Biol Chem 267, 21: 14616–14621, 1992

    Google Scholar 

  42. 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 203: 1127–1133, 1988

    Google Scholar 

  43. Trifaró J-M, Vitale ML: Cytoskeleton dynamics during neurotransmitter release. TINS 16, 11: 466–471, 1993

    Google Scholar 

  44. Trifaró J-M, Vitale ML, Rodríguez Del Castillo A: Scinderin and chromaffin cell actin network dynamics during neurotransmitter release. J Physiol 87:89–106, 1993

    Google Scholar 

  45. Andre 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 263: 722–728, 1988

    Google Scholar 

  46. Ampe C, Vandekerchove 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 Phisarum actin. EMBO J 6: 4149–4157, 1987

    Google Scholar 

  47. Way M, Pope B, Weeds AG: Evidence for functional homology in the F-actin binding domains of gelsolin and alfa actinin: implications for the requirements of severing and capping. J Cell Biol 119: 835–842, 1992

    Google Scholar 

  48. Pope B, Way M, Weeds AG: Two of the three actin binding domains of gelsolin bind to the same subdomain of actin. FEBS Lett 280: 70–74, 1991

    Google Scholar 

  49. Huber R, Schneider M, Mayr J, Römisch J, Paques EP: The calcium binding sites in human annexin V by crystal structure analysis at 2.0 Å resolution. FEBS Lett 275: 15–24, 1990

    Google Scholar 

  50. Chaponnier C, Janmey P, Yin H: The actin filament severing domain of plasma gelsolin. J Cell Biol 103:1473–1481, 1986

    Google Scholar 

  51. Bryan J: Gelsolin has 3 actin binding sites. J Cell Biol 106: 1553–1562

  52. Lassing, I., and U Lindberg 1985. Specific interation between phosphatidylinositol 4,5 biphosphate and profilactin. Nature 314: 472–474, 1988

    Google Scholar 

  53. Janmey PA, Stossell TP: Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature 325: 362–364, 1987

    Google Scholar 

  54. Yin HL, Janmey PA, Schleicher M: Severin is a gelsolin prototype. FEBS Lett 264: 78–80, 1990

    Google Scholar 

  55. Isenberg G: Actin binding proteins-lipid interactions. J Muscle Res Cell Motil 12: 136–144, 1991

    Google Scholar 

  56. Janmey PA, Lamb J, Allen PG, Matsudaira PT: Phosphoinositide binding peptides derived from the sequences of gelsolin and villin. J Biol Chem 267: 11818–11823, 1992

    Google Scholar 

  57. Kwiatowski DJ, Stossel TP, Orkin SH, Mole JE, Colten HR, Yin HL: Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin binding domain. Nature 323: 455–458, 1986

    Google Scholar 

  58. Bazari WL, Matsudaira P, Wallek M, Smeal T, Jakes R, Ahmed Y: Villin sequence and peptide map identify six homologous domains. Proc Natl Acad Sci USA 85: 4986–4990, 1988

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Secretory Process Research Program, Department of Pharmacology, University of Ottawa, 451 Smyth Road, K1H 8M5, Ottawa, Ontario, Canada

    M. G. Marcu, A. Rodríguez Del Castillo, M. L. Vitale & J. -M. Trifaró

Authors
  1. M. G. Marcu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Rodríguez Del Castillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. L. Vitale
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. -M. Trifaró
    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

Marcu, M.G., Rodríguez Del Castillo, A., Vitale, M.L. et al. Molecular cloning and functional expression of chromaffin cell scinderin indicates that it belongs to the family of Ca2+-dependent F-actin severing proteins. Mol Cell Biochem 141, 153–165 (1994). https://doi.org/10.1007/BF00926179

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00926179

Key words

Search

Navigation