Skip to main content
SpringerLink
Log in

The 135 kDa actin-bundling protein fromLilium longiflorum pollen is the plant homologue of villin

  • Rapid Communication
  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

Actin microfilaments, which are essential for cell growth and cytoplasmic streaming in pollen tubes, are closely dependent on actin-binding proteins for their organization and regulation. We have purified the plant 135 kDa actin-bundling protein (P-135-ABP) fromLilium longiflorum pollen and determined that its amino acid composition is highly similar to members of the villin-gelsolin family of proteins. We used antibodies against P-135-ABP to probe an expression cDNA library ofL. longiflorum pollen and isolated a full-length clone (ABP135) that corresponds to a 106 kDa polypeptide. The deduced amino acid sequence ofABP135 shows homology with members of the villin-gelsolin family of proteins and contains the characteristic six repeats of this family, as well as an extended carboxy-terminal domain that includes the villin headpiece preceded by a highly variable region. Using two-dimensional polyacrylamide gel electrophoresis we detected at least 5 isoforms of P-135-ABP, with isoelectric points (pI) ranging between 5.6 to 5.9. The most abundant P-135-ABP isoform has a pI of 5.8, closely approximating the pI predicted from the deducedABP135 amino acid sequence. These data, together with the partial amino acid sequence from a proteolytic peptide of the protein, indicate that P-135-ABP is a plant villin. Immuno-detection of Lilium villin in rapidly frozen pollen tubes localized it to actin bundles. Lilium villin is also ubiquitously expressed in all tissues tested. Since villins, like gelsolins, are also Ca2+-dependent severing, capping, and nucleating proteins, Lilium villin may participate in F-actin fragmentation and nucleation in the apex of the pollen tube where there is steep Ca2+ gradient.

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

BMM:

butyl methyl-methacrylate

PPI:

polyphos-phoinositides

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  • Baskin TI, Miller DD, Vos JW, Wilson JE, Hepler PK (1996) Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy. J Microsc 182: 149–161

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Bretscher A, Weber K (1979) Villin: the major microfilament-associated protein of the intestinal microvillus. Proc Natl Acad Sci USA 76: 2321–2325

    PubMed  Google Scholar 

  • Burtnick LD, Koepf EK, Grimes J, Jones EY, Stuart DI, McLaughlin PJ, Robinson RC (1997) The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation. Cell 90: 661–670

    PubMed  Google Scholar 

  • Cai G, Moscatelli A, Cresti M (1997) Cytoskeletal organization and pollen tube growth. Trends Plant Sci 2: 86–91

    Google Scholar 

  • Cant K, Knowles BA, Mahajan-Miklos S, Heintzelman M, Cooley L (1998)Drosophila fascin mutants are rescued by overexpression of the villin-like protein, quail. J Cell Sci 111: 213–221

    PubMed  Google Scholar 

  • Costa de Beauregard MA, Pringault E, Robine S, Louvard D (1995) Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells. EMBO J 14: 409–421

    PubMed  Google Scholar 

  • De Corte V, Gettemans J, De Ville Y, Waelkens E, Vandekerckhove J (1996) Fragmin, a microfilament regulatory protein fromPhysarum polycephalum, is phosphorylated by casein kinase II-type enzymes. Biochemistry 35: 5472–5480

    PubMed  Google Scholar 

  • Doering DS, Matsudaira P (1996) Cysteine scanning mutagenesis at 40 of 76 positions in villin headpiece maps the F-actin binding site and structural features of the domain. Biochemistry 35: 12677–12685

    PubMed  Google Scholar 

  • Franck Z, Footer M, Bretscher A (1990) Microinjection of villin into cultured cells induces rapid and long-lasting changes in cell morphology but does not inhibit cytokinesis, cell motility, or membrane ruffling. J Cell Biol 111: 2475–2485

    PubMed  Google Scholar 

  • Friederich E, Huet C, Arpin M, Louvard D (1989) Villin induces microvilli growth and actin redistribution in transfected fibroblasts. Cell 59: 461–475

    PubMed  Google Scholar 

  • —, Vancompernolle K, Huet C, Goethals M, Finidori J, Vandekerckhove J, Louvard D (1992) An actin-binding site containing a conserved motif of charged amino acid residues is essential for the morphogenic effect of villin. Cell 70: 81–92

    PubMed  Google Scholar 

  • Glenney JR Jr, Geisler N, Kaulfus P, Weber K (1981) Demonstration of at least two different actin-binding sites in villin, a calciumregulated modulator of F-actin organization. J Biol Chem 256: 8156–8161

    PubMed  Google Scholar 

  • Hesterkamp T, Weeds AG, Mannherz HG (1993) The actin monomers in the ternary gelsolin: 2 actin complex are in an antiparallel orientation. Eur J Biochem 218: 507–513

    PubMed  Google Scholar 

  • Hofmann A, Noegel AA, Bomblies L, Lottspeich F, Schleicher M (1993) The 100 kDa F-actin capping protein ofDictyostelium amoebae is a villin prototype (‘protovillin’). FEBS Lett 328: 71–76

    PubMed  Google Scholar 

  • Kim SR, Kim Y, An G (1993) Molecular cloning and characterization of anther-preferential cDNA encoding a putative actindepolymerizing factor. Plant Mol Biol 21: 39–45

    PubMed  Google Scholar 

  • Kinkema M, Wang H, Schiefelbein J (1994) Molecular analysis of the myosin gene family inArabidopsis thaliana. Plant Mol Biol 26: 1139–1153

    PubMed  Google Scholar 

  • Kohno T, Shimmen T (1987) Ca2+-induced fragmentation of actin filaments in pollen tubes. Protoplasma 141: 177–179

    Google Scholar 

  • Kwiatkowski DJ, Janmey PA, Yin HL (1989) Identification of critical functional and regulatory domains in gelsolin. J Cell Biol 108: 1717–1726

    PubMed  Google Scholar 

  • Lancelle SA, Hepler PK (1992) Ultrastructure of freeze-substituted pollen tubes ofLilium longiflorum. Protoplasma 167: 215–230

    Google Scholar 

  • Lopez I, Anthony RG, Maciver SK, Jiang CJ, Khan S, Weeds AG, Hussey PJ (1996) Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins. Proc Natl Acad Sci USA 93: 7415–7420

    PubMed  Google Scholar 

  • Luck A, D'Haese J, Hinssen H (1995) A gelsolin-related protein from lobster muscle: cloning, sequence analysis and expression. Biochem J 305: 767–775

    PubMed  Google Scholar 

  • Markus MA, Matsudaira P, Wagner G (1997) Refined structure of villin 14T and a detailed comparison with other actin-severing domains. Protein Sci 6: 1197–1209

    PubMed  Google Scholar 

  • McCurdy DW, Kim M (1998) Molecular cloning of a novel fimbrinlike cDNA fromArabidopsis thaliana. Plant Mol Biol 36: 23–31

    PubMed  Google Scholar 

  • McKnight CJ, Matsudaira PT, Kim PS (1997) NMR structure of the 35-residue villin headpiece subdomain. Nat Struct Biol 4: 180–184

    PubMed  Google Scholar 

  • McLaughlin PJ, Gooch JT, Mannherz HG, Weeds AG (1993) Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature 364: 685–692

    PubMed  Google Scholar 

  • Messerli M, Robinson KR (1997) Tip localized Ca2+ pulses are coincident with peak pulsatile growth rates in pollen tubes ofLilium longiflorum. J Cell Sci 110: 1269–1278

    PubMed  Google Scholar 

  • Miller DD, Lancelle SA, Hepler PK (1996) Actin microfilaments do not form a dense meshwork inLilium longiflorum pollen tube tips. Protoplasma 195: 123–132

    Google Scholar 

  • Northrop J, Weber A, Mooseker MS, Franzini-Armstrong C, Bishop MF, Dubyak GR, Tucker M, Walsh TP (1986) Different calcium dependence of the capping and cutting activities of villin. J Biol Chem 261: 9274–9281

    PubMed  Google Scholar 

  • Pinson KI, Dunbar L, Samuelson L, Gumucio DL (1998) Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli. Dev Dyn 211: 109–121

    PubMed  Google Scholar 

  • Pollard TD, Cooper JA (1986) Actin and actin-binding proteins: a critical evaluation of mechanisms and functions. Annu Rev Biochem 55: 987–1035

    PubMed  Google Scholar 

  • Schnuchel A, Wiltscheck R, Eichinger L, Schleicher M, Holak TA (1995) Structure of severin domain 2 in solution. J Mol Biol 247: 21–27

    PubMed  Google Scholar 

  • Sun HQ, Wooten DC, Janmey PA, Yin HL (1994) The actin sidebinding domain of gelsolin also caps actin filaments: implications for actin filament severing. J Biol Chem 269: 9473–9479

    PubMed  Google Scholar 

  • Taylor LP, Hepler PK (1997) Pollen germination and tube growth. Annu Rev Plant Physiol Plant Mol Biol 48: 461–491

    PubMed  Google Scholar 

  • T'Jampens D, Meerschaert K, Constantin B, Bailey J, Cook LJ, De Corte V, De Mol H, Goethals M, Van Damme J, Vandekerckhove J, Gettemans J (1997) Molecular cloning, over-expression, developmental regulation and immunolocalization of fragminP, a gelsolin-related actin-binding protein fromPhysarum polycephalum plasmodia. J Cell Sci 110: 1215–1226

    PubMed  Google Scholar 

  • Valenta R, Duchene M, Pettenburger K, Sillaber C, Valent P, Bettelheim P, Breitenbach M, Rumpold H, Kraft D, Scheiner O (1991) Identification of profilin as a novel pollen allergen: IgE autoreactivity in sensitized individuals. Science 253: 557–560

    PubMed  Google Scholar 

  • Vidali L, Hepler PK (1997) Characterization and localization of profilin in pollen grains and tubes ofLilium longiflorum. Cell Motil Cytoskeleton 36: 323–338

    PubMed  Google Scholar 

  • —, Perez HE, Lopez VV, Noguez R, Zamudio F, Sanchez F (1995) Purification, characterization, and cDNA cloning of profilin fromPhaseolus vulgaris. Plant Physiol 108: 115–123

    PubMed  Google Scholar 

  • Wilkins MR, Williams KL (1997) Cross-species protein identification using amino acid composition, peptide mass fingerprinting, isoelectric point and molecular mass: a theoretical evaluation. J Theor Biol 186: 7–15

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Yokota E, McDonald AR, Liu B, Shimmen T, Palevitz BA (1995) Localization of a 170 kDa myosin heavy chain in plant cells. Protoplasma 185: 178–187

    Google Scholar 

  • —, Takahara K, Shimmen T (1998) Actin-bundling protein isolated from pollen tubes of lily: biochemical and immunocytochemical characterization. Plant Physiol 116: 1421–1429

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biology Department, University of Massachusetts, 01003, Amherst, MA, USA

    L. Vidali & P. K. Hepler

  2. Biochemistry and Molecular Biology Department, University of Massachusetts, Amherst

    A. Y. Cheung

  3. Molecular and Cellular Biology Program, University of Massachusetts, Amherst

    L. Vidali, A. Y. Cheung & P. K. Hepler

  4. Plant Biology Program, University of Massachusetts, Amherst

    A. Y. Cheung & P. K. Hepler

  5. Himeji Institute of Technology, Horima Science Park City, Hyogo

    E. Yokota & T. Shimmen

Authors
  1. L. Vidali
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Yokota
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Y. Cheung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T. Shimmen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P. K. Hepler
    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

Vidali, L., Yokota, E., Cheung, A.Y. et al. The 135 kDa actin-bundling protein fromLilium longiflorum pollen is the plant homologue of villin. Protoplasma 209, 283–291 (1999). https://doi.org/10.1007/BF01453456

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01453456

Keywords

Search

Navigation