Advertisement

Protoplasma

, Volume 194, Issue 3–4, pp 215–230 | Cite as

Covisualization by computation optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

  • J. Scott Gens
  • Christophe Reuzeau
  • Keith W. Doolittle
  • James G. McNally
  • Barbara G. PickardEmail author
Article

Summary

Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal β1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not β1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specificity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105–110 and 115–125 kDa. These bands are again recognized by the visualizationi antibody, which was raised against the extracellular domain of chicken β1 integrin, and are also reconized by an antibody against the intracellular domain of chicken β1 integrin. Because β1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronectin are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesioni sites in animals.

Keywords

Adhesion sites Allium cepa Wide-field computational optical-sectioning microscopy Fibronectin antibodies Integrin Vitronectin antibodies 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Agard DA (1984) Optical sectioning microscopy: cellular architecture in three dimensions. Annu Rev Biophys Bioeng 13: 191–219PubMedGoogle Scholar
  2. Bock E (1991) Cell-cell adhesion molecules. Biochem Soc Trans 19: 1077–1080Google Scholar
  3. Carrington WA, Fogarty KE, Lifschitz L, Fay FS (1989) Three-dimensional imaging on confocal and wide-field microscopes. In: Pawley J (ed) The handbook of biological confocal microscopy. IMR Press, Madison, pp 151–161Google Scholar
  4. Cheresh DA, Mecham RP (1994) Integrins: molecular and biological responses to the extracellular matrix. Academic Press, San DiegoGoogle Scholar
  5. Clark EA, Brugge JS (1995) Integrins and signal transduction pathways: the road taken. Science 268: 233–239PubMedGoogle Scholar
  6. Conchello J-A (1994) Super-resolution and point spread function sensitivity analysis of the expectation-maximization algorithm for computational optical sectioning microscopy. In: Schulz TJ, Snyder DL (eds) Image reconstruction and restoration. Proc Int Soc Opt Eng 2302: 369–378Google Scholar
  7. ——, McNally JG (1996) Fast regularization technique for expectation maximization algorithm for computational optical sectioning microscopy. Proc Int Soc Opt Eng 2655: 199–208Google Scholar
  8. ——, Hansen EW (1990) Enhanced 3-D reconstruction from confocal scanning microscope images. 1: Deterministic and maximum likelihood reconstructions. Appl Opt 29: 3795–3804Google Scholar
  9. ——, Kim JJ, Hansen EW (1994) Enhanced 3-D reconstruction from confocal scanning microscope images. 2: Depth discrimination vs signal-to-noise ratio in partially confocal images. Appl Opt Info Process 33: 3740–3750Google Scholar
  10. Dempster AD, Laird NM, Rubin DB (1977) Maximum likelihood from incomplete data via the EM algorithm. J R Statist Soc B 39: 1–37Google Scholar
  11. Diekmann W, Venis MA, Robinson DG (1995) Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts. Proc Natl Acad Sci USA 92: 3425–3429PubMedGoogle Scholar
  12. Ding JP, Pickard BG (1993a) Mechanosensory calcium-selective cation channels in epidermal cells. Plant J 3: 83–110Google Scholar
  13. ——, Pickard BG (1993b) Modulation of mechanosensitive calciumselective channels by temperature. Plant J 3: 713–720PubMedGoogle Scholar
  14. ——, Badot P-M, Pickard BG (1993a) Aluminum and hydrogen ions inhibit a mechanosensory calcium-selective channel. Aust J Plant Physiol 20: 771–778PubMedGoogle Scholar
  15. - Gens JS, Pont-Lezica RF, McNally JG, Pickard BG (1993b) The plasmalemmal control center model. In: Symposium: Cell wall-plasma membrane interaction. XV International Botanical Congress Abstracts, p 83Google Scholar
  16. Doolittle KW, Reddy I, McNally JG (1995) 3D analysis of cell movement during normal and myosin-II-null cell morphogenesis inDietyostelium. Dev Biol 167: 118–129PubMedGoogle Scholar
  17. Edwards KL, Pickard BG (1987) Detection and transduction of physical stimuli in plants. In: Wagner E, Greppin H, Millet B (eds) The cell surface and signal transduction. Springer, Berlin Heidelberg New York Tokyo, pp 45–66Google Scholar
  18. Felding-Habermann B, Cheresh DA (1993) Vitronectin and its receptors. Curr Opin Cell Biol 5: 864–868PubMedGoogle Scholar
  19. Gens JS, McNally JG, Pickard BG (1993) Resolution of binding sites for antibodies to integrin, vitronectin, and fibronectin on onion epidermis protoplasts and depectinated walls. ASGSB Bull 7: 2Google Scholar
  20. ——, Doolittle KW, McNally JG, Pickard BG (1994) Binding sites for antibodies to animal integrin, vitronectin and fibronectin in a plant model for mechanosensing. Biophys J 66: A169Google Scholar
  21. Green PB (1994) Connecting gene and hormone action to form, pattern and organogenesis: biophysical transductions. J Exp Bot 45: 1775–1788Google Scholar
  22. Grenningloh G, Bieber AJ, Rehm EJ, Snow PM, Traquina ZR, Hortsch M, Patel NH, Goodman CS (1990) Molecular genetics of neuronal recognition inDrosophila: evolution and function of immunoglobulin superfamily cell adhesion molecules. Cold Spring Harbor Symp Quant Biol 55: 327–340PubMedGoogle Scholar
  23. Gumbiner BM (1993) Proteins associated with the cytoplasmic surface of adhesion molecules. Neuron 11: 551–564PubMedGoogle Scholar
  24. He Z-H, Fujiki M, Kohorn BD (1996) A cell wall associated, receptor-like protein kinase. J Biol Chem 271: 19789–19793PubMedGoogle Scholar
  25. Ingber DE, Dike L, Hansen L, Karp S, Liley H, Manictis A, McNamee H, Mooney D, Plopper G, Sims J, Wang N (1994) Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. Int Rev Cytol 150: 173–224PubMedGoogle Scholar
  26. Ito Y, Abe S, Davies E (1992) Co-localization of cytoskeleton proteins and polysomes with a membrane fraction from peas. J Exp Bot 45: 253–259Google Scholar
  27. Hynes RO (1990) Fibronectins. Springer, Berlin Heidelberg New York TokyoGoogle Scholar
  28. —— (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11–25PubMedGoogle Scholar
  29. Joshi S, Miller MI (1993) Maximum a posterior estimation with Good's roughness for three-dimensional optical-sectioning microscopy. J Opt Soc Am A 10: 1078–1085PubMedGoogle Scholar
  30. Kaminskyj SGW, Heath IB (1995) Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth. J Cell Sci 108: 849–856PubMedGoogle Scholar
  31. Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10: 203–209PubMedGoogle Scholar
  32. Laemmli EK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685PubMedGoogle Scholar
  33. Lord EM, Sanders LC (1992) Roles for the extracellular matrix in plant development and pollination: a special case of cell movement in plants. Dev Biol 153: 16–28PubMedGoogle Scholar
  34. McNally JG, Preza C, Conchello J-A, Thomas LJ Jr (1994) Artifacts in computational optical-sectioning microscopy. J Opt Soc Am A 11: 1056–1067Google Scholar
  35. Marcantonio E, Hynes RO (1988) Antibodies to the conserved cytoplasmic domain of the integrin β1 subunit react with proteins in vertebrates, invertebrates, and fungi. J Cell Biol 106: 1765–1772PubMedGoogle Scholar
  36. Oparka KJ, Prior DAM, Crawford JW (1994) Behavior of plasma membrane, cortical ER and plasmodesmata during plasmolysis of onion epidermal cells. Plant Cell Environ 17: 163–171Google Scholar
  37. Pennell RI, Janniche L, Kjellbom P, Scofield GN, Pert JM, Roberts K (1991) Developmental regulation of a plasma membrane arabinogalactan protein epitope in oilseed rape flowers. Plant Cell 3: 1317–1326PubMedGoogle Scholar
  38. Pickard BG (1994) Contemplating the plasmalemmal control center model. Protoplasma 182: 1–9PubMedGoogle Scholar
  39. ——, Ding JP (1993) The mechanosensory calcium-selective ion channel: key component of a plasmalemmal control centre? Aust J Plant Physiol 20: 439–459PubMedGoogle Scholar
  40. ——, McNally JG, Reuzeau C (1995) The endomembrane sheath — a “new” component of the plant cell? ASGSB Bull 9: 29Google Scholar
  41. Pont-Lezica RF, McNally JG, Pickard BG (1993) Wall-to-membrane linkers in onion epidermis: some hypotheses. Plant Cell Environ 16: 111–123Google Scholar
  42. Preissner KT (1991) Structure and biological role of vitronectin. Anon Rev Cell Biol 7: 275–310Google Scholar
  43. Preza C, Ollinger JM, McNally JG, Thomas LJ Jr (1992a) Pointspread sensitivity analysis for computational optical-sectioning microscopy. Micron Microsc Acta 23: 501–513Google Scholar
  44. ——, Miller MI, Thomas LJ Jr, McNally JG (1992b) Regularized linear method for reconstruction of three-dimensional microscopic objects from optical sections. J Opt Soc Am A 9: 219–228PubMedGoogle Scholar
  45. Quatrano RS, Brian L, Aldridge J, Schulz T (1991) Polar axis fixation inFucus zygotes: components of the cytoskeleton and extracellular matrix. Development Suppl 1: 11–16Google Scholar
  46. Reuzeau C, Doolittle KW, McNally JG, Pickard BG (1995a) Injected antibodies against animal vinculin, ankyrin, talin and spectrin form punctate arrays connected by a fine “lacework” in living onion cells. J Cell Biochem Suppl 21A: 465Google Scholar
  47. ——, McNally JG, Pickard BG (1995b) β1 integrin in the endomembrane system of onion epidermal cells is colocalized with spectrin and actin. ASGSB Bull 9: 29Google Scholar
  48. Russ JC (1992) The image processing handbook. CRC Press, Boca RatonGoogle Scholar
  49. Sanders LC, Wang C-S, Walling LL, Lord EM (1991) A homolog of the substrate adhesion molecule vitronectin occurs in four species of flowering plants. Plant Cell 3: 629–635PubMedGoogle Scholar
  50. Schindler M, Meiners S, Cheresh DA (1989) RGD-dependent linkage between plant cell wall and plasma membrane: consequences for growth. J Cell Biol 108: 1955–1965PubMedGoogle Scholar
  51. Schulz M, Janßen M, Knop M, Schnabl H (1994) Stress and age related spots with immunoreactivity to ubiquitin-antibody at protoplast surfaces. Plant Cell Physiol 35: 551–556Google Scholar
  52. Tamkun JW, DeSimone DW, Fonda D, Patel RS, Buck C, Horwitz AF, Hynes RO (1986) Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell 42: 271–282Google Scholar
  53. Tuckwell DS, Weston SA, Humphries MJ (1993) Integrins: a review of their structure and mechanisms of ligand binding. In: Jones G, Wigley C, Warn RS (eds) Cell behavior: adhesion and motility. Company of Biologists, Cambridge, pp 107–136 (Society of Experimental Biology symposium 47)Google Scholar
  54. Wagner VT, Matthysse AG (1992) Involvement of a vitronectin-like protein in attachment ofAgrobacterium tumefaciens to carrot suspension culture cells. J Bacteriol 174: 5999–6003PubMedGoogle Scholar
  55. ——, Brian L, Quatrano RS (1992) Role of a vitronectin-like molecule in embryo adhesion of the brown algaFucus. Proc Natl Acad Sci USA 89: 3644–3648PubMedGoogle Scholar
  56. Wang C-S, Walling LL, Gu YQ, Ware CF, Lord EM (1994) Two classes of proteins and mRNAs inLillium longiflorum L. identified by human vitronectin probes. Plant Physiol 104: 711–717PubMedGoogle Scholar
  57. Wang J-L, Walling LL, Jauh GY, Gu Y-Q, Lord EM (1996) Lily cofactor-independent phosphoglycerate mutase (PGAM-i): purification, partial sequencing, and immunolocalization. Planta (in press)Google Scholar
  58. Wang N, Butler JP, Ingber DE (1993) Mechanotransduction across the cell surface and through the cytoskeleton. Science 260: 1124–1127PubMedGoogle Scholar
  59. Wayne R, Staves MP, Leopold AC (1992) The contribution of the extracellular matrix to gravisensing in characean cells. J Cell Sci 101: 611–623PubMedGoogle Scholar
  60. Wyatt SE, Carpita NC (1993) The plant cytoskeleton-cell-wall continuum. Trends Cell Biol 3: 413–417PubMedGoogle Scholar
  61. Zhang S-D, Kassis J, Olde B, Mellerick DM, Odenwald WF (1996) Pollux, a novelDrosophila adhesion molecule, belongs to a family of proteins expressed in plants, yeast, nematodes, and man. Genes Dev 10: 1108–1119PubMedGoogle Scholar
  62. Zhu J-K, Shi J, Singh U, Wyatt SE, Bressan RA, Hasegawa PM, Carpita NC (1993) Enrichment of vitronectin- and fibronectin-like proteins in NaCl-adapted plant cells and evidence for their involvement in plasma membrane-cell wall adhesion. Plant 13: 637–646Google Scholar
  63. ——, Damsz B, Kononowicz AK, Bressan RA, Hasegawa PM (1994) A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongation factor-1α. Plant Cell 6: 393–404PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • J. Scott Gens
    • 1
  • Christophe Reuzeau
    • 1
  • Keith W. Doolittle
    • 1
  • James G. McNally
    • 1
  • Barbara G. Pickard
    • 1
    Email author
  1. 1.Biology DepartmentWashington UniversitySt. Louis

Personalised recommendations