, Volume 199, Issue 3–4, pp 173–197 | Cite as

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

  • Christophe Reuzeau
  • Keith W. Doolittle
  • James G. McNally
  • Barbara G. PickardEmail author
Original Papers


Covisualizations with wide-field computational opticalsectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is antigenicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-β1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of β1 integrin and an antibody against the matrix region of β1 integrin. The latter antibody also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.


Actin Adhesion sites Allium cepa Endomembrane system Integrin Spectrin 



3,3′-dihexyloxacarbocyanine iodide


Gaussian filtering


maximum likelihood (algorithm or method)


phosphate-buffered saline


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Allen NS, Brown DT (1988) Dynamics of the endoplasmic reticulum in living onion epidermal cells in relation to microtubules, microfilaments, and intracellular particle movement. Cell Motil Cytoskeleton 10: 153–163Google Scholar
  2. An Y-Q, McDowell JM, Huang S, McKinney EC, Chambliss S, Meagher RB (1996) Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10: 107–121Google Scholar
  3. Ayala J (1994) Transport and internal organization of membranes: vesicles, membrane networks and GTP-binding proteins. J Cell Sci 107: 753–763Google Scholar
  4. Beck KA, Buchanan JA, Malhotra V, Nelson WJ (1994) Golgi spectrin: identification of an erythroid β-spectrin homolog associated with the Golgi complex. J Cell Biol 127: 707–723Google Scholar
  5. Bennett V, Gilligan DM (1993) The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Annu Rev Cell Biol 9: 27–66Google Scholar
  6. 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, WI, pp 151–161Google Scholar
  7. Cheresh DA, Mecham RP (1994) Integrins: molecular and biological responses to the extracellular matrix. Academic Press, San DiegoGoogle Scholar
  8. Chrombach A, Rodbard D (1971) Polyacrylamide gel electrophoresis. Science 172: 440–450Google Scholar
  9. Cleary AL (1995) F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin. Protoplasma 185: 152–165Google Scholar
  10. —, Gunning BES, Wasteneys GO, Hepler PK (1992) Microtubule and F-actin dynamics at the division site in livingTradescantia stamen hair cells. J Cell Sci 103: 977–988Google Scholar
  11. Conchello JA, McNally JG (1966) Fast regularization technique for expectation maximization algorithm for computational optical sectioning microscopy. In: Cogswell CJ, Kino GS, Wilson T (eds) Three-dimensional microscopy: image acquisition and processing. Proc SPIE 2655: 199–208Google Scholar
  12. Craig S, Staehelin LA (1988) High pressure freezing of intact plant tissues: evaluation and characterization of novel features of the endoplasmic reticulum and associated membrane systems. Eur J Cell Biol 46: 80–93Google Scholar
  13. de Ruijter N, Emons A (1993) Immunodetection of spectrin antigens in plant cells. Cell Biol Int Rep 17: 169–182Google Scholar
  14. Ding JP, Pickard BG (1993) Mechanosensory calcium-selective cation channels in epidermal cells. Plant J 3: 83–110Google Scholar
  15. Enomoto-Iwamoto M, Menko AS, Philp N, Boettiger D (1993) Evaluation of integrin molecules involved in substrate adhesion. Cell Adhes Commun 1: 191–202Google Scholar
  16. Fairbairn DJ, Goodbody KC, Lloyd CW (1994) Simultaneous labelling of microtubules and fibrillar bundles in tobacco BY-2 cells by the anti-intermediate filament antibody, ME 101. Protoplasma 182: 160–169Google Scholar
  17. Faraday CD, Spanswick RM (1993) Evidence for a membrane skeleton in higher plants: a spectrin-like polypeptide co-isolates with rice root plasma membrane. FEBS Lett 318: 313–316Google Scholar
  18. Fuhrmann C, Bereiter-Hahn J, Brändle K (1990) Influence of the cytoskeleton, energy supply, and protein synthesis on the structure of the endoplasmic reticulum. Protoplasma 158: 53–65Google Scholar
  19. Geiger B, Ayalon O (1992) Cadherins. Annu Rev Cell Biol 8: 307–332Google Scholar
  20. Gens JS, Reuzeau C, Doolittle KW, McNally JG, Pickard BG (1996) Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts. Protoplasma 194: 215–230Google Scholar
  21. —, Fitriani T, McNally JG, Pickard BG (1997) Wall-associated receptor-like kinase 1 (WAK1) comingles with antigens recognized by antibodies against β1 integrin and other adhesion site proteins in wall/membrane puncta of BY2 tobacco cells. Plant Physiol 114 Suppl: 14–15Google Scholar
  22. Gilmore AP, Burridge K (1996) Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4-5-bisphosphate. Nature 381: 531–535Google Scholar
  23. Goddard RH, Wick SM, Silflow CD, Snustad DP (1994) Microtubule components of the plant cytoskeleton. Plant Physiol 104: 1–6Google Scholar
  24. Gomez S, Morgans C (1993) Interaction between band 3 and ankyrin begins in early compartments of the secretory pathway and is essential for band 3 processing. J Biol Chem 268: 19593–19597Google Scholar
  25. Grabski S, Feijter AW de, Schindler M (1993) Endoplasmic reticulum forms a dynamic continuum for lipid diffusion between contiguous soybean root cells. Plant Cell 5: 25–38Google Scholar
  26. Gumbiner BM (1993) Proteins associated with the cytoplasmic surface of adhesion molecules. Neuron 11: 551–564Google Scholar
  27. Gunning BES, Steer MW (1996) Plant cell biology. Jones and Bartlett Publishers, Sudbury, MAGoogle Scholar
  28. Hartwig JH (1994) Actin-binding proteins 1: spectrin superfamily. Protein Profile 1: 715–749Google Scholar
  29. Haugland RP (1992) Handbook of fluorescent probes and research chemicals, 5th edn. Molecular Probes, Inc., Eugene, ORGoogle Scholar
  30. Hepler P, Palevitz BA, Lancelle SA, McCauley MM, Lichtscheidl I (1990) Cortical endoplasmic reticulum in plants. J Cell Sci 96: 355–373Google Scholar
  31. Hitt AL, Luna EJ (1994) Membrane interactions with the actin cytoskeleton. Curr Opin Cell Biol 6: 120–130Google Scholar
  32. 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
  33. Jockusch BM, Bubeck P, Giehl K, Kroemker M, Moschner J, Rothkegel M, Rüdiger M, Schlüter K, Stanke G, Winkler J (1995) The molecular architecture of focal adhesions. Annu Rev Cell Biol 11: 379–416Google Scholar
  34. Kieffer JD, Plopper G, Ingber DE, Hartwig JH, Kupper TS (1995) Direct binding of F-actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro. Biochem Biophys Res Commun 217: 466–474Google Scholar
  35. Knebel W, Quader H, Schnepf E (1990) Mobile and immobile endoplasmic reticulum in onion bulb epidermis cells: short- and longterm observations with a confocal laser scanning microscope. Eur J Cell Biol 52: 328–340Google Scholar
  36. Laemmli EK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685Google Scholar
  37. Li Y-X, Keizer J, Stojilkovic SS, Rinzel J (1995) Ca2+ excitability of the ER membrane: an explanation for IP3-induced Ca2+ oscillations. Am J Physiol 269: C1079-C1092Google Scholar
  38. Lichtscheidl IK, Url WG (1990) Organization and dynamics of cortical endoplasmic reticulum in inner epidermal cells of onion bulb scales. Protoplasma 157: 203–215Google Scholar
  39. —, Lancelle SA, Hepler PK (1990) Actin-endoplasmic reticulum complexes inDrosera: their structural relationship with the plasmalemma, nucleus, and organelles in cells prepared by high pressure freezing. Protoplasma 155: 116–126Google Scholar
  40. Liebe S, Menzel D (1995) Actomyosin-based motility of endoplasmic reticulum and chloroplasts inVallisneria mesophyll cells. Biol Cell 85: 207–222Google Scholar
  41. —, Quader H (1994) Myosin in onion (Allium cepa) bulb scale epidermal cells: involvement in dynamics of organelles and endoplasmic reticulum. Physiol Plant 90: 114–124Google Scholar
  42. 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: 1765–1772Google Scholar
  43. McNulty AK, Saunders MJ (1992) Purification and immunological detection of pea nuclear intermediate filaments: evidence for plant nuclear lamins. J Cell Sci 103: 407–414Google Scholar
  44. Michaud D, Guillet G, Rogers PA, Charest PM (1991) Identification of a 220 kDa membrane-associated plant cell protein immunologically related to human b-spectrin. FEBS Lett 294: 77–80Google Scholar
  45. Miller DD, Scordilis SP, Hepler PK (1995) Identification and localization of three classes of myosins in pollen tubes ofLilium longiflorum andNicotiana alata. J Cell Sci 108: 2549–2563Google Scholar
  46. Mizuno K (1995) A cytoskeletal 50 kDa protein in higher plants that forms intermediate-sized filaments and stabilizes microtubules. Protoplasma 186: 99–112Google Scholar
  47. Morré DJ, Keenan TW (1994) Golgi apparatus buds — vesicles or coated ends of tubules? Protoplasma 179: 1–4Google Scholar
  48. Oparka KJ, Read ND (1994) The use of fluorescent probes for studies of living plant cells. In: Harris N, Oparka KJ (eds) Plant cell biology: a practical approach. Oxford University Press, Oxford, pp 27–50Google Scholar
  49. —, Murphy R, Derrick PM, Prior DAM, Smith JAC (1991) Modification of the pressure-probe technique permits controlled intracellular microinjection of fluorescent probes. J Cell Sci 98: 539–544Google Scholar
  50. —, 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
  51. Pardi R, Bossi G, Inverardi L, Rovida E, Bender JR (1995) Conserved regions in the cytoplasmic domains of the leukocyte integrin α1β2 are involved in endoplasmic reticulum retention, dimerization, and cytoskeletal association. J Immunol 155: 1252–1263Google Scholar
  52. Penman S (1995) Rethinking cell structure. Proc Natl Acad Sci USA 92: 5251–5257Google Scholar
  53. Pickard BG (1994) Contemplating the plasmalemmal control center model. Protoplasma 182: 1–9Google Scholar
  54. —, Ding JP (1993) The mechanosensory calcium-selective ion channel: key component of a plasmalemmal control centre? Aust J Plant Physiol 20: 439–459Google Scholar
  55. —, Reuzeau C, Doolittle K, McNally JG (1994) High resolution visualization in onion of distribution patterns of spectrin, talin and vinculin antigenicities. ASGSB Bull 8: 54Google Scholar
  56. —, McNally JG, Reuzeau C (1995) The endomembrane sheath — a “new” component of the plant cell? ASGSB Bull 9: 29Google Scholar
  57. Pollard TD, Almo S (1994) Structure of actin binding proteins: insights about function at atomic resolution. Annu Rev Cell Biol 10: 207–249Google Scholar
  58. Pont-Lezica RF, McNally JG, Pickard BG (1993) Wall-to-membrane linkers in onion epidermis: some hypotheses. Plant Cell Environ 16: 111–123Google Scholar
  59. Porter KR(1984) The cytomatrix: a short history of its study. J Cell Biol 99 Suppl: 3s-12sGoogle Scholar
  60. Pruss RM, Mirsky R, Raff MC, Thorpe R, Dowding AJ, Anderton BH (1981) All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell 27: 419–428Google Scholar
  61. Quader H (1990) Formation and disintegration of cisternae of the endoplasmic reticulum visualized in live cells by conventional fluorescence and confocal laser scanning microscopy: evidence for the involvement of calcium and the cytoskeleton. Protoplasma 155: 166–175Google Scholar
  62. —, Fast H (1990) Influence of cytosolic pH changes on the organization of the endoplasmic reticulum in epidermal cells of onion bulb scales: acidification by loading with weak organic acids. Protoplasma 157: 216–224Google Scholar
  63. —, Liebe S (1995) Actin filament-independent formation of tubular endoplasmic reticulum in onion epidermis cells. J Plant Physiol 145: 71–77Google Scholar
  64. —, Schnepf E (1986) Endoplasmic reticulum and cytoplasmic streaming: fluorescence microscopical observations in adaxial epidermis cells of onion bulb scales. Protoplasma 131: 250–253Google Scholar
  65. —, Hofmann A, Schnepf E (1987) Shape and movement of the endoplasmic reticulum in onion bulb epidermis cells: possible involvement of actin. Eur J Cell Biol 44: 17–26Google Scholar
  66. — — — (1989) Reorganization of the endoplasmic reticulum in epidermal cells of onion bulb scales after cold stress: involvement of cytoskeletal elements. Planta 177: 273–280Google Scholar
  67. Quatrano RS, Brian L, Aldridge J, Schultz T (1991) Polar axis fixation inFucus zygotes: components of the cytoskeleton and extracellular matrix. Development Suppl 1: 11–16Google Scholar
  68. Quinlan R, Hutchison C, Lane B (1994) Intermediate filament proteins. Protein Profile 1: 779–800Google Scholar
  69. Reuzeau C, Pont-Lezica RF (1995) Comparing plant and animal extracellular matrix-cytoskeleton connections — are they alike? Protoplasma 186: 113–121Google Scholar
  70. —, 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
  71. — — — — (1995b) Spectrin surrounds the endomembrane system of onion epidermal cells. Mol Biol Cell 6 Suppl: 370AGoogle Scholar
  72. —, McNally JG, Pickard BG (1995c) β1 Integrin in the endomembrane system of onion epidermal cells is colocalized with spectrin and actin. ASGSB Bull 9: 29Google Scholar
  73. —, Fichter CR, McNally JG, Pickard BG (1997a) Colocalization of injected antibodies against spectrin, ankyrin, and members of the anion exchange family in onion epidermal cells. Plant Physiol 114 Suppl: 88–89Google Scholar
  74. - - McNally JG, Pickard BG (1997b) The endomembrane sheath: a key structure for understanding the plant cell? Protoplasma (in press)Google Scholar
  75. Ruetz S, Lindsey AE, Ward CL, Kopito RR (1993) Functional activation of plasma membrane anion exchangers occurs in a pre-Golgi compartment. J Cell Biol 121: 37–48Google Scholar
  76. Schindler M, Meiners S, Cheresh DA (1989) RGD-dependent linkage between plant cell wall and plasma membrane: consequences for growth. J Cell Biol 108: 1955–1965Google Scholar
  77. Schmit A-C, Lambert A-M (1990) Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells. Plant Cell 2: 129–138Google Scholar
  78. Schwanz MA, Ingber DE (1994) Integrating with integrins. Mol Biol Cell 5: 3t89–93Google Scholar
  79. Sikorski AP, Swat W, Brzezińska M, Wróblewski Z, Bisikirska B (1993) A protein cross-reacting with anti-spectrin antibodies is present in higher plant cells. Z Naturforsch 48c: 580–583Google Scholar
  80. Staiger CJ, Yuan M, Valenta R, Shaw PJ, Warn RM, Lloyd CW (1994) Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments. Curr Biol 4: 215–219Google Scholar
  81. Stickens D, Verbelen J-P (1996) Spatial structure of mitochondria and ER denotes changes in cell physiology of cultured tobacco protoplasts. Plant J 9: 85–92Google Scholar
  82. Terasaki M, Reese TS (1992) Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanines. J Cell Sci 101: 315–322Google Scholar
  83. Thimann KV, Reese K, Nachmias VT (1992) Actin and the elongation of plant cells. Protoplasma 171: 153–166Google Scholar
  84. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354Google Scholar
  85. Traas JA, Doonan JH, Rawlins DJ, Shaw PJ, Watts J, Lloyd CW (1987) An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus. J Cell Biol 105: 387–395Google Scholar
  86. Wachtler F, Stahl A (1993) The nucleolus: a structural and functional interpretation. Micron 24: 473–505Google Scholar
  87. Wang N, Ingber DE (1995) Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry. Biochem Cell Biol 73: 327–335Google Scholar
  88. —, Butler JP, Ingber DE (1993) Mechanotransduction across the cell surface and through the cytoskeleton. Science 260: 1124–1127Google Scholar
  89. Wang Y, Yan L (1988) The membrane protein of leaf cell membrane ofVicia faba. Chin Sci Bull 33: 231–235Google Scholar
  90. — — (1991) Immunochemical identification of spectrins on the plasma membrane of leaf cells ofVicia faba. Chin Sci Bull 36: 862–866Google Scholar
  91. Wayne R, Staves MP, Leopold AC (1992) The contribution of the extracellular matrix to gravisensing in characean cells. J Cell Sci 101: 611–623Google Scholar
  92. Williamson RE (1993) Organelle movements. Annu Rev Plant Physiol Plant Mol Biol 44: 181–202Google Scholar
  93. Yang C, Xing L, Zhai Z (1992) Intermediate filaments in higher plant cells and their assembly in a cell-free system. Protoplasma 171: 44–54Google Scholar

Copyright information

© Springer-Verlag 1997

Authors and Affiliations

  • Christophe Reuzeau
    • 1
  • Keith W. Doolittle
    • 1
  • James G. McNally
    • 1
  • Barbara G. Pickard
    • 1
    Email author
  1. 1.Biology DepartmentWashington UniversitySaint LouisUSA

Personalised recommendations