Skip to main content
SpringerLink
Log in

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Summary

The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with ≥20 mM NaOH or ≥0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).

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

References

  • Ahkong QF, Blow AM, Botham GM, Launder, JM, Quirk SJ, Lucy JA (1978) Proteinase and cell fusion. FEBS Lett 95:147–159

    Google Scholar 

  • Baker TS, Caspar DLD, Hollingshead CJ, Goodenough DA (1983) Gap junction structures. IV. Asymmetric features revealed by low-irradiation microscopy. J Cell Biol 96:204–216

    Google Scholar 

  • Barondes SH, Haywood-Reid PL, Cooper DNW (1985) Discoidin 1, an endogenous lectin, is externalized from Dictyostelium discoideum in multilamellar bodies. J Cell Biol 100:1825–1833

    Google Scholar 

  • Benedetti EL, Emmelot P (1965) Electron microscope observations on negatively stained plasma membranes isolated from rat liver. J Cell Biol 26:299–305

    Google Scholar 

  • Bennett MVL, Goodenough DA (1978) Gap junctions, electrotonic coupling and intercellular communication. Neurosci Res Progr Bull 16:377–488

    Google Scholar 

  • Berdan RC (1985) Crayfish heptopancreas cell junctions: Subcellular fractionation, ultrastructural and biochemical anlysis. Ph D Thesis Baylor College of Medicine, Houston, TX

    Google Scholar 

  • Berdan RC (1987) Intercellular communication in arthropods: Biophysical, ultrastructural and biochemical approaches In: WC DeMello (ed) Intercellular Communication — New Perspectives. Plenum Press, New York, pp 299–370

    Google Scholar 

  • Berdan RC, Caveney S (1985) Gap junction ultrastructure in three states of conductance. Cell Tissue Res 239:111–122

    Google Scholar 

  • Berdan RC, Shivers RR, Bulloch AGM (1987) Chemical synapses, particle arrays, pseudo-gap junctions and gap junctions of neurons and glia in the buccal ganglion of Helisoma. Synapse (inpress)

  • Berezny R (1979) Effect of protease inhibitors on matrix proteins and the association of replication DNA. Exp Cell Res 123:411–414

    Google Scholar 

  • Burnette WN (1981) Western Blotting: Electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203

    Google Scholar 

  • Caveney S (1976) The insect epidermis: A functional syncytium. In: Hepurn HR (ed) The insect integument. Elsevier, Amsterdam, pp 259–264

    Google Scholar 

  • Caveney S (1986) Intercellular communication In: Kerkuta GA, Gilber LI (eds) Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 2. Pergamon press, Toronto, pp 319–370

    Google Scholar 

  • Couch CB, Strittmatter WJ (1983) Rat myoblast fusion requires metalloendoprotease activity. Cell 32:257–265

    Google Scholar 

  • Couch CB, Strittmatter WJ (1984) Specific blockers of myoblast fusion inhibit a soluble and not the membrane-associated metaloendoprotease in myoblasts. J Biol Chem 98:5396–5399

    Google Scholar 

  • Culvenor JG, Evans HW (1977) Preparation of hepatic gap (communicating) junctions. Biochem J 168:475–481

    Google Scholar 

  • Elder JM, Pickett RA, Hamilton J, Lerner RA (1977) Radio-iodination of proteins in single polyacrylamide gel slices. J Biol Chem 252:6510–6515

    Google Scholar 

  • Ehrhart JC (1981) Further purification of mouse liver gap junctions with deoxycholate and protein composition. Cell Biol Int Rep 5:1055–1061

    Google Scholar 

  • Epstein ML, Gilula NB (1977) A study of communication specificity between cells in culture. J Cell Biol 75:769–787

    Google Scholar 

  • Finbow M, Yancey BS, Johnson R, Revel J-P (1980) Independent lines of evidence suggesting a major gap junctional protein with a molecular weith of 26,000. Proc Natl Acad Sci USA 77:970–974

    Google Scholar 

  • Finbow ME, Eldridge T, Bumltjens J, Lane NJ, Shuttleworth J, Pitts JD (1984) Isolation and characterization of arthropod gap junctions. EMBO J 3:2271–2278

    Google Scholar 

  • Furshpan EJ, Potter DD (1959) Transmission at the giant motor synapse of the crayfish. J Physiol 145:289–325

    Google Scholar 

  • Gilula NB, Satir P (1971) Septate and gap junctions in molluscan gill epithelium. J Cell Biol 51:869–872

    Google Scholar 

  • Gilula NB, Epstein ML, Beers WH (1978) Cell-to-cell communication and ovulation. J Cell Biol 78:58–75

    Google Scholar 

  • Goodenough DA (1974) The isolation of mouse hepatocyte gap junctions. J Cell Biol 54:646–656

    Google Scholar 

  • Goodman CS, Spitzer NC (1979) Embryonic development of identified neurones: Differentiation from neuroblast to neurone. Nature 280:208–214

    Google Scholar 

  • Hawkes R, Niday E, Gordon J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119:142–147

    Google Scholar 

  • Henderson D, Eibl H, Weber K (1979) Structure and biochemistry of mouse hepatic gap junctions. J Mol Biol 132:193–218

    Google Scholar 

  • Hertzberg EL (1984) A detergent-independent procedure for the isolation of gap junctions from rat liver. J Biol Chem 259:9936–9943

    Google Scholar 

  • Hertzberg EL, Gilula NB (1979) Isolation and characterization of gap junctions from rat liver. J Biol Chem 254:2138–2147

    Google Scholar 

  • Hertzberg EL, Gilula NB (1982) Liver gap junctions and lens fiber junctions: Comparative analysis and calmodulin interaction. Cold Spring Harbor Symposia on Quantitiative Biology, XLVI, pp 639–654

    Google Scholar 

  • Hertzberg EL, Skibbens RV (1984) A protein homologous to the 27,000 dalton liver gap junction protein is present in a wide variety of species and tissues. Cell 39:61–69

    Google Scholar 

  • Hertzberg EL, Lawrence T, Gilula NB (1981) Gap junctional communciation. Ann Rev Physiol 43:479–491

    Google Scholar 

  • Hertzberg EL, Anderson DJ, Friedlander M, Gilula NB (1982) Comparative analysis of the major polypeptides from liver gap junctions and lens fiber junctions. J Cell Biol 92:53–59

    Google Scholar 

  • Hertzberg El, Spray DC, Bennett MVL (1985) Reduction of gap junctional conductance by microinjection of antibodies against the 27-KD liver gap junction polypeptide. Proc Natl Acad Sci USA 82:2412–2416

    Google Scholar 

  • Johnson DA, Gautsch JW, Sportsman JR, Elder JH (1984) Improved technique utilizing non-fat dry milk for analysis of proteins and nucleic acid transferred to nitrocellulose. Gene Anal Techn 1:3–8

    Google Scholar 

  • Johnson GD, de C Nogueira Araujo GM (1981) A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43:349–350

    Article  CAS  PubMed  Google Scholar 

  • Kensler RW, Goodenough DA (1980) Isolation of mouse myocardial gap junctions. J Cell Biol 86:755–764

    Google Scholar 

  • Kistler J, Bullivant S (1980) Lens gap junctions and othogonal arrays are unrelated. FEBS Lett 111:73–78

    Google Scholar 

  • Kumar NM, Gilula NB (1986) Cloning and characterization of human and liver cDNAs coding for a gap junction protein. J Cell Biol 103:767–776

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227:680–685

    PubMed  Google Scholar 

  • Lane NJ, Swales LS (1980) Dispersal of junctional particles, not internalization, during the in vivo disappearance of gap junctions. Cell 19:579–586

    Google Scholar 

  • Larsen WJ (1983) Biological implications of gap junction structure, distribution and composition: A review. Tissue Cell 51:645–671

    Google Scholar 

  • Lawson D, Raff MC, Gomperts B, Fewtrell C, Gilula NB (1977) Molecular events during membrane fusion. J Cell Biol 72:242–259

    Google Scholar 

  • Lees-Miller JP, Caveney S (1982) Drugs that block calmodulin activity inhibit cell-to-cell communication in the epidermis of Tenebrio molitor. J Membr Biol 69:233–245

    Google Scholar 

  • Loewenstein WR (1981) Junctional intercellular communication: The cell-to-cell membrane channel. Physiol Rev 61:829–913

    Google Scholar 

  • Loewenstein WR, Kanno Y (1964) Studies on an epthelial (gland) cell junction. I Modifications of surface membrane permeability. J Cell Biol 22:565–568

    Google Scholar 

  • Lopresti V, Macagno ER, Levinthal C (1974) Structure and development of neuronal connections in isogenic organisms: Transient gap junctions between growing optic axons and lamina neuroblasts. Proc Natl Acad Sci USA 71:1098–1102

    Google Scholar 

  • Lowry OM, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Manjunath CK, Goings GE, Page E (1982) Isolation and protein composition of gap junctions from rabbit hearts. Biochem J 205:189–194

    Google Scholar 

  • Manjunath CK, Goings GE, Page E (1984) Detergent sensitivity and splitting of isolated liver gap junctions. J Membr Biol 78:147–155

    Google Scholar 

  • Markham R, Frey S, Hills GJ (1963) Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology 20:88–102

    Google Scholar 

  • McGookey DJ, Anderson RGW (1983) Morphological characterization of the cholesterol ester cycle in cultured mouse macrophage foam cells. J Cell Biol 97:1156–1168

    Google Scholar 

  • McVicar LK, Shivers RR (1985) Gap junctions and intercellular communication in the hepatopancreas of the crayfish (Orconectes propinguus) during molt. Cell Tissue Res 240:261–269

    Google Scholar 

  • Mundy DI, Strittmatter WJ (1985) Requirement for metalloendoprotease in exocytosis: Evidence in mast cells and adrenal chromaffin cells. Cell 40:645–656

    Google Scholar 

  • Neutra M, Schaeffer SF (1977) Membrane interactions between adjacent mucous secretion granules. J Cell Biol 74:983–991

    Google Scholar 

  • Nicholson BJ, Takemoto LJ, Hunkapiller MW, Hood LE, Revel J-P (1983) Differences between liver gap junctions and lens MP26 from rat: Implications for tissue specificity of gap junctions. Cell 22:967–978

    Google Scholar 

  • Oakley BR, Kirsch DR, and Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105:361–363

    CAS  PubMed  Google Scholar 

  • Palka J (1982) Genetic manipulation of sensory pathways in Drosophila In: Spitzer NC (ed) Neuronal Development. Plenum Press, New York, pp 121–170

    Google Scholar 

  • Paul DL (1986) Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol 103:123–134

    Google Scholar 

  • Peracchia C (1980) Structural correlates of gap junction permeation. Int Rev Cytol 66:81–146

    Google Scholar 

  • Peterson GL (1977) A simplification of the protein method of Lowry et al. which is more generally applicable. Anal Biochem 83:346–356

    CAS  PubMed  Google Scholar 

  • Revel JP, Nicholson BJ, Yancey SB (1985) Chemistry of gap junctions. Ann Rev Physiol 47:263–279

    Google Scholar 

  • Ryerse J, Nagel BA (1984) Gap junction distribution in the Drosophila wing disc mutants vg, 1 gd, 1 c43, and 1 g1. Dev Biol 105:396–403

    Google Scholar 

  • Sikerwar SS, Malhotra SK (1983) A structural characterization of gap junctions isolated from mouse liver. Cell Biol Int Rep 7:879–903

    Google Scholar 

  • Specian RD, Neutra MR (1980) Nechanism of rapid mucous secretion in goblet cells stimulated by acetylcholine. J Cell Biol 85:626–640

    Google Scholar 

  • Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43

    CAS  PubMed  Google Scholar 

  • Tanaka Y, DeCamilli P, Meldolesi J (1980) Membrane interactions between secretion granules and plasmalemma in three exocrine glands. J Cell Biol 84:438–453

    Google Scholar 

  • Thomas JB, Wyman RJ (1984) Mutations altering synaptic connectivity between identified neurons in Drosophila. J Neurosci 4:530–538

    Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    CAS  PubMed  Google Scholar 

  • Van Eldik LJ, Hertzberg EL, Berdan RC, Gilula NB (1985) Interaction of calmodulin and other calcium-modulated proteins with mammalian and arthropod junctional membrane proteins. Biochem Biophys Res Commun 126:825–832

    Google Scholar 

  • Warner AE, Guthrie SC, Gilula NB (1984) Antibodies to gap junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature 311:127–131

    Google Scholar 

  • Weir MP, Lo CW (1984) Gap-junctional communication compartments in the Drosophila wing imaginal disk. Dev Biol 102:130–146

    Google Scholar 

  • Wray W, Boulikas T, Wray VP, Hancock R (1981) Silver staining of proteins in polyacrylamide gels. Anal Biochem 118:197–203

    CAS  PubMed  Google Scholar 

  • Wyman RJ, Thomas JB (1983) What genes are necessary to make an identified synapse? In: Cold Spring Harbor Symposia on Quantitative Biology, Vol XLVIII. Cold Spring Harbor Laboratory, pp 641–652

  • Young JD, Cohn ZA, Gilula NB (1987) Functional assembly of gap junction conductance in lipid bilayers: Demonstration that the major 27 kd protein forms the junctional channel. Cell 48:733–743

    Google Scholar 

  • Zampighi G, Simon SA, Robertson JD, McIntosh TJ, Costello MJ (1982) On the structural organization of isolated bovine lens fiber junctions. J Cell Biol 93:175–189

    Google Scholar 

  • Zervos AS, Hope J, Evans WH (1985) Preparation of a gap junction fraction from uteri of pregnant rats: the 28-Kd polypeptides of uterus, liver and heart gap junctions are homologous. J Cell Biol 101:1363–1370

    Google Scholar 

Download references

Author information

Author notes

  1. Norton B. Gilula

    Present address: Department of Molecular Biology, Research Institute of Scripps Clinic, 92037, La Jolla, California, USA

Authors and Affiliations

  1. Department of Cell Biology, Baylor College of Medicine, Houston, Texas, USA

    Robert C. Berdan & Norton B. Gilula

Authors
  1. Robert C. Berdan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Norton B. Gilula
    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

Berdan, R.C., Gilula, N.B. The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis. Cell Tissue Res. 251, 257–274 (1988). https://doi.org/10.1007/BF00215833

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00215833

Key words

Search

Navigation