Skip to main content
SpringerLink
Log in

Decreased gap junctional communication in neurobiotin microinjected lens epithelial cells after taxol treatment

  • Original Article
  • Published:
Anatomy and Embryology Aims and scope Submit manuscript

Abstract

The aim of the study was to examine gap-junction-mediated intercellular communication after experimentally induced aggregations of microtubules in cultured bovine lens epithelial cells. Intercellular communication between lens cells appears to be crucial for normal lens homeostasis. However, investigations on the maintenance of direct ion and metabolite exchange via gap junctions and its quantified dependency of cytoskeletal microtubules have not been available under conditions leading to bundling of microtubules. Thus, metabolic coupling of neighboring lens epithelial cells was quantified following microinjections of neurobiotin into single cells under various conditions. In controls, intensive gap-junction-mediated intercellular communication could be documented by dye-spreading of microinjected neurobiotin. In contrast, taxol treatment for 1–3 days impaired, but did not completely block gap-junction-mediated intercellular communication. After depletion of taxol, a complete recovery of intercellular communication was achieved. In addition, confocal laser scanning microscopy and rapid-freeze deep-etch electron microscopy revealed a displacement of actin-filaments from the perinuclear cytoplasm, accompanied by an abnormal aggregation of microtubules after taxol treatment, including impeded translocation of connexin 43 from the cytoplasm into the plasma membrane. Incubation of cells with nocodazole destroyed the microtubule network, accompanied by a clear reduction of plasma-membrane-integrated connexin 43 and significant impairment of dye spreading. Thus, in lens epithelial cells intercellular communication at gap junctions made by connexin 43 depends on the integrity of the microtubule network through the translocation of connexins to the plasma membrane.

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

Fig. 1a–j
Fig. 2a–f
Fig. 3
Fig. 4a–f
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Berthoud VM, Cook AJ, Beyer EC (1994) Characterization of the gap junction protein connexin56 in the chicken lens by immunofluorescence and immunoblotting. Invest Ophthalmol Vis Sci 35:4109–4117

    CAS  PubMed  Google Scholar 

  • Berthoud VM, Bassnett S, Beyer EC (1999) Cultured chicken embryo lens cells resemble differentiating fiber cells in vivo and contain two kinetic pools of connexin 56. Exp Eye Res 68:475–484

    Article  CAS  PubMed  Google Scholar 

  • Beyer EC, Kistler J, Paul DL, Goodenough DA (1989) Antisera directed against connexin 43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol 108:595–605

    Article  CAS  PubMed  Google Scholar 

  • Bloomfield SA, Xin D, Persky SE (1995) A comparison of receptive field and tracer coupling size of horizontal cells in the rabbit retina. Vis Neurosci 12:985–999

    CAS  PubMed  Google Scholar 

  • Breipohl W, Leyendecker M, von Tiesenhausen K, Grus FH (1995) Effects of UV-B on the growth pattern of bovine passage I and II lens epithelial cells in vitro. Ophthalmic Res 27:62–68

    PubMed  Google Scholar 

  • Dahm R, van Marle J, Prescott AR, Quinlan RA (1999) Gap junctions containing alpha8-connexin (MP70) in the adult mammalian lens epithelium suggests a re-evaluation of its role in the lens. Exp Eye Res 69:45–56

    Article  CAS  PubMed  Google Scholar 

  • Gao Y, Spray DC (1998) Structural changes in lenses of mice lacking the gap junction protein connexin43. Invest Ophthalmol Vis Sci 39: 1198–1209

    CAS  PubMed  Google Scholar 

  • George CH, Kendall JM, Evans WH (1999) Intracellular trafficking pathways in the assembly of connexins into gap junctions. J Biol Chem 274:8678–8685

    Article  CAS  PubMed  Google Scholar 

  • Giepmans BN, Verlaan I, Hengeveld T, Janssen H, Calafat J, Falk MM, Moolenaar WH (2001) Gap junction protein connexin 43 interacts directly with microtubules. Curr Biol 11:1364–1368

    Article  CAS  PubMed  Google Scholar 

  • Giessmann D, Theiss C, Breipohl W, Meller K (2003) Microinjection of anti-actin antibodies impaired gap junctional intercellular communication in lens epithelial cells in vitro. Curr Eye Res 27: 157–164

    Article  PubMed  Google Scholar 

  • Goldberg GS, Lampe PD, Nicholson BJ (1999) Selective transfer of endogenous metabolites through gap junctions composed of different connexins. Nat Cell Biol 1:457–459

    Article  CAS  PubMed  Google Scholar 

  • Gong X, Li E, Klier G, Huang Q, Wu Y, Lei H, Kumar NM, Horwitz J, Gilula NB (1997) Disruption of alpha3 connexin gene leads to proteolysis and cataractogenesis in mice. Cell 91:833–843

    Article  CAS  PubMed  Google Scholar 

  • Goodenough DA (1979) Lens gap junctions: a structural hypothesis for nonregulated low-resistance intercellular pathways. Invest Ophthalmol Vis Sci 18:1104–1122

    CAS  PubMed  Google Scholar 

  • Goodenough DA (1992) The crystalline lens. A system networked by gap junctional intercellular communication. Semin Cell Biol 3:49–58

    CAS  PubMed  Google Scholar 

  • Graham RC, Karnovsky MJ (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubles of mouse kidney; ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291–302

    CAS  PubMed  Google Scholar 

  • Hernandez Galindo EE, Theiss C, Steuhl KP, Meller D (2003) Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane. Exp Eye Res 76:303–314

    Article  CAS  PubMed  Google Scholar 

  • Jiang JX, White TW, Goodenough DA (1995) Changes in connexin expression and distribution during chick lens development. Dev Biol 168:649–661

    Article  CAS  PubMed  Google Scholar 

  • Johnson RG, Meyer RA, Li XR, Preus DM, Tan L, Grunenwald H, Paulson AF, Laird DW, Sheridan JD (2002) Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules. Exp Cell Res 275:67–80

    Article  CAS  PubMed  Google Scholar 

  • Kinsey VE, Reddy DVN (1964) Studies of the crystalline lens, XI: the relative role of the epithelium and capsule in transport. Invest Ophthalmol 4:104–116

    Google Scholar 

  • Kistler JB, Kirkland B, Bullivant S (1985) Identification of a 70,000-D protein in lens membrane junctional domains. J Cell Biol 101:28–35

    Article  CAS  PubMed  Google Scholar 

  • Kumar NM, Gilula NB (1996) The gap junction communication channel. Cell 84:381–388

    Article  CAS  PubMed  Google Scholar 

  • Laird DW, Puranam KL, Revel JP (1991) Turnover and phosphorylation dynamics of connexin 43 gap junction protein in cultured cardiac myocytes. Biochem J 273:67–72

    CAS  PubMed  Google Scholar 

  • Lauf U, Giepmans BN, Lopez P, Braconnot S, Chen SC, Falk MM (2002) Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells. Proc Natl Acad Sci USA 99:10446–10451

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Lonchampt MO, Laurent M, Coutois Y, Trenchev P, Hughes RC (1976) Microtubules and microfilaments of bovine lens epithelial cells: electron microscopy and immunofluorescence staining with specific antibodies. Exp Eye Res 23:505–518

    Article  CAS  PubMed  Google Scholar 

  • Martin PEM, Blundell G, Ahmad S, Errington RJ, Evans WH (2001) Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels. J Cell Sci 114:3845–3855

    CAS  PubMed  Google Scholar 

  • Masurovsky EB, Peterson ER, Crain SM, Horwitz SB (1981) Microtubule arrays in taxol-treated mouse dorsal root ganglion-spinal cord cultures. Brain Res 217:392–398

    Article  CAS  PubMed  Google Scholar 

  • Masurovsky EB, Peterson ER, Crain SM, Horwitz SB (1983) Morphological alterations in dorsal root ganglion neurons and supporting cells of organotypic mouse spinal cord-ganglion cultures exposed to taxol. Neuroscience 10:491–509

    Article  CAS  PubMed  Google Scholar 

  • Mathias RT, Rae JL, Baldo GJ (1997) Physiological properties of the normal lens. Physiol Rev 77:21–50

    CAS  PubMed  Google Scholar 

  • Meller K (1989) Chromatolysis of dorsal root ganglion cells studied by cryofixation. Cell Tissue Res 256:59–66

    Article  Google Scholar 

  • Meller K (1992) Axoplasmic transport of horseradish peroxidase in single neurons of the dorsal root ganglion studied in vitro by microinjection. Cell Tissue Res 270:139–148

    Article  CAS  PubMed  Google Scholar 

  • Meller K (1994) Intracellular injection of transport markers and antibodies into in vitro cultured nerve cells. Neurosci Protocols 50:1–11

    Google Scholar 

  • Miller TM, Goodenough DA (1985) Gap junction structures after experimental alteration of junctional channel conductance. J Cell Biol 101:1741–1748

    Article  CAS  PubMed  Google Scholar 

  • Mills SL, Massey SC (2000) A series of biotinylated tracers distinguishes three types of gap junction in retina. J Neurosci 20:8629–8636

    CAS  PubMed  Google Scholar 

  • Musil LS, Beyer EC, Goodenough DA (1990) Expression of the gap junction protein connexin 43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J Membr Biol 116:63–175

    Google Scholar 

  • Parekh H, Simpkins H (1997) The transport and binding of taxol. Gen Pharmacol 29:167–172

    CAS  PubMed  Google Scholar 

  • Paul DL, Ebihara L, Takemoto LJ, Swenson KI, Goodenough DA (1991) Connexin 46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol 115:1077–1089

    Article  CAS  PubMed  Google Scholar 

  • Paulson AF, Lampe PD, Meyer RA, TenBroek E, Atkinson MM, Walseth TF, Johnson RG (2000) Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking. J Cell Sci 113:3037–3049

    CAS  PubMed  Google Scholar 

  • Peracchia C (1978) Calcium effects on gap junction structure and cell coupling. Nature 271:669–671

    CAS  PubMed  Google Scholar 

  • Piatigorsky J, Webster H, Wollberg M (1972) Cell elongation in the cultured embryonic chick lens epithelium with and without protein synthesis. Involvement of microtubules. J Cell Biol 55:82–92

    Article  CAS  PubMed  Google Scholar 

  • Rup DM, Veenstra RD, Wang HZ, Brink PR, Beyer EC (1993) Chick connexin-56, a novel lens gap junction protein. Molecular cloning and functional expression. J Biol Chem 268:706–712

    CAS  PubMed  Google Scholar 

  • Saez JC, Connor JA, Spray DC, Bennett MV (1989) Hepatocyte gap junctions are permeable to the second mess inositol 1,4,5-trisphosphate, and to calcium ions. Proc Natl Acad Sci USA 86:2708–2712

    CAS  PubMed  Google Scholar 

  • Saez JC, Nairn AC, Czernik AJ, Spray DC, Hertzberg EL, Greengard P, Bennett MVL (1990) Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. Eur J Biochem 192:263–273

    Article  CAS  PubMed  Google Scholar 

  • Saleh SM, Takemoto DJ (2000) Overexpression of protein kinase C gamma inhibits gap junctional intercellular communication in the lens epithelial cells. Exp Eye Res 71:99–102

    Article  CAS  PubMed  Google Scholar 

  • Schiff PB, Horwitz SB (1980) Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci USA 77:1561–1565

    CAS  PubMed  Google Scholar 

  • Spray DC, White RL, Campos de Carvalho A, Harris AL, Bennett MVL (1984) Gating of gap junction channels. Biophys J 45:219–230

    CAS  PubMed  Google Scholar 

  • TenBroek EM, Johnson R, Louis CF (1994) Cell-to-cell communication in a differentiating ovine lens culture system. Invest Ophthalmol Vis Sci 35:215–228

    CAS  PubMed  Google Scholar 

  • Theiss C, Meller K (2000) Taxol impairs anterograde axonal transport of microinjected horse radish peroxidase in dorsal root ganglia neurons in vitro. Cell Tissue Res 299:213–224

    CAS  PubMed  Google Scholar 

  • Theiss C, Meller K (2002a) Aluminum impairs gap junctional intercellular communication between astroglial cells in vitro. Cell Tissue Res 310: 143–154

    Article  CAS  PubMed  Google Scholar 

  • Theiss C, Meller K (2002b) Microinjected anti-actin antibodies impaired cell-communication in astrocytes in vitro. Exp Cell Res 281:197–204

    Article  CAS  PubMed  Google Scholar 

  • White TW, Bruzzone R, Goodenough DA, Paul DL (1992) Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fibre protein MP70. Mol Biol Cell 3:711–720

    CAS  PubMed  Google Scholar 

  • White TW, Goodenough DA, Paul DL (1998) Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts. J Cell Biol 143:815–825

    Article  CAS  PubMed  Google Scholar 

  • Zampighi GA, Simon SA, Hall JE (1992) The specialized junctions of the lens. Int Rev Cytol 136:185–225

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors especially thank M. Leyendecker for her help in setting up the initial tissue culture conditions and A. Ambrosat for secretarial work. We also wish to thank Dr. Schmidt and Dr. Masalme for providing eyes from slaughtered cattle.

Author information

Authors and Affiliations

  1. Institut für Anatomie, Abteilung für Cytologie, Medizinische Fakultät, Ruhr-Universität Bochum, 44780, Bochum, Germany

    Daniel Giessmann, Carsten Theiss & Karl Meller

  2. Abteilung für Experimentelle Ophthalmologie, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany

    Winrich Breipohl

Authors
  1. Daniel Giessmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carsten Theiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Winrich Breipohl
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karl Meller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carsten Theiss.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Giessmann, D., Theiss, C., Breipohl, W. et al. Decreased gap junctional communication in neurobiotin microinjected lens epithelial cells after taxol treatment. Anat Embryol 209, 391–400 (2005). https://doi.org/10.1007/s00429-005-0456-z

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00429-005-0456-z

Keywords

Search

Navigation