Hostname: page-component-7c8c6479df-995ml Total loading time: 0 Render date: 2024-03-29T01:22:17.241Z Has data issue: false hasContentIssue false

Quantitative Analysis of ZO-1 Colocalization with Cx43 Gap Junction Plaques in Cultures of Rat Neonatal Cardiomyocytes

Published online by Cambridge University Press:  12 May 2005

Ching Zhu
Affiliation:
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA
Ralph J. Barker
Affiliation:
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA
Andrew W. Hunter
Affiliation:
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA
Yuhua Zhang
Affiliation:
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA
Jane Jourdan
Affiliation:
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA
Robert G. Gourdie
Affiliation:
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425, USA
Get access

Abstract

The gap junction (GJ) is an aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules of less than 1000 Da between cells. In excitable organs such as heart and brain, GJs configure extended intercellular pathways for stable and long-term propagation of action potential. In a previous study in adult rat heart, we have shown that the Drosophila disks-large related protein ZO-1 shows low to moderate colocalization at myocyte borders with the GJ protein Cx43. In the present study, we detail a protocol for characterizing the pattern and level of colocalization of ZO-1 with Cx43 in cultures of neonatal myocytes at the level of individual GJ plaques. The data indicate that ZO-1 shows on average a partial 26.6% overlap (SD = 11.3%) with Cx43 GJ plaques. There is a strong positive correlation between GJ plaque size and area of ZO-1 colocalization, indicating that the level of associated ZO-1 scales with the area of the GJ plaque. Qualitatively, the most prominent colocalization occurs at the plaque perimeter. These studies may provide insight into the presently unknown biological function of ZO-1 interaction with Cx43.

Type
Research Article
Copyright
© 2005 Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Angst, B.D., Khan, L.U., Severs, N.J., Whitely, K., Rothery, S., Thompson, R.P., Magee, A.I., & Gourdie, R.G. (1997). Dissociated spatial patterning of GJs and cell adhesion junctions during postnatal differentiation of ventricular myocardium. Circ Res 80, 88.Google Scholar
Barker, R.J. & Gourdie, R.G. (2002). Connexin interacting proteins. In Cell-Cell Coupling and Impulse Propagation in Health and Disease, DeMello, W.C. (Ed.), pp. 2553. New York: Kluwer Publications.
Barker, R.J., Price, R.L., & Gourdie, R.G. (2002). Increased association of ZO-1 with connexin43 during remodeling of cardiac GJs. Circ Res 90, 317324.Google Scholar
Duffy, H.S., Sorgen, P.L., Girvin, M.E., O'Donnell, P., Coombs, W., Taffet, S.M., Delmar, M., & Spray, D.C. (2002). pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains. J Biol Chem 277, 3670636714.Google Scholar
Dupont, E., Matsushita, T., Kaba, R.A., Vozzi, C., Coppen, S.R., Khan, N., Kaprielian, R., Yacoub, M.H., & Severs, N.J. (2001). Altered connexin expression in human congestive heart failure. J Mol Cell Cardiol 33, 359371.Google Scholar
Fonseca, C.G., Green, C.R., & Nicholson, L.F. (2002). Upregulation in astrocytic connexin 43 GJ levels may exacerbate generalized seizures in mesial temporal lobe epilepsy. Brain Res 929, 105116.Google Scholar
Giepmans, B.N.G. & Moolenaar, W.H. (1998). The GJ protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr Biol 8, 931934.Google Scholar
Gonzalez-Mariscal, L., Betanzos, A., Nava, P., & Jaramillo, B.E. (2003). Tight junction proteins. Prog Biophys Mol Biol 81, 144.Google Scholar
Goodenough, D.A. & Paul, D.L. (2003). Beyond the gap: Functions of unpaired connexon channels. Nat Rev Mol Cell Biol 4, 285294.Google Scholar
Gourdie, R.G., Green, C.R., & Severs, N.J. (1991). GJ distribution in adult mammalian myocardium revealed by an anti-peptide antibody and laser scanning confocal microscopy. J Cell Sci 99, 4155.Google Scholar
Green, C.R., Peters, N.S., Gourdie, R.G., Rothery, S., & Severs, N.J. (1993). Validation of immunohistochemical quantification in confocal scanning laser microscopy: A comparative assessment of GJ size with confocal and ultrastructural techniques. J Histochem Cytochem 41, 13391349.Google Scholar
Gros, D.B. & Jongsma, H.J. (1996). Connexins in mammalian heart function. Bioessays 18, 719730.Google Scholar
Kumar, N.M. & Gilula, N.B. (1996). The GJ communication channel. Cell 84, 382388.Google Scholar
Lo, C.W. (2000). Role of GJs in cardiac conduction and development: Insights from the connexin knockout mice. Circ Res 87, 346348.Google Scholar
Sepp, R., Severs, N.J., & Gourdie, R.G. (1997). Altered patterns of intercellular junction distribution in hypertrophic cardiomyopathy. Heart 76, 412417.Google Scholar
Simpson, D.G., Terracio, L., Terracio, M., Price, R.L., Turner, D.C., & Borg, T.K. (1994). Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix. J Cell Phys 161, 89105.Google Scholar
Smith, J.H., Green, C.R., Peters, N.S., Rothery, S., & Severs, N.J. (1991). Altered patterns of GJ distribution in ischemic heart disease: An immunohistochemical study of human myocardium using laser scanning confocal microscopy. Am J Pathol 139, 801821.Google Scholar
Spach, M.S. (1997). Discontinuous cardiac conduction: Its origin in cellular connectivity with long-term adaptive changes that cause arrhythmias. In Discontinuous Conduction in the Heart, Soonper, P.M., Joyner, R.W. & Jalife, J. (Eds.), pp. 551. New York: Futura Publishing Company, Inc.
Toyofuku, T., Yabuki, M., Otsu, K., Kuzuya, T., Hori, M., & Tada, M. (1998). Direct association of the GJ protein connexin 43 with ZO-1 in cardiac myocytes. J Biol Chem 273, 1272512731.Google Scholar