Gap junctions are intercellular channels which play a major role in synchronizing cellular activities and are involved in the control of growth and development. Because they synchronize heart and other muscle cells, junction defects may be involved in arrhythmias and problems in parturition. There needs to be a better understanding of how they are built and how the physiological factors which causes them to open and close, exert their action. We have carried out preliminary work and shown that we can study details of gap junction structure by using a relatively new tool, scanning (atomic) force microscopy. We now propose to extend this work, capitalizing on the fact that we have just succeeded to examine gap junctions to reveal very fine detail. Using a combination of techniques which includes the use of immunolabeling we would analyze the topology of connexin molecules. By combining an atomic force microscope with fluorescence microscopy we hope to identify gap junction precursors either on cell surfaces, or if this does not succeed on membranes derived from cell surface. We know such precursors exist because we have obtained evidence that the extracellular domains of connexins are exposed before junctions form. We want to visualize the precursor structures in the AFM and compare them to what should be the corresponding structures revealed by dissection in mature junctions. We also hope to get some understanding of gap junction biogenesis by studying the organization of connexins in intracellular organelles.
| Derose, J A; Revel, J (1999) A comparative study of colloidal particles as imaging standards for microscopy. J Microsc 195:64-78 |
