Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model.
Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction--the cardiac gap junction--is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart. Freeze-fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision-related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasi-crystalline arrangement, not a 'random' arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap-junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap-junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131-142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three-dimensional precision mapping of gap junctions through thick slices of cardiac tissue.(ABSTRACT TRUNCATED AT 400 WORDS)
Severs
N J
NJ
Department of Cardiac Medicine, National Heart and Lung Institute, London, U.K.
Gourdie
R G
RG
Harfst
E
E
Peters
N S
NS
Green
C R
CR
D000818:Animals; D001145:Arrhythmias, Cardiac / Q000473:pathology; D005614:Freeze Fracturing / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000473:pathology / Q000502:physiology / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods; D009206:Myocardium / Q000166:cytology* / Q000473:pathology; D011817:Rabbits; D051381:Rats