Severs N J, Gourdie R G, Harfst E, Peters N S, Green C R
Department of Cardiac Medicine, National Heart and Lung Institute, London, U.K.
J Microsc. 1993 Mar;169(Pt 3):299-328. doi: 10.1111/j.1365-2818.1993.tb03308.x.
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)
细胞间连接对于细胞间的相互作用至关重要。借助这些连接，构成组织的单个细胞的活动得以协调，使每个组织系统能够作为一个整体发挥功能。在本综述中，作者关于一种特定类型的连接——心脏缝隙连接——的研究工作作为一个案例模型呈现，以说明一系列显微镜方法作为多学科方法的一部分，如何有助于扩展我们对细胞连接及其功能的理解。在心脏中，缝隙连接形成了快速冲动传导和传播的低电阻通路，使心肌细胞收缩能够同步刺激。缝隙连接还形成了直接细胞间通讯的通路，这一功能在发育过程中的形态发生信号传导中尤为重要。所讨论的工作展示了电子显微镜、免疫细胞化学和共聚焦扫描激光显微镜技术在理解正常成年心脏、发育中的心脏和患病心脏中缝隙连接功能的结构基础方面的一些应用。通过快速冷冻技术制备心脏组织的冷冻断裂电子显微镜检查，其中将与切除相关的细胞结构损伤降至最低或避免，使得推断体内功能性缝隙连接的结构成为可能。在活体动物中正常跳动直至冷冻瞬间的心脏中的缝隙连接由以准晶体排列组织的连接子（跨膜通道）组成，而不是关于缝隙连接功能结构相关性的原始假设中提出的“随机”排列。连接子排列的改变会因缺血和缺氧而发生，尽管这些与缝隙连接通透性的关系是间接的。为了获得用于绘制心脏组织中缝隙连接分布的探针，制备了针对与心脏中报道的主要缝隙连接蛋白连接蛋白43序列部分匹配的合成肽的多克隆抗血清。通过斑点印迹、蛋白质印迹以及对分离的缝隙连接进行免疫金标记来确认抗血清的特异性。发现一种抗血清（针对第131 - 142位残基产生的）作为细胞化学探针特别有效。开发了一种用于共聚焦扫描激光显微镜的免疫荧光标记程序，以实现通过心脏组织厚切片对缝隙连接进行三维精确绘图。（摘要截取自400字）
