Lopez P, Balicki D, Buehler L K, Falk M M, Chen S C
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Cell Commun Adhes. 2001;8(4-6):237-42. doi: 10.3109/15419060109080730.
To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.
为了研究活细胞中缝隙连接的结构组成和动力学，我们用自发荧光示踪剂绿色荧光蛋白（GFP）及其青色（CFP）和黄色（YFP）变体标记了它们的亚基蛋白，即连接蛋白。标记的连接蛋白能正常组装，通道也具有功能。由CFP和YFP标记的连接蛋白组装而成的缝隙连接斑的高分辨率荧光图像显示，通道的分布模式严格取决于连接蛋白亚型。观察到了共分布以及分隔到完全分离的区域的现象。基于相关研究，我们提出通道分布受内在的、连接蛋白亚型特异性信号调控。高分辨率延时图像显示，与之前的预期相反，缝隙连接是通道的动态组装体。簇内的通道以及簇本身都是可移动的，并且不断经历结构重排。运动很复杂，使得通道能够移动，这与其他未锚定在细胞骨架元件上的质膜蛋白类似。然而，综合分析表明，缝隙连接通道的运动并非由推动质膜蛋白运动的扩散所驱动。相反，最近的研究表明，缝隙连接通道的运动是间接的，主要由代谢性内吞和外排导致的质膜脂质流动所推动。
