Guo Yan-Ming, Chen Shiuhwei, Shetty Premnath, Zheng Genhua, Lin Rueyling, Li Wen-hong
Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390, USA.
Nat Methods. 2008 Sep;5(9):835-41. doi: 10.1038/nmeth.1238.
To study the physiological regulation and function of cell-cell gap junction communication in vivo, we developed a bioconjugate of caged dye, named dextran-CANPE-HCC, for imaging cell coupling in small model organisms. In vitro, the compound was photolyzed efficiently with robust fluorescence enhancement. Dextran-CANPE-HCC delivered into Caenorhabditis elegans oocytes was retained in cells throughout development. Using local uncaging, we photolyzed dextran-CANPE-HCC to release the small HCC dye and imaged the dynamics of intercellular dye transfer through gap junction channels, a technique we named Trojan-local activation of molecular fluorescent probes (LAMP). Early during embryonic development, the pattern of cell coupling undergoes dramatic remodeling and imaging revealed that the germ cell precursors, P2, P3 and P4, were isolated from the somatic cell communication compartment. As dextran-CANPE-HCC is chemically and metabolically stable, Labeled worms showed very bright signal upon photoactivation after hatching, which allowed us to examine cell coupling in living worms noninvasively.
为了研究体内细胞间缝隙连接通讯的生理调节和功能，我们开发了一种名为葡聚糖 - CANPE - HCC的笼锁染料生物共轭物，用于在小型模式生物中成像细胞偶联。在体外，该化合物经光解后荧光显著增强。注入秀丽隐杆线虫卵母细胞的葡聚糖 - CANPE - HCC在整个发育过程中都保留在细胞内。我们利用局部解笼技术，光解葡聚糖 - CANPE - HCC以释放小分子HCC染料，并对通过缝隙连接通道的细胞间染料转移动力学进行成像，这项技术我们称为特洛伊木马 - 分子荧光探针局部激活（LAMP）。在胚胎发育早期，细胞偶联模式经历了显著重塑，成像显示生殖细胞前体P2、P3和P4与体细胞通讯区室分离。由于葡聚糖 - CANPE - HCC在化学和代谢上都很稳定，标记的线虫在孵化后光激活时显示出非常明亮的信号，这使我们能够无创地检测活线虫中的细胞偶联。
