title: Tracking Dynamic Gap Junctional Coupling in Live Cells by Local Photoactivation and Fluorescence Imaging.
abstract: Intercellular communication through gap junction channels is crucial for maintaining cell homeostasis and synchronizing physiological functions of tissues and organs. In this chapter, we present a noninvasive fluorescence imaging assay termed LAMP (local activation of a molecular fluorescent probe) that consists of the following steps: loading cells with a caged and cell permeable coumarin probe (NPE-HCCC2/AM), locally photolyzing the caged coumarin in one or a subpopulation of coupled cells, monitoring cell-cell dye transfer by digital fluorescence microscopy, and post-acquisition analysis to quantify the rate of junction dye transfer using Fick's equation. The LAMP assay can be conveniently carried out in fully intact cells to assess the extent and degree of cell coupling, and is compatible with other fluorophores emitting at different wavelengths to allow multicolor imaging. Moreover, by carrying out multiple photo-activations in a coupled cell pair, LAMP assay can track changes in cell coupling strength between coupled cells, hence providing a powerful method for investigating the regulation of junctional coupling by cellular biochemical changes.
authors.affiliation: State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Center for R&D of Fine Chemicals, Guizhou University, Huaxi St., Guiyang, 550025, China.
authors.forename: Song
authors.initials: S
authors.lastname: Yang
authors.affiliation: Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-9039, USA. wen-hong.li@utsouthwestern.edu.
authors.forename: Wen-Hong
authors.initials: WH
authors.lastname: Li
mesh_terms: D002450:Cell Communication / Q000502:physiology*; D018031:Connexin 43 / Q000378:metabolism*; D003374:Coumarins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D017629:Gap Junctions / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D010782:Photolysis; D061251:Primary Cell Culture
chemical_list: D018031:Connexin 43; D003374:Coumarins; D005456:Fluorescent Dyes; C500216:GJA1 protein, human; D049452:Green Fluorescent Proteins
keywords: Caged coumarin; Caged dye; Gap junction communication; Infrared-LAMP assay; LAMP assay; NPE-HCCC2/AM; Photoactivation