title: Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics.
abstract: Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.
authors.affiliation: National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA. gsosinsky@ucsd.edu
authors.forename: Gina E
authors.initials: GE
authors.lastname: Sosinsky
authors.forename: Guido M
authors.initials: GM
authors.lastname: Gaietta
authors.forename: Galen
authors.initials: G
authors.lastname: Hand
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.forename: Areum
authors.initials: A
authors.lastname: Han
authors.forename: Mason
authors.initials: M
authors.lastname: Mackey
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Adams
authors.forename: James
authors.initials: J
authors.lastname: Bouwer
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002863:Chromogenic Compounds / Q000737:chemistry; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry*; D017629:Gap Junctions / Q000378:metabolism / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron
chemical_list: D002863:Chromogenic Compounds; D018031:Connexin 43; D005452:Fluoresceins; D003545:Cysteine