Multicolor and electron microscopic imaging of connexin trafficking.
Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.
Gaietta
Guido
G
National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Deerinck
Thomas J
TJ
Adams
Stephen R
SR
Bouwer
James
J
Tour
Oded
O
Laird
Dale W
DW
Sosinsky
Gina E
GE
Tsien
Roger Y
RY
Ellisman
Mark H
MH
D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001152:Arsenicals / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D018031:Connexin 43 / Q000096:biosynthesis / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004705:Endocytosis; D005089:Exocytosis; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D009942:Organometallic Compounds / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D001152:Arsenicals; D018031:Connexin 43; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D010078:Oxazines; C454115:ReAsH-EDT2 compound; D011994:Recombinant Proteins; D015100:3,3'-Diaminobenzidine; D003545:Cysteine