Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living cells.
We report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br). The dye readily translocated to the NE at physiological temperature where it exhibited enhanced fluorescence when excited at 620-650 nm in contrast to 480-520 nm excitation in the endocytic pathway and in the endoplasmic reticulum (ER). In vitro data indicated that the dye reveals an enrichment of negatively charged lipids, presumably due to local phospholipid synthesis. Dual-excitation imaging of FM4-64 in relation to lamina-associated polypeptide-1-green fluorescent protein during mitosis suggested that the disassembly of NE preserves microscale lipid complexes in the ER. Convolutions of NE in cancer or primary cells were readily visualized, and killing of tumor cells by T cells was marked by sudden loss of the long-wavelength excited fluorescence in the NE coincident with apoptosis. This report of FM4-64 as the first vital dye sensitive to the NE environment opens new ways for real-time visualization and functional studies of the NE.
Zal
Tomasz
T
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA. tzal@mdanderson.org
Zal
M Anna
MA
Lotz
Carina
C
Goergen
Craig J
CJ
Gascoigne
Nicholas R J
NR
D002470:Cell Survival; D002478:Cells, Cultured; D003902:Detergents; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000737:chemistry* / Q000187:drug effects; D011726:Pyridinium Compounds / Q000737:chemistry* / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000737:chemistry* / Q000494:pharmacology
D003902:Detergents; C092350:FM 4-64; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds