Properties of fluorescently labeled Xenopus lamin A in vivo.
Wild type Xenopus lamin A, a lamin A mutant lacking the carboxy-terminal cysteine (C662-->S), and human vimentin were expressed in bacteria and fluorescently labeled with 5-iodoacetamidofluorescein (5-IAF). In vitro reconstitution experiments and microinjection of both lamins into living cells revealed that they were indistinguishable from the non-fluorescently labeled proteins. When the 5-IAF lamin A was microinjected into the cytoplasm of 3T3 cells it was rapidly transported into the nucleus, giving rise within 1 h to a strong lamina fluorescence, whereas the lamin A mutant formed dotlike intranuclear aggregates. 5-IAF lamin A associated with the nuclear envelope of microinjected 3T3 cells and 5-IAF vimentin which was incorporated into the preexisting vimentin filaments of this cell line, were analyzed by photobleaching employing two different methods, (i) scanning microphotolysis using a modified laser scanning microscope, and (ii) the conventional photobleaching technique in which the integral fluorescence of a single spot was measured by photon counting. A low but significant fluorescence recovery was measured within 10 min for both 5-IAF-labeled intermediate filament proteins, lamin A and vimentin, in bleached areas of the nuclear envelope and the cytoplasmic intermediate filaments, respectively.
Schmidt
M
M
Division of Electron Microscopy, Theodor Boveri Institute, University of Würzburg, Germany.
Tschödrich-Rotter
M
M
Peters
R
R
Krohne
G
G
D016475:3T3 Cells; D000818:Animals; D005452:Fluoresceins; D034904:Lamin Type A; D034882:Lamins; D051379:Mice; D009685:Nuclear Envelope / Q000378:metabolism* / Q000528:radiation effects; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry; D014981:Xenopus
D005452:Fluoresceins; D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; C015296:5-iodoacetamidofluorescein