Abstract

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.