PROPERTIES OF FLUORESCENTLY LABELED XENOPUS LAMIN-A IN-VIVO

Wild type Xenopus lamin A, a Iamin A mutant lacking the carboxy-termin al cysteine (C662 --> S), and human vimentin were expressed in bacteri a and fluorescently labeled with 5-iodoacetamidofluorescein (5-IAF). I n vitro reconstitution experiments and microinjection of both lamins i nto living cells revealed that they were indistinguishable from the no n-fluorescently labeled proteins. When the 5-IAF lamin A was microinje cted into the cytoplasm of 3T3 cells it was rapidly transported into t he nucleus, giving rise within 1 h to a strong lamina fluorescence, wh ereas the lamin A mutant formed dotlike intranuclear aggregates. 5-IAF lamin A associated with the nuclear envelope of microinjected 3T3 cel ls and 5-IAF vimentin which was incorporated into the preexisting vime ntin filaments of this cell Line, were analyzed by photobleaching empl oying two different methods, (i) scanning microphotolysis using a modi fied laser scanning microscope, and (ii) the conventional photobleachi ng technique in which the integral fluorescence of a single spot was m easured by photon counting. A low but significant fluorescence recover y was measured within 10 min for both 5-IAF-labeled intermediate filam ent proteins, Iamin A and vimentin, in bleached areas of the nuclear e nvelope and the cytoplasmic intermediate filaments, respectively.