IN-VIVO ASSEMBLY KINETICS OF FLUORESCENTLY LABELED XENOPUS LAMIN-A MUTANTS

The assembly kinetics of Xenopus wild type lamin A and 7 mutants were analyzed by the microinjection of renatured 5-iodoacet-amidofluorescei n (5-IAF)-labeled protein into mouse 3T3 cells. This experimental syst em has the advantage that the masking of the mutant phenotype by the f ormation of heterooligomeric complexes with newly synthesized lamins o f the microinjected cell is negligibly small. The wild type protein an d all mutants containing a nuclear localization signal were transporte d within 10 min into the nucleus. The wild type protein exhibited a st rong lamina fluorescence 30 min after microinjection, whereas mutant m olecules showed a delayed but complete, a delayed and incomplete, or n o lamina assembly at all. The lamin A mutant lacking the carboxy-termi nal cysteine of the CxxM-motif exhibited a delayed but complete assemb ly, whereas previously performed transfection experiments demonstrated no significant interaction of this mutant with the lamina. According to present knowledge this phenotype in transfected cells can be explai ned by an overexpression of this mutant. We have identified an additio nal domain in the carboxy-terminal tail of lamin A that promotes its a ssembly into the lamina. In vitro this domain is required for the chro matin binding of lamin A. Lamin A molecules lacking the non-helical am ino-terminal head domain showed no significant lamina staining, wherea s point mutations in conserved regions of the alpha helix resulted in an incomplete assembly.