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-iodoacetamidofluorescein (5-IAF)-labeled protein into mouse 3T3 cells. This experimental system has the advantage that the masking of the mutant phenotype by the formation of heterooligomeric complexes with newly synthesized lamins of the microinjected cell is negligibly small. The wild type protein and all mutants containing a nuclear localization signal were transported within 10 min into the nucleus. The wild type protein exhibited a strong lamina fluorescence 30 min after microinjection, whereas mutant molecules showed a delayed but complete, a delayed and incomplete, or no lamina assembly at all. The lamin A mutant lacking the carboxy-terminal cysteine of the CxxM-motif exhibited a delayed but complete assembly, whereas previously performed transfection experiments demonstrated no significant interaction of this mutant wih the lamina. According to present knowledge this phenotype in transfected cells can be explained by an overexpression of this mutant. We have identified an additional domain in the carboxy-terminal tail of lamin A that promotes its assembly into the lamina. In vitro this domain is required for the chromatin binding of lamin A. Lamin A molecules lacking the non-helical amino-terminal head domain showed no significant lamina staining, whereas point mutations in conserved regions of the alpha helix resulted in an incomplete assembly.
Schmidt
M
M
Division of Electron Microscopy, Theodor Boveri Institute, University of Würzburg, Germany.
Krohne
G
G
D016475:3T3 Cells / Q000648:ultrastructure; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005452:Fluoresceins; D007700:Kinetics; D007797:Laminin / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D017384:Sequence Deletion; D014981:Xenopus
D005452:Fluoresceins; D007797:Laminin; C015296:5-iodoacetamidofluorescein