Investigation of nuclear architecture with a domain-presenting expression system.
We have investigated the topogenic properties of the nucleus by ectopic expression of chimeric proteins consisting of a NLS-modified cytoplasmic filament-forming protein, Xenopus laevis vimentin, and domains of inner nuclear membrane proteins. Whereas the "carrier" without cargo, the NLS-vimentin alone, is deposited in a few nuclear body-type structures (J.M. Bridger, H. Herrmann, C. Münkel, P. Lichter, J. Cell Sci., 111, 1241-1253), the distribution is entirely changed upon coupling with the evolutionarily conserved domain of the lamin B tail, the entire lamin B tail, the amino-terminal nucleoplasmic segment of the lamin B receptor (LBR), and the LEM domain of emerin, respectively. Remarkably, every individual chimeric protein exhibits a completely different distribution. Therefore, we assume that the chimeric parts are specifically recognized by factors engaged in nucleus-specific topogenesis. Thus, the conserved domain of the lamin B tail results in the formation of many small accumulations spread all over the nucleus. The chimera with the complete lamin B tail is deposited in short fibrillar aggregates within the nucleus. It does not mediate the integration of the chimeric protein into the nuclear membrane in cultured cells, indicating that the lamin tail alone is not sufficient to direct the integration of a protein into the lamina in vivo. In contrast, in the nuclear assembly system of Xenopus laevis the recombinant NLS-vimentin-lamin tail protein is concentrated at the nuclear membrane. The LBR chimera is arranged in a "beaded-chain"-type fashion, quite different from the more random deposition of NLS-vimentin alone. To our surprise, the LEM domain of emerin induces the retention of most of the chimeric proteins within the cytoplasm. Hence, it appears to be engaged in a strong cytoplasmic interaction that overrides the nuclear localization signal. Finally, the lamin chimera with the conserved part of the lamin B tail is shown to recruit LBR to the nuclear vimentin bodies and, vice versa, the LBR chimera attracts lamin B in transfected cells, thereby demonstrating their bona fide interaction in vivo.
Dreger
Christine K
CK
Division for Cell Biology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, Germany.
König
Alexandra R
AR
Spring
Herbert
H
Lichter
Peter
P
Herrmann
Harald
H
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001695:Biology / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D002474:Cell-Free System; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006801:Humans; D034882:Lamins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis; D000097682:Lamin B Receptor
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D034882:Lamins; D008164:Luminescent Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D000097682:Lamin B Receptor; C054562:yellow fluorescent protein, Bacteria