Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells.
The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.
Izumi
M
M
Biodesign Research Group, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, 351-0198, Japan.
Vaughan
O A
OA
Hutchison
C J
CJ
Gilbert
D M
DM
D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D004261:DNA Replication; D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection
D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C440308:lamin C; D013752:Tetracycline