Targeting of RCC1 to chromosomes is required for proper mitotic spindle assembly in human cells.
Ran GTPase is involved in several aspects of nuclear structure and function, including nucleocytoplasmic transport and nuclear envelope formation. Experiments using Xenopus egg extracts have shown that generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 also plays roles in mitotic spindle assembly. Here, we have examined the localization and function of RCC1 in mitotic human cells. We show that RCC1, either the endogenous protein or that expressed as a fusion with green fluorescent protein (GFP), is localized predominantly to chromosomes in mitotic cells. This localization requires an N-terminal lysine-rich region that also contains a nuclear localization signal and is enhanced by interaction with Ran. Either mislocalization of GFP-RCC1 by removal of the N-terminal region or the expression of dominant Ran mutants that perturb the GTP/GDP cycle causes defects in mitotic spindle morphology, including misalignment of chromosomes and abnormal numbers of spindle poles. These results indicate that the generation of Ran-GTP in the vicinity of chromosomes by RCC1 is important for the fidelity of mitotic spindle assembly in human cells. Defects in this system may result in abnormal chromosome segregation and genomic instability, which are characteristic of many cancer cells.
Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, United Kingdom.
William
W
Moore
Chuanmao
C
Zhang
Paul R
PR
Clarke
D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007399:Interphase / Q000502:physiology; D008938:Mitosis / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C062521:RCC1 protein, human; D011993:Recombinant Fusion Proteins; D020931:ran GTP-Binding Protein