Changes in mobility account for camptothecin-induced subnuclear relocation of topoisomerase I.
DNA topoisomerase I is a nucleolar protein, which relocates to the nucleoplasm in response to drugs stabilizing topoisomerase I.DNA intermediates (e.g. camptothecin). Here we demonstrate that this phenomenon is solely caused by the drug's impact on the interplay between mobility and localization of topoisomerase I in a living cell nucleus. We show by photobleaching of cells expressing biofluorescent topoisomerase I-chimera that the enzyme moves continuously between nucleoli and nucleoplasm. Complex kinetics of fluorescence recovery after photobleaching indicates that two enzyme fractions with different mobility coexist in nucleoli and nucleoplasm. However, the whole complement of topoisomerase I is in continuous flux between these compartments and nucleolar accumulation can plausibly explained by the enzyme's 2-fold lesser overall mobility in nucleoli versus nucleoplasm. Upon addition of camptothecin, topoisomerase I relocates within 30 s from the nucleoli to radial nucleoplasmic structures. At these sites, the enzyme becomes retarded in a dose-dependent manner. Inside nucleoli the mobility of topoisomerase I is much less affected by camptothecin. Thus, the enzyme's distribution equilibrium is shifted toward the nucleoplasm, which causes nucleolar delocalization. In general, topoisomerase I is an entirely mobile nuclear component, unlikely to require specific signaling for movements between nuclear compartments.
Christensen
Morten O
MO
Department of Clinical Chemistry, Medizinische Poliklinik, University of Würzburg, Klinikstrasse 6-8, D-97070 Würzburg, Germany.
Barthelmes
Hans U
HU
Feineis
Silke
S
Knudsen
Birgitta R
BR
Andersen
Anni H
AH
Boege
Fritz
F
Mielke
Christian
C
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D002453:Cell Cycle; D002460:Cell Line; D002466:Cell Nucleolus / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D004622:Embryo, Mammalian; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin