Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis.
A-type cyclin-dependent kinases (CDKs), also known as cdc2, are central to the orderly progression of the cell cycle. We made a functional Green Fluorescent Protein (GFP) fusion with CDK-A (Cdc2-GFP) and followed its subcellular localization during the cell cycle in tobacco cells. During interphase, the Cdc2-GFP fusion protein was found in both the cytoplasm and the nucleus, where it was highly resistant to extraction. In premitotic cells, a bright and narrow equatorial band appeared on the cell surface, resembling the late preprophase band, which disintegrated within 10 min as followed by time-lapse images. Cdc2-GFP was not found on prophase spindles but left the chromatin soon after this stage and associated progressively with the metaphase spindle in a microtubule-dependent manner. Arresting cells in mitosis through the stabilization of microtubules by taxol further enhanced the spindle-localized pool of Cdc2-GFP. Toward the end of mitosis, Cdc2-GFP was found at the midzone of the anaphase spindle and phragmoplast; eventually, it became focused at the midline of these microtubule structures. In detergent-extracted cells, the Cdc2-GFP remained associated with mitotic structures. Retention on spindles was prevented by pretreatment with the CDK-specific inhibitor roscovitine and was enhanced by the protein phosphatase inhibitor okadaic acid. Furthermore, we demonstrate that both the endogenous CDK-A and Cdc2-GFP were cosedimented with taxol-stabilized plant microtubules from cell extracts and that Cdc2 activity was detected together with a fraction of polymerized tubulin. These data provide evidence that the A-type CDKs associate physically with mitotic structures in a microtubule-dependent manner and may be involved in regulating the behavior of specific microtubule arrays throughout mitosis.
Weingartner
M
M
Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
Binarova
P
P
Drykova
D
D
Schweighofer
A
A
David
J P
JP
Heberle-Bors
E
E
Doonan
J
J
Bögre
L
L
D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008938:Mitosis; D010947:Plants, Toxic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology*
D002843:Chromatin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016203:CDC2 Protein Kinase