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Vol. 19, Issue 8, 3426-3441, August 2008

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Rapid Cycling and Precocious Termination of G1 Phase in Cells Expressing CDK1AF

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305-5174

Submitted February 19, 2008; Revised April 25, 2008; Accepted May 6, 2008
Monitoring Editor: Mark Solomon

In Xenopus embryos, the cell cycle is driven by an autonomous biochemical oscillator that controls the periodic activation and inactivation of cyclin B1-CDK1. The oscillator circuit includes a system of three interlinked positive and double-negative feedback loops (CDK1 -> Cdc25 -> CDK1; CDK1 Wee1 CDK1; and CDK1 Myt1 CDK1) that collectively function as a bistable trigger. Previous work established that this bistable trigger is essential for CDK1 oscillations in the early embryonic cell cycle. Here, we assess the importance of the trigger in the somatic cell cycle, where checkpoints and additional regulatory mechanisms could render it dispensable. Our approach was to express the phosphorylation site mutant CDK1AF, which short-circuits the feedback loops, in HeLa cells, and to monitor cell cycle progression by live cell fluorescence microscopy. We found that CDK1AF-expressing cells carry out a relatively normal first mitosis, but then undergo rapid cycles of cyclin B1 accumulation and destruction at intervals of 3–6 h. During these cycles, the cells enter and exit M phase-like states without carrying out cytokinesis or karyokinesis. Phenotypically similar rapid cycles were seen in Wee1 knockdown cells. These findings show that the interplay between CDK1, Wee1/Myt1, and Cdc25 is required for the establishment of G1 phase, for the normal 20-h cell cycle period, and for the switch-like oscillations in cyclin B1 abundance characteristic of the somatic cell cycle. We propose that the HeLa cell cycle is built upon an unreliable negative feedback oscillator and that the normal high reliability, slow pace and switch-like character of the cycle is imposed by a bistable CDK1/Wee1/Myt1/Cdc25 system.

* Present address: Department of Biology and The Biocomplexity Institute, Indiana University, 212 S. Hawthorne Dr., Simon Hall (SI-MSB), Room 043F, Bloomington, IN 47405-7003.

Address correspondence to: James E. Ferrell, Jr. (james.ferrell{at}stanford.edu)

Abbreviations used: ADB, antibody-diluting buffer; APC, anaphase-promoting complex; CDK1, cyclin-dependent kinase 1; CFP, cyan fluorescent protein; d-siRNA, diced small interfering RNA; GFP, green fluorescent protein; MBS, mitotic biosensor; NEB, nuclear envelope breakdown; NER, nuclear envelope reformation; PCNA, proliferating cell nuclear antigen; pHH3, phospho-histone H3; RFP, red fluorescent protein; RT, room temperature; YFP, yellow fluorescent protein.

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