Interregulation of CDKA/CDK1 and the Plant-Specific Cyclin-Dependent Kinase CDKB in Control of the Chlamydomonas Cell Cycle

Abstract

Short title: CDK regulation of the Chlamydomonas cell cycleOne-sentence summary: Chlamydomonas CDKA and CYCA1 are dispensable for mitosis, but the plant-specific CDKB, probably in complex with CYCB1, promotes most events of mitosis and downregulates CDKA following cell division.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Frederick R. Cross (fcross@mail.rockefeller.edu). ABSTRACTThe cyclin-dependent kinase CDK1 is essential for mitosis in fungi and animals. Plant genomes contain the CDK1 ortholog CDKA, and a plant kingdom-specific relative, CDKB. The green alga Chlamydomonas reinhardtii has a long G1 growth period followed by rapid cycles of DNA replication and cell division. We show that null alleles of CDKA extend the growth period prior to the first division cycle and modestly extend the subsequent division cycles, but do not prevent cell division, indicating at most a minor role for the CDK1 ortholog in mitosis in Chlamydomonas. A null allele of cyclin A has a similar though less extreme phenotype. In contrast, both CDKB and cyclin B are essential for mitosis. CDK kinase activity measurements imply that the predominant in vivo complexes are probably cyclin A-CDKA and cyclin B-CDKB. We propose a negative feedback loop: CDKA activates cyclin B-CDKB. Cyclin B-CDKB in turn promotes mitotic entry and inactivates cyclin A-CDKA. Cyclin A-CDKA and cyclin B-CDKB may redundantly promote DNA replication. We show that the anaphase-promoting complex is required for inactivation of both CDKA and CDKB and is essential for anaphase. These results are consistent with findings in Arabidopsis and may delineate the core of plant kingdom cell cycle control which, compared to the well-studied yeast and animal systems, exhibits deep conservation in some respects and striking divergence in others.

Conservation and divergence of DNA replication control inChlamydomonas reinhardtii

“…Since the transgene was expressed in the context of the endogenous ts-lethal mutation, cell cycle progression was dependent on the fluorescent fusion protein in each case. CDKB1-mCherry was first expressed at 10 hrs, maximized around 14 hrs and declined by 24 hrs as previously shown [41]. These times correlate with the period of multiple fission cycles.…”

“…We created universal tagging plasmids containing mCherry, Venus or GFP to be used in Chlamydomonas . The plasmid backbone was generated from CDKB1 plasmid which contains mCherry-tagged CDKB1 gene [41]. The final plasmids contain aphVIII CDS for paramycin resistance that is useful for drug selection in Chlamydomonas , an Ampicillin resistant gene for bacterial selection, and mCherry, GFP or Venus followed by the CDKB1 3’UTR, as well as multiple cloning sites for insertions of desired promoters and coding sequences (Figure 4A).…”

“…All transgenes were constructed with their native promoters, so that we could analyze accumulation of MCM6-mCherry, CDC45-mCherry, ORC1-Venus and CDC6-mCherry fusion proteins at endogenous levels throughout the cell cycle. We included a strain expressing CDKB1-mCherry as a positive control since it is known to accumulate specifically in division-phase cells [41]. Cells were arrested at G1 phase as described above, then released into complete media at 33°C.…”

“…We used a cdc27 cell cycle mutant in order to examine Mcm6 localization in metaphase-blocked cells. CDC27 is a part of the A naphase P romoting C omplex (APC) [42], required for the metaphase-anaphase transition [35, 41]. MCM6 was diffused from the nucleus in metaphase-arrested cdc27 cells, and co-staining of microtubules showed that these cells contained mitotic spindles (Figure 7B).…”

Cleavage-furrow formation without F-actin inChlamydomonas

“…Three different methods were used for cell-cycle synchronization in this study: (i) the 12L:12D/liquid TP method was essentially as described by Fang et al (119) except that TP medium at 26°C was used in place of HSM; (ii) the 12L:12D/TAP agar method was as described previously (120), except that it was carried out at 26°C; and (iii) the -N method was exactly as described previously using a combination of 21°C and 33°C (121). Although overall synchrony and the timing of mitosis and cytokinesis as determined by microscopic examination varied slightly depending on the method, we observed no significant qualitative or quantitative difference in the cells’ response to F-actin perturbation introduced by LatB addition before the onset of cytokinesis.…”