Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo

Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others.     

Structure of yellow lupine genes coding for mitotic cyclins

Cell cycle progression in eukaryotes is controlled by complexes of p34 protein kinases and cyclins. For the first time in plants, we have established the sequence of four yellow lupine mitotic cyclin B1 genes. Their coding regions and expression pattern were also characterised recently. Structure of all the four lupine genes is similar: they consist of nine exons and eight introns, analogously located, except Luplu;CycB1;3 lacking 7th intron. Analysis of 5'-regulatory sequences of two of them showed that both comprise M-specific activators (MSA), common to plant genes induced in late G2 and early M. Putative repressor binding sites CDE/CHR found in animal G2-specific promoters can also be detected in lupine genes. Controlling region of Luplu;CycB1;4 gene that is highly activated by IAA, contains up to 7 auxin response elements, while insensible to IAA Luplu;CycB1;4 gene have no such motifs. Further studies should be undertaken to determine precisely the functions of putative regulatory elements in the expression of lupine mitotic cyclins.     

Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.     

The division of Drosophila germline stem cells and their precursors requires a specific cyclin

A fundamental yet essentially unexplored question in stem cell biology is whether the stem cell cycle has specific features. Three B-cyclins in Drosophila, Cyclins (Cyc) A, B, and B3, associate with CDK1 and play partially redundant roles in embryogenic mitosis . Here, we show that the division of Drosophila GSCs and their precursors, the primordial germ cells (PGCs), specifically requires CycB. CycB is ubiquitously expressed in both germline and somatic lineages. However, CycB mutation does not have obvious effect on somatic development but causes PGCs to severely under proliferate. Moreover, both female and male CycB mutant GSCs fail to be maintained properly. Removing Cyclin B specifically from female GSCs causes the same defect, confirming the direct and cell-autonomous function of Cyclin B for GSC division. In contrast, two other G2 cyclins, CycA and CycB3, are also expressed in PGCs and GSCs, but overexpressing CycA cannot rescue the CycB mutant defects. These results indicate that the requirement of CycB for PGC and GSC divisions unlikely reflects the insufficient level of G2 cyclins in the CycB mutant but is in favor of a distinct function of CycB in these cells. Our results indicate that stem cells may use specific cell cycle regulators for their division.     

Requirements of cyclin a for mitosis are independent of its subcellular localization

Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. We show that interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Here, we tested if the unique mitotic function of CycA requires nuclear accumulation. We fused subcellular localization signals to CycA and tested their mitotic capability. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA was able to induce mitosis. We noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16(th) embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein.