The spindle checkpoint in the dinoflagellate Crypthecodinium cohnii.

Dinoflagellates are a major group of organisms with an extranuclear spindle. As the purpose of the spindle checkpoint is to ensure proper alignment of the chromosomes on the spindle, dinoflagellate cell cycle control may be compromised to accomodate the extranuclear spindle. In the present study, we demonstrated that nocodazole reversibly prolonged the G2 + M phase of the dinoflagellate cell cycle, in both metaphase and anaphase. The regulation of the spindle checkpoint involves the activation and inhibition of the anaphase promoting complex (APC), which in turn degrades specific cell cycle regulators in the metaphase to anaphase transition. In Crypthecodinium cohnii, nocodazole was also able to induce a prolongation of the degradation of mitotic cyclins and a delay in the inactivation of p13(suc1)-associated histone kinase activities. In addition, cell extracts prepared from C. cohnii in G1 phase and G2/M phase (or nocodazole treated) were able to activate and inhibit, respectively, the degradation of exogenous human cyclin B1 in vitro. The present study thus demonstrated the presence of the spindle checkpoint and APC-mediated cyclin degradation in dinoflagellates. This is discussed in relation to a possible role of the nuclear membrane in mitosis in dinoflagellates.

Cyclins in a dinoflagellate cell cycle.

The dinoflagellates are distinct eukaryotes in having and extranuclear spindle and permanently condensed chromosomes. These cytologic features implicate special adaptations to the molecular mechanisms of cell cycle control. We have demonstrated the presence of cyclin-box-containing polypeptides in dinoflagellates by immunoblotting using peptide-generated antibodies. We identified four major cyclin-box-containing polypeptides. The cell cycle dynamics of these polypeptides were also investigated in synchronized populations, using a newly developed method. Of the four major cyclin-box-containing polypeptides detected, a triplex with apparent molecular weight of 75 kDa did not change appreciably during the cell cycle. For two other cyclin-box-containing polypeptides with apparent molecular weights of 50 and 65 kDa, we observed an early expression in the cell cycle, with the level accumulating and eventually being degraded on the exit of mitosis. At least on cyclin-box-containing polypeptide (50 kDa) was also observed in a protein complex bound to p13suc1 beads. The bound complex head associated histone kinase activity. Variation of this activity corresponded well with the periodic expression of the 50-kDa cyclin-box-containing polypeptide during the cell cycle of Crypthecodinium cohnii. This demonstrates the presence of cyclins and cyclin-dependent kinases in dinoflagellates.