Treatment ofVicia fabaroot tip cells with specific inhibitors to cyclin‐dependent kinases leads to abnormal spindle formation

Abstract

Many events during cell division are triggered by an evolutionary conserved regulator, the cyclin‐dependent kinase (Cdk). Here we used two novel drugs, the purine analogues bohemine and roscovitine, to study the role of Cdks in cell cycle progression and microtubule organisation inVicia fabaroot tip cells. Both drugs inhibited the activity of immunopurifiedVicia fabaand alfalfa Cdc2‐kinase. The transcript levels of an A‐ and B‐type cyclin, as well as of thecdc2genes, declined in treated root tips, while the mRNA level of a D‐type cyclin gene was not affected. An observed transient arrest at the G1/S and G2/M regulatory points indicated that inhibition of the Cdc2‐kinase had an effect on both transitions. In contrast to the regular bipolar spindle in untreated cell, in drug‐treated metaphase cells abnormally short and dense kinetochore microtubule fibres were observed. These microtubules were randomly arranged in the vicinity of the kinetochores and connected the chromosomes. Thus, the chromosomes were not aligned on the metaphase plate but were arranged in a circle, with kinetochores pointing inwards and chromosome arms pointing outwards. γ‐Tubulin, which plays a role in microtubule nucleation, also localised to the centre of the monopolar spindle. The observed abnormalities in mitosis, after inhibition of Cdc2‐kinase by specific drugs, suggest a role for this enzyme in regulating some of the steps leading to a bipolar spindle structure.