To confirm and further detail the effect of a microtubule-poisoning drug on the centriole growth, we tested the effect of BAY 43-9006 nocodazole on centrosome overduplication induced by Plk4 overexpression in S phase at a concentration that disrupts the microtubule network. In order to have a sensitive read-out for centriole overduplication after Plk4 overexpression, we quantified the number of newly formed procentrioles per mother centriole. Indeed, the inducible expression of Plk4 in a U2OS/plk4 cell line results in the accumulation of Plk4 at the parental centriole which LY294002 drives the formation of variable numbers of centrioles ranging from 2 to 9 as indicated by the staining of the centriolar marker centrin-2. The induction of Plk4 overexpression promotes the accumulation of centrosome proteins such as hSAS-6, CPAP, CP110 or Centrin-2 at the parental centriole forming a ring or a halo initiating the sprouting of procentrioles. Consistent with previous work, application of nocodazole during the centriole overduplication decreased the proportion of cells with more than three procentrioles when compared to the control cells. Concomitantly, the proportion of cells with no or one procentriole increased. Interestingly, mother centrioles without daughter centriole still recruited Plk4, and the formation of a halo as indicated by the accumulation of Centrin-2 was still apparent suggesting that while the initial events of the centriole duplication take place in the presence of nocodazole, procentriole growth may be defective. The disruption of the microtubule network by nocodazole is unlikely to be responsible for this inhibition because the inactivation of the dynein mediated transport by a dominant negative approach has no effect on centrosome duplication. Thus, these observations suggest that nocodazole may directly inhibit centriole overduplication by blocking the growth of centriolar tubules. Our previous work showed that the growth of procentrioles start between 6 and 16 hours after induction of Plk4. To determine whether nocodazole depolymerizes centriolar tubules, we added the drug 12 hours after the induction of Plk4 to allow for the initiation of centriolar tubule growth. Surprinsingly, drug addition at this stage had no effect on centriole overduplication indicating that the nocodazole did not depolymerize centriolar tubules. Together with the observation that nocodazole inhibits centriole duplication, our results indicate that nocodazole inhibits the polymerization of centriolar tubules early during the procentriole assembly process. We next tested the effect of nocodazole during the normal oneround duplication of the centrosome in the same cell line. U2OS cells in G1 were treated with nocodazole and the duplication state of the centrosome were analysed 15 hours later by staining the centrosome with the centriole marker CP110 which is recruited at growing procentrioles.