Background information: Centrosome duplication normally parallels with DNA replication and is responsible for correct segregation of replicated DNA into the daughter cells. Although geminin interacts with Cdt1 to prevent loading of MCMs (minichromosome maintenance proteins) on to the replication origins, inactivation of geminin nevertheless causes centrosome over-duplication in addition to the re-replication of the genome, suggesting that geminin may play a role in centrosome duplication. However, the exact mechanism by which loss of geminin affects centrosomal duplication remains unclear and the possible direct interaction of geminin with centrosomal-localized proteins is still unidentified.
Results: We report in the present study that geminin is physically localized to the centrosome. This unexpected geminin localization is cell-cycle dependent and mediated by the actin-related protein, Arp1, one subunit of the dynein-dynactin complex. Disruption of the integrity of the dynein-dynactin complex by overexpression of dynamitin/p50, a well-characterized inhibitor of dynactin, reduces the centrosomal localization of both geminin and Arp1. Enrichment of geminin on centrosomes was enhanced when cellular ATP production was suppressed in the ATP-inhibitor assay, whereas the accumulation of geminin on the centrosome was disrupted by depolymerization of the microtubules using nocodazole. We further demonstrate that the coiled-coil motif of geminin is required for its centrosomal localization and the interaction of geminin with Arp1. Depletion of geminin by siRNA (small interfering RNA) in MDA-MB-231 cells led to centrosome over-duplication. Conversely, overexpression of geminin inhibits centrosome over-duplication induced by HU in S-phase-arrested cells, and the coiled-coil-motif-mediated centrosomal localization of geminin is required for its inhibition of centrosome over-duplication. Centrosomal localization of geminin is conserved among mammalian cells and geminin might perform as an inhibitor of centrosome duplication.
Conclusions: The results of the present study demonstrate that a fraction of geminin is localized on the centrosome, and the centrosomal localization of geminin is Arp1-mediated and dynein-dynactin-dependent. The coiled-coil motif of geminin is required for its targeting to the centrosome and inhibition of centrosome duplication. Thus the centrosomal localization of geminin might perform an important role in regulation of proper centrosome duplication.