Role for microtubules in centrosome doubling in Chinese hamster ovary cells

The centrosome must be replicated once, and only once, during each cell cyc le. To achieve this somatic cells need to synthesize centrosome proteins, t arget those centrosome proteins to the parental centrosome, and then assemb le the centrosome subunits into a functional organelle. The mechanisms that underlie each of these processes are not known. Studies were performed to investigate whether cellular microtubules are involved in centrosome doubli ng events. For these experiments, CHO cells were arrested in either hydroxy urea (HU) alone or in HU plus a microtubule inhibitor for 36-40 h. The cell s then were induced to enter mitosis and the numbers of spindle poles/centr osomes were counted following processing of the cells for immunofluorescenc e microscopy using anticentrosome antiserum. These studies demonstrated tha t centrosome replication events occurred in cells arrested with either HU a lone or HU and taxol while centrosome replication did not occur in cells tr eated with HU and either nocodazole or colcemid. Immunoblot analysis determ ined that centrosome proteins were synthesized in HU/nocodazole-arrested ce lls and demonstrated that the role of microtubules in the centrosome replic ation process is not to ensure the synthesis of centrosome subunits. Rather , our results suggest that microtubules may be involved in the transport/ta rgeting of centrosome subunits to the parental centrosome during duplicatio n events. For microtubules to contribute to the transport of centrosome sub units during centrosome doubling, centrosome subunits would need to be able to bind to microtubules. To test this, co-sedimentation studies were perfo rmed and it was determined that the centrosome proteins, though overproduce d under these conditions, remained soluble in HU/nocodazole-treated cells a nd co-pelleted with taxol-stabilized microtubules in the presence of GTP an d AMP-PNP. Moreover, co-sedimentation of one of the centrosome proteins, PC M-1, with microtubules could be inhibited by pre-incubation of extracts wit h antibodies against dynactin. Together, these data suggest that during cen trosome replication in somatic mammalian cells, PCM-1, and perhaps other ce ntrosome components, are targeted to the centrosome via transport along mic rotubules by motor complexes that include dynein/dynactin. Cell Motil. Cyto skeleton 42:60-72, 1999. (C) 1999 Wiley-Liss, Inc.