Role for microtubules in centrosome doubling in Chinese hamster ovary cells.

The centrosome must be replicated once, and only once, during each cell cycle. To achieve this somatic cells need to synthesize centrosome proteins, target those centrosome proteins to the parental centrosome, and then assemble 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 doubling events. For these experiments, CHO cells were arrested in either hydroxyurea (HU) alone or in HU plus a microtubule inhibitor for 3640 h. The cells then were induced to enter mitosis and the numbers of spindle poles/centrosomes were counted following processing of the cells for immunofluorescence microscopy using anticentrosome antiserum. These studies demonstrated that centrosome replication events occurred in cells arrested with either HU alone or HU and taxol while centrosome replication did not occur in cells treated with HU and either nocodazole or colcemid. Immunoblot analysis determined that centrosome proteins were synthesized in HU/nocodazole-arrested cells and demonstrated that the role of microtubules in the centrosome replication process is not to ensure the synthesis of centrosome subunits. Rather, our results suggest that microtubules may be involved in the transport/targeting of centrosome subunits to the parental centrosome during duplication events. For microtubules to contribute to the transport of centrosome subunits during centrosome doubling, centrosome subunits would need to be able to bind to microtubules. To test this, co-sedimentation studies were performed and it was determined that the centrosome proteins, though overproduced under these conditions, remained soluble in HU/nocodazole-treated cells and co-pelleted with taxol-stabilized microtubules in the presence of GTP and AMP-PNP. Moreover, co-sedimentation of one of the centrosome proteins, PCM-1, with microtubules could be inhibited by pre-incubation of extracts with antibodies against dynactin. Together, these data suggest that during centrosome replication in somatic mammalian cells, PCM-1, and perhaps other centrosome components, are targeted to the centrosome via transport along microtubules by motor complexes that include dynein/dynactin.

Localization of autoepitopes on the PCM-1 autoantigen using scleroderma sera with autoantibodies against the centrosome.

Characterization of epitope domains of autoantigens is important for deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portion of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434-1465, and 1661-1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434-1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.