Characterization of the early events of centrosome duplication in mammalian cells and the roles of tektin in centrosome duplication, midbody formation and cytokinesis.

Centrosome duplication normally occurs once per cell cycle. While centriole number determines the number of centrosomes, it remains unclear whether or not centrosomes can duplicate in the absence of centriole elongation. To test this, Chinese hamster ovary cells were arrested in S-phase, and thus were capable of undergoing repeated rounds of duplication without coordinate cell cycle progression. Microtubule polymerization was subsequently inhibited, allowing the centrosome duplication cycle to continue but preventing the elongation of centrioles. By immunofluorescence, it was found that after 60h of S-phase arrest in the absence of microtubule polymerization, multiple Ìøå¤-tubulin-containing structures form, often close to the nuclear envelope. By electron microscopy, it was verified that these structures lacked centrioles. After colcemid is removed, these structures assembled morphologically and functionally normal centrosomes. Importantly, the number of centrosomes that assemble following colcemid removal is similar to the number of true centrioles that assemble during S-phase arrest alone. Therefore, the reduplication of centrosomes during S-phase arrest does not require an intact microtubule network. In addition, there is a duplication event that repeatedly occurs during S-phase that does not require the formation or elongation of daughter centrioles. Such a duplication event could represent the formation of a centrosome precursor structure, which has the capacity to assemble a complete centrosome. Tektins represent a conserved family of proteins are thought to confer structural stability to the axonemal doublets. However, the role of tektins in somatic cells is unclear. Here, the possible roles of tektins in centrosome duplication and cytokinesis are examined in mammalian cells. To determine the role of tektin in centrosome duplication, siRNA of tektin 1 or 3 in S-phase arrested CHO cells was shown to prevent centrosome reduplication, indicating that that these proteins are required for the centrosome to duplicate. Additionally, siRNA of tektin 2 in mitotic cells prevented the formation of the midbody, and caused the mislocalization of known midbody matrix proteins. Therefore, a key role for tektins in the centrosome duplication cycle has been shown. In addition, a novel function for tektin 2 maintaining the midbody and completing cytokinesis has been shown.