Centrosome duplication is tightly coupled with the cell cycle and neither too many nor too few centrosomes are induced in a normal cell. To study how centrosome assembly is regulated, we analyzed the abnormal process of multiple centrosome replications in Chinese hamster ovary (CHO) cells induced by hydroxyurea (HU), which is known to uncouple the centrosome cycle from the cell cycle. Green fluorescent protein (GFP)-tagged centrin2 expressed in CHO cells labels both centrioles and the pericentriolar material (PCM). Counting fluorescent spots of GFP-centrin in synchronized cells showed that in G1/S-arrested cells, centrioles are initially duplicated in a template manner. Further treatment with HU overrides the suppression of excess centriole/centrosome replication in a cell where the full complement of centrioles/centrosomes already exists. Time-lapse fluorescence microscopy revealed that small centrin-containing foci emerged in the cytoplasm during HU treatment. These foci are surrounded by a PCM cloud and their number continuously increases as cells are exposed to HU for longer periods of time. Both the centrosome and cytoplasmic foci are highly mobile, continuously changing their position in a manner dependent on microtubules/microtubule dynamics. The centrosome number increases as small foci grow in size and resolve into recognizable centrosomes. As this occurs in a random fashion, the cells arrested longer with HU induced highly heterogeneous numbers of centrosomes.
- CHO cells
- Pericentriolar material
- Time-lapse fluorescence microscopy