Dynamics of Chromatin and the Nucleolus in Arrested Cells.

<p>(A) Time-lapse images of chromatin and the nucleolus. A <i>MET3-CDC20</i> strain expressing the tagged histone, Htb2-mRFP, and the tagged nucleolar protein, Gar1-GFP, was arrested by addition of methionine for 4 hours before imaging. Note that the chromatin mass (red) shifted between the mother (M) and bud (B) domains repeatedly and changed shape in doing so. When most of the chromatin was in the bud domain, a narrow tail (T) extended across the mother domain. When the chromatin was retracting from the bud domain, the trailing edge often had a flattened surface (F) (rectangle). The nucleolus (Nu—green) always remained in the mother domain. In the inserts, the green signal is absent so that the red can be more uniformly detected. The blue images are bright-field. Strain: ATY3175. (B) As in Fig 2A except that the nucleolus and chromatin in the mother domain were further extended. In this situation, the tail in the mother domain defined a narrow ribbon that ends in a red patch (*) at its extremity. Strain: ATY3175. (C) Time-lapse images of chromatin, the spindle and the spindle pole body. A <i>MET3-CDC20</i> strain expressing Htb2-mRFP, Tub1-GFP and the SPB protein, Spc42-CFP, was arrested by addition of methionine for 4 hours and then imaged. The orientation of the spindle (Sp) shifted and the entire spindle frequently moved from one domain to the other along with chromatin. As shown in the lower images, the SPBs (arrow) remained associated with both extremities of the spindle. Strain: ATY7135. (D-E) Localization of centromeres (D) and kinetochores (E). The tagged variant histone, Cse4-GFP, and the tagged kinetochore protein, Ndc10-GFP, were localized in cycling cells (upper panels) and in a <i>MET3-CDC20</i> strain after arrest for 4 hours by addition of methionine (lower panels). Cycling cells: In most unbudded cells (Unb), both Cse4-GFP and Ndc10-GFP formed single clusters, while they had doubled in cells with small buds (Sm) or medium-sized buds (Md) and in cells in anaphase (Ana). Arrested cells: Note the frequent presence of additional fluorescent foci (fragments) for both Cse4-GFP and Ndc10-GFP. Both cells expressing Cse4-GFP with the tag at an internal position and cells with the tag at the C-terminus exhibited comparable fragmentation. Strains: ATY6196, ATY6882, ATY6833. (F) Quantitation of the number of foci detected in preparations equivalent to Fig 2D and 2E. Strains: ATY6196 and ATY6882.