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. 2007 Aug 13;178(4):583–594. doi: 10.1083/jcb.200610112

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Copyright © 2007, The Rockefeller University Press

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Figure 4. — Behavior and mitotic effects of NudE fragments. (a) Diagram of NudE-binding domains and deletion fragments used in this study: LIS1-binding domain (aa 56–166), CENP-F–binding domain (aa 192–323), and dynein-binding domain (aa 256–344). (b) Kinetochore association of full-length and truncated NudE versus LIS1. GFP-tagged full-length NudE and its deletion constructs were expressed in COS7 cells, treated with nocodazole, and stained with anti-GFP, anti-LIS1, and DAPI. The C-terminal (N189) but not N-terminal (C188) NudE fragment associated with kinetochores. (c) Microtubule organization in LLC-PK1 cells overexpressing NudE fragments revealed severe defects in spindle pole organization after C188 overexpression. No effect was seen in N189-overexpressing cells in comparison with controls. Insets show GFP markers indicating transfection. (d) An LLC-PK1 cell overexpressing a C-terminal fragment of NudE (N189; inset) responsible for kinetochore targeting became arrested in metaphase, as revealed by phase-contrast time-lapse analysis. The chromosomes became pyknotic by 120 min, which is indicative of cell death. (e) Cells overexpressing a C-terminal NudE fragment (N189) showed clear defects in mitotic progression, including mitotic arrest and subsequent cell death (44%), as well as delayed anaphase onset (44%; n = 16). (f) Quantitation of the mitotic index for each deletion fragment showed a 15-fold decrease in mitotic cells after the overexpression of NudE-N189 in comparison with controls. No substantial effect was seen with the overexpression of full-length NudE or NudE-C188. Error bars represent the SEM. Bars (b and c), 5 μM; (d) 10 μM.