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Fig. 5. The cell-cycle regulation of microtubule plus-end localisation. (A,B) S2 cells were stained simultaneously for EB1 and CLIP-190. (A) The CLIP-190 signal at microtubule plus-ends (either interphase or astral microtubules) is strong in interphase but very weak in mitosis. By contrast, the EB1 signal is constant in both mitosis and interphase. Bar, 10 µm. (B) Higher magnification images of interphase (upper panels) and mitotic cells (lower) marked by the squares in A. (C) Cell-cycle change of CLIP-190 and EB1 localisation to microtubule plus-ends. The plus-end signal-intensity relative to background was measured for each cell-cycle stage (a total of ten microtubules from three different cells were scored for each mitotic stage) and is shown as a circle with the standard deviation represented by vertical bars. CLIP-190 dissociates from microtubule plus-ends during mitosis. (D) Levels of EB1 and CLIP-190 at microtubule plus-ends in mitotic cells after control (n=30 microtubules from three different cells) and Dhc (n=18 microtubules from three different cells) RNAi. In Dhc-depleted cells, CLIP-190 does not localise to kinetochores, but still dissociates from microtubule plus-ends during mitosis. (E) Cell-cycle regulation of CLIP-190 localisation. Molecular requirements for CLIP-190 localisation during the cell cycle are illustrated. During interphase, CLIP-190 localises to microtubule plus-ends in an EB1-dependent manner. The association of CLIP-190, but not EB1, to microtubule ends is greatly reduced during mitosis. Instead, CLIP-190 is localised to unattached kinetochores in mitosis. This localisation depends on dynein-dynactin and Lis1. Dynein-dynactin and Lis1 localisation are mutually dependent on each other, and both depend on Rod. Upon attachment to microtubules, CLIP-190 is removed from kinetochores by the motor-activity of dynein.
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