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Fig. 4. The plus-end localisation of CLIP-190 depends on EB1. S2 cells were immunostained with antibodies against EB1, CLIP-190 and {alpha}-tubulin. (A) Colocalisation of CLIP-190 and EB1 to the microtubule plus-ends during interphase. Both proteins accumulate near microtubule plus-ends and weakly localise along the microtubules. The region marked with a rectangular box is magnified below each panel. (B) Distinct localisation of CLIP-190 and EB1 in mitosis. CLIP-190 localises to the kinetochores at prometaphase, whereas EB1 localises to spindle and aster microtubules without accumulation at kinetochores. (C) Conserved domain structure of CLIP-190. CLIP-190 has two CAP-Gly domains in its N-terminus, followed by a coiled-coil region and two zinc finger motifs in its C-terminus. Subregions of CLIP-190 shown here were expressed in bacteria as GST-fusion proteins. (D) Physical interaction between CLIP-190 and EB1. GST-tagged subdomains of CLIP-190 and GST alone were expressed in bacteria and bound to glutathione beads. After washing, the beads were incubated in bacterial lysates containing MBP-EB1 or MBP alone (to control for the MBP-part of the fusion protein). The glutathione beads were washed again and analysed by immunoblotting using an MBP antibody. The N-terminal domain (CAP-Gly domain) of CLIP-190 directly interacts with EB1. (E) Delocalisation of CLIP-190 in EB1-depleted cells. S2 cells were immunostained for CLIP-190, {alpha}-tubulin and EB1 after EB1 depletion. The region marked with a box is magnified below each panel. Localisation of CLIP-190 to microtubule plus-ends was abolished (compare with A). (F) The proportion of cells with CLIP-190 at microtubule plus-ends after control and EB1 RNAi (n=100 cells scored each). Grey boxes indicate robust localisation of CLIP-190 to microtubule plus-ends, whereas open boxes indicate a greatly reduced signal. (G) The localization of CLIP-190 to unattached kinetochores is unaffected in EB1 RNAi. S2 cells were immunostained for DNA, CLIP-190 and EB1 after EB1 depletion. CLIP-190 was still be seen on unattached kinetochores in EB1 RNAi (arrowheads; compare with B). Bars, 10 µm.