Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends

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Abstract
CLIP-170 was the first microtubule plus-end-tracking protein to be described, and is implicated in the regulation of microtubule plus-ends and their interaction with other cellular structures. Here, we have studied the cell-cycle-dependent mechanisms which localise the sole Drosophila melanogaster homologue CLIP-190. During mitosis, CLIP-190 localises to unattached kinetochores independently of spindle-checkpoint activation. This localisation depends on the dynein-dynactin complex and Lis1 which also localise to unattached kinetochores. Further analysis revealed a hierarchical dependency between the proteins with respect to their kinetochore localisation. An inhibitor study also suggested that the motor activity of dynein is required for the removal of CLIP-190 from attached kinetochores. In addition, we found that CLIP-190 association to microtubule plus-ends is regulated during the cell cycle. Microtubule plus-end association is strong in interphase and greatly attenuated during mitosis. Another microtubule plus-end tracking protein, EB1, directly interacts with the CAP-Gly domain of CLIP-190 and is required to localise CLIP-190 at microtubule plus-ends. These results indicate distinct molecular requirements for CLIP-190 localisation to unattached kinetochores in mitosis and microtubule ends in interphase.