title: Detection and quantification of microtubule detachment from centrosomes and spindle poles.
abstract: Microtubule detachment from microtubule organizing centers is an important cellular process required for normal cell proliferation. When cells enter mitosis, microtubule turnover increases along with a concurrent increase in microtubule detachment. MCAK, a kinesin-related protein whose abundance is highest during the early stages of mitosis, has been shown to regulate microtubule detachment. Abnormal increases or decreases in the frequency of detachment interfere with spindle function and inhibit cell division. It has been shown that drugs able to promote microtubule assembly (e.g., paclitaxel, epothilones) prevent cell division by suppressing microtubule detachment from centrosomes. Conversely, cytotoxic concentrations of microtubule destabilizing drugs (e.g., vinblastine, nocodazole), tubulin mutations that cause paclitaxel resistance, and specific β-tubulin isotypes increase the frequency of microtubule detachment. In this chapter, we describe a method to calculate the frequency of microtubule detachment by transfecting cells with EGFP-MAP4 and directly observing detachment by live cell imaging.
authors: Department of Physiology and Pharmacology, Snyder Institute for Chronic Disease, University of Calgary, Calgary, Alberta, Canada.
authors: Anutosh
authors: A
authors: Ganguly
authors: Hailing
authors: H
authors: Yang
authors: Fernando
authors: F
authors: Cabral
mesh_terms: D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism*; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D016547:Kinesins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D022101:Microtubule-Organizing Center / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D012235:Rhodamines / Q000378:metabolism; D064047:Spindle Poles / Q000378:metabolism*; D013194:Staining and Labeling; D014747:Vinblastine / Q000494:pharmacology
chemical_list: C091489:KIF2C protein, human; D008869:Microtubule-Associated Proteins; D012235:Rhodamines; D049452:Green Fluorescent Proteins; D014747:Vinblastine; D016547:Kinesins; D017239:Paclitaxel
keywords: Centrosome; Katanin; MAP4; MCAK; Microtubule fragments; Microtubule release; Paclitaxel; Spindle poles; Tubulin isotypes; Tubulin mutations