Dynamic reorganization of Eg 5 in the mammalian spindle throughout mitosis requires dynein and TPX 2

Kinesin-5 is an essential mitotic motor. However, how its spatial–temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification–tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end–directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end–directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2. INTRODUCTION Molecular motors play essential roles in mitosis, but how they are localized and regulated within the spindle during mitosis remains poorly understood. Eg5, a member of the kinesin-5 family, was one of the first mitotic motors identified and has subsequently been shown to be essential during mitosis in diverse cells (Le Guellec et al., 1991; Hoyt et al., 1992; Roof et al., 1992; Sawin et al., 1992; Heck et al., 1993; Blangy et al., 1995; Reddy and Day, 2001; Bannigan et al., 2007). Structurally, Eg5 forms bipolar homotetramers, with motor domains located at each end of an elongated molecule (Cole et al., 1994; Blangy et al., 1995; Kashina et al., 1996). This organization allows Eg5 to cross-link and slide adjacent parallel and antiparallel microtubules, a behavior that has been directly visualized in vitro (Kapitein et al., 2005; van den Wildenberg et al., 2008). Compared with other kinesin motor proteins, Eg5 is relatively slow and weakly processive (Sawin et al., 1992; Cole et al., 1994; Kwok et al., 2006; Korneev et al., 2007). Functional regulation of Eg5 may be mediated through its phosphorylation by cyclin-dependent kinase 1 in its tail domain (Blangy et al., 1995), which increases Eg5 binding to microtubules in vitro and is required for localizing the motor to spindle microtubules in cells (Blangy et al., 1995; Sawin and Mitchison, 1995; Cahu et al., 2008). Additionally, Eg5 is regulated by the microtubuleassociated protein TPX2, which localizes the motor to spindle microtubules and modulates motor activity (Ma et al., 2011). Monitoring Editor Kerry S. Bloom University of North Carolina Received: Sep 28, 2011 Revised: Feb 3, 2012 Accepted: Feb 9, 2012 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E11-09-0820) on February 15, 2012. Address correspondence to: P. Wadsworth (patw@bio.umass.edu). © 2012 Gable et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: BAC, bacterial artificial chromosome; CC1, coiled coil-1; EGFP, enhanced GFP; FBS, fetal bovine serum; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; HRP, horseradish peroxidase; IgG, immunoglobulin G; LAP, localization and affinity purification; ROI, region of interest; shRNA, short hairpin RNA; STLC, S-trityl-l-cysteine; TIRF, total internal reflection fluorescence.