title: Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2.
abstract: 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.
authors.affiliation: Department of Biology, University of Massachusetts Amherst, Amherst, MA 01003, USA.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Gable
authors.forename: Minhua
authors.initials: M
authors.lastname: Qiu
authors.forename: Janel
authors.initials: J
authors.lastname: Titus
authors.forename: Sai
authors.initials: S
authors.lastname: Balchand
authors.forename: Nick P
authors.initials: NP
authors.lastname: Ferenz
authors.forename: Nan
authors.initials: N
authors.lastname: Ma
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Collins
authors.forename: Carey
authors.initials: C
authors.lastname: Fagerstrom
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Ross
authors.forename: Ge
authors.initials: G
authors.lastname: Yang
authors.forename: Patricia
authors.initials: P
authors.lastname: Wadsworth
mesh_terms: D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D018374:LLC-PK1 Cells; D051379:Mice; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D013552:Swine
chemical_list: D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C521052:Kif11 protein, mouse; D004398:Dyneins; D016547:Kinesins