title: TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule.
abstract: The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼ 200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays.
authors.affiliation: the Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003.
authors.forename: Sai K
authors.initials: SK
authors.lastname: Balchand
authors.affiliation: the Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Mann
authors.affiliation: the Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003.
authors.forename: Janel
authors.initials: J
authors.lastname: Titus
authors.affiliation: the Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Physics and.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Ross
authors.affiliation: the Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003 From the Departments of Biology and.
authors.forename: Patricia
authors.initials: P
authors.lastname: Wadsworth
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
chemical_list: D018797:Cell Cycle Proteins; C470243:KIF11 protein, human; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C122167:TPX2 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016547:Kinesins
keywords: Eg5; TPX2; kinesin; microtubule; microtubule-associated protein (MAP); mitosis; mitotic spindle; spindle