The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly.
Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin family member that uses the energy of ATP hydrolysis to depolymerize microtubules. We demonstrated previously that XKCM1 is essential for mitotic-spindle assembly in vitro and acts by regulating microtubule dynamics as a pure protein, in extracts and in cells. A portion of the XKCM1 pool is specifically localized to centromeres during mitosis and may be important in chromosome movement. To selectively analyze the function of centromere-bound XKCM1, we generated glutathione-S-transferase (GST) fusion proteins containing the N-terminal globular domain (GST-NT), the centrally located catalytic domain (GST-CD), and the C-terminal alpha-helical tail (GST-CT) of XKCM1. The GST-NT protein targeted to centromeres during spindle assembly, suggesting that the N-terminal domain of XKCM1 is sufficient for centromere localization. Addition of GST-NT prior to or after spindle assembly replaced endogenous XKCM1, indicating that centromere targeting is a dynamic process. Loss of endogenous XKCM1 from centromeres caused a misalignment of chromosomes on the metaphase plate without affecting global spindle structure. These results suggest that centromere bound XKCM1 has an important role in chromosome positioning on the spindle.
Walczak
Claire E
CE
Medical Sciences, Indiana University, Bloomington, IN 47405, USA. cwalczak@indiana.edu
Gan
Eugene C
EC
Desai
Arshad
A
Mitchison
Timothy J
TJ
Kline-Smith
Susan L
SL
D000595:Amino Acid Sequence; D000818:Animals; D002503:Centromere; D005982:Glutathione Transferase / Q000378:metabolism; D016547:Kinesins / Q000502:physiology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus; D014981:Xenopus; D029867:Xenopus Proteins / Q000502:physiology*
C088335:KIF11 protein, Xenopus; D029867:Xenopus Proteins; D005982:Glutathione Transferase; D016547:Kinesins