Hec1 contributes to mitotic centrosomal microtubule growth for proper spindle assembly through interaction with Hice1.
Previous studies have stipulated Hec1 as a conserved kinetochore component critical for mitotic control in part by directly binding to kinetochore fibers of the mitotic spindle and by recruiting spindle assembly checkpoint proteins Mad1 and Mad2. Hec1 has also been reported to localize to centrosomes, but its function there has yet to be elucidated. Here, we show that Hec1 specifically colocalizes with Hice1, a previously characterized centrosomal microtubule-binding protein, at the spindle pole region during mitosis. In addition, the C-terminal region of Hec1 directly binds to the coiled-coil domain 1 of Hice1. Depletion of Hice1 by small interfering RNA (siRNA) reduced levels of Hec1 in the cell, preferentially at centrosomes and spindle pole vicinity. Reduction of de novo microtubule nucleation from mitotic centrosomes can be observed in cells treated with Hec1 or Hice1 siRNA. Consistently, neutralization of Hec1 or Hice1 by specific antibodies impaired microtubule aster formation from purified mitotic centrosomes in vitro. Last, disruption of the Hec1/Hice1 interaction by overexpressing Hice1DeltaCoil1, a mutant defective in Hec1 interaction, elicited abnormal spindle morphology often detected in Hec1 and Hice1 deficient cells. Together, the results suggest that Hec1, through cooperation with Hice1, contributes to centrosome-directed microtubule growth to facilitate establishing a proper mitotic spindle.
Wu
Guikai
G
Department of Biological Chemistry, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA.
Wei
Randy
R
Cheng
Eric
E
Ngo
Bryan
B
Lee
Wen-Hwa
WH
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D018385:Centrosome / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; C000628759:HAUS8 protein, human; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; C490197:NUF2 protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins