Identification of two novel components of the human NDC80 kinetochore complex.
Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.
Bharadwaj
Rajnish
R
Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Qi
Wei
W
Yu
Hongtao
H
D000595:Amino Acid Sequence; D017209:Apoptosis; D002118:Calcium / Q000737:chemistry; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D003080:Cold Temperature; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D018386:Kinetochores / Q000378:metabolism / Q000648:ultrastructure*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus; D013997:Time Factors; D014162:Transfection
D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; C501384:MAD1L1 protein, human; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C484695:SPC24 protein, human; C484694:SPC25 protein, human; D002118:Calcium