The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity.
We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.
McCleland
Mark L
ML
Department of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, Virginia 22908, USA.
Gardner
Richard D
RD
Kallio
Marko J
MJ
Daum
John R
JR
Gorbsky
Gary J
GJ
Burke
Daniel J
DJ
Stukenberg
P Todd
PT
D000705:Anaphase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D029721:Drosophila Proteins; D000072159:Dynactin Complex; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018816:Genes, cdc; D019476:Insect Proteins / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism / Q000648:ultrastructure*; D007976:Leupeptins / Q000494:pharmacology; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008938:Mitosis; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D008941:Spindle Apparatus / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; C070352:BUB3 protein, S cerevisiae; C489446:Bub3 protein, Xenopus; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D000072159:Dynactin Complex; D005656:Fungal Proteins; D019476:Insect Proteins; D007976:Leupeptins; C501382:MAD1 protein, S cerevisiae; C577695:MAD2 protein, S cerevisiae; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D008869:Microtubule-Associated Proteins; C112472:NDC80 protein, S cerevisiae; C087340:NUF2 protein, S cerevisiae; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; C076055:Zw10 protein, Drosophila; D011494:Protein Kinases; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde