Protein architecture of the human kinetochore microtubule attachment site.
Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.
Wan
Xiaohu
X
Department of Biology, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599, USA.
O'Quinn
Ryan P
RP
Pierce
Heather L
HL
Joglekar
Ajit P
AP
Gall
Walt E
WE
DeLuca
Jennifer G
JG
Carroll
Christopher W
CW
Liu
Song-Tao
ST
Yen
Tim J
TJ
McEwen
Bruce F
BF
Stukenberg
P Todd
PT
Desai
Arshad
A
Salmon
E D
ED
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000737:chemistry* / Q000378:metabolism*; D008677:Metaphase; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D009687:Nuclear Proteins
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C472643:Knl1 protein, human; C492664:MIS12 protein, human; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins