Molecular architecture of a kinetochore-microtubule attachment site.
Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes. Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore-microtubule attachment using quantitative fluorescence microscopy of GFP-tagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone, the copy number ranges from one or two for inner kinetochore proteins such as Mif2p, to 16 for the DAM-DASH complex at the kinetochore-microtubule interface. These counts allow us to visualize the overall arrangement of a kinetochore-microtubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments.
Joglekar
Ajit P
AP
Department of Biology, University of North Carolina, 607 Fordham hall, CB# 3280, Chapel Hill, NC 27599, USA.
Bouck
David C
DC
Molk
Jeffrey N
JN
Bloom
Kerry S
KS
Salmon
Edward D
ED
D001665:Binding Sites; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry; D049452:Green Fluorescent Proteins; D018386:Kinetochores / Q000737:chemistry* / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry; D008941:Spindle Apparatus
C092746:CSE4 protein, S cerevisiae; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C083768:MIF2 protein, S cerevisiae; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins