Molecular architecture of a kinetochore–microtubule attachment site

Abstract

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 [1][2][3][4][5][6][7][8][9] . 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 GFPtagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone 1 , the copy number ranges from one or two for inner kinetochore proteins such as Mif2p 2 , to 16 for the 9 at the kinetochoremicrotubule interface. These counts allow us to visualize the overall arrangement of a kinetochoremicrotubule 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.Accurate segregation of sister chromosomes during mitosis depends on the assembly of structural proteins at the kinetochore that link spindle microtubule plus-ends to centromeric DNA (CEN DNA). The structural arrangement of these proteins within the kinetochore underlies its function in force generation. It may also influence how the spindle assembly checkpoint senses kinetochore-microtubule attachment, and how errors in attachment are corrected to prevent chromosome mis-segregation. Although serial-section transmission electron microscopy has revealed the overall three-dimensional architecture of vertebrate kinetochores, the structure of individual kinetochore-microtubule attachment remains poorly characterized. Consequently, a mechanistic model of kinetochore function that integrates the details of its structure cannot currently be constructed. To understand the molecular architecture of a kinetochore-microtubule attachment site, we focused on counting the copy number for the core structural kinetochore proteins and protein complexes that are necessary for stable kinetochore-microtubule attachment. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Localization of antibodies to Ndc80 in vertebrate cells suggests that the Ndc80p-Nuf2p end of the NDC80 complex localizes proximal to the microtubule attachment site, whereas the other end localizes proximal to the inner centromere7 , 15. In budding yeast, the NDC80 complex and the microtubule associated protein complex, DAM-DASH, are both necessary for microtubule attachment10 , 11. The DAM-DASH complex is a heterodecamer and contains the protein Ask1p. Purified DAM-DASH complexes assemble into rings aro...

Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins

“…As mentioned earlier, the yeast kinetochore is built around a single centromeric nucleosome that contains two Cse4p molecules (Collins et al, 2004;Joglekar et al, 2006;Meluh et al, 1998). Thus, the signal from a kinetochore cluster expressing Cse4p-GFP represents the fluorescence of 32 GFP molecules (16 kinetochores with 2 Cse4p-GFP molecules per kinetochore).…”

Condensin Regulates the Stiffness of Vertebrate Centromeres

“…The quantification was determined by quantitative fluorescence of CENP-H: GFP (in cells where GFP was knocked into the endogenous single copy CENP-H gene) relative to the copy number of 8 Ndc80 molecules per budding yeast kinetochore according to the method previously described (Joglekar et al, 2006).…”

“…CENP-A is a modified histone H3 specific for inner kinetochore chromatin (Warburton et al, 1997;Marshall et al, 2008), and CENP-H purifies with CENP-A containing mononucleosomes in vitro (Foltz et al, 2006). 2) Although we cannot exclude that levels of some kinetochore proteins may differ in condensin-depleted kinetochores, loss of condensin had no detectable effect on the absolute number of CENP-H molecules per kinetochore (29 in SMC2 ON ; 31 in SMC2 OFF ) measured by quantitative fluorescence (Figure 2e) relative to the amount of Ndc80 at budding yeast kinetochores (Joglekar et al, 2006). 3) The ratio of CENP-A to CENP-H was unaltered in the presence and absence of condensin ( Figure 2e).…”

Rings, bracelets, sleeves, and chevrons: new structures of kinetochore proteins

“…In yeast, the Dam1 complex has been measured by immunoblot analysis to be present at 650 copies per cell, just enough for one 16-mer ring around each of 32 kinetochore microtubules (8). In fact, fluorescence microscopy measures 16-20 Dam1 complexes in each budding yeast kinetochore at metaphase (18). The quantification is consistent with a single ring around each kinetochore microtubule, but a ring has never been seen near the plus-end of the microtubule even when examined by electron tomography with a resolution of ~5 nm.…”

“…The ring may be sensitive to freeze substitution and therefore not visualized by these methods. At anaphase, each kinetochore only contains 10 -11 Dam1 complexes, suggesting the 16-mer ring is not needed during anaphase when microtubules undergo rapid depolymerization (18), a time when a ring would seem to be most important. The nature of the Dam1 structure in cells remains to be determined and could be altered by binding to the Ndc80 complex and other kinetochore components.…”