Figure 4. The synthetic kinetochore requires many components of natural kinetochores.

A) Dissection of the genetic requirements for the synthetic kinetochore. Cells carrying temperature-sensitive mutations in a variety of kinetochore proteins were analyzed for their ability to biorient a chromosome carrying a synthetic kinetochore at 37 °C. The control (Syn. Kt.) contains the synthetic kinetochore but does not have any kinetochore temperature-sensitive mutations. Graphs in part A and B show the mean values ± s.d. of 3 experiments where at least 200 cells per genotype were counted per experiment. The values for the percent with 2 separated dots are the following: syn. kt. 55 ± 2, dam1-1 15 ± 5, ndc80-1 11 ± 6, nuf2-61 5 ± 3, spc24-1 5 ± 3, nsl1-54 14 ± 9, dsn1-7 7 ± 1, mtw1-1 11 ± 5, okp1-5 17 ± 11, ctf13-30 50 ± 3, ndc10-1 41 ± 5. B) Mutants that allow the synthetic kinetochore to biorient prevent biorientation of natural kinetochores. A natural kinetochore was analyzed for its ability to biorient with either an ndc10-1 or ctf13-30 temperature sensitive mutation. The wildtype control (wt kt) does not have any kinetochore temperature-sensitive mutations. The values for the percent with 2 separated dots are the following: wt. kt. 74 ± 1, ndc10-1 18 ± 1, ctf13-30 24 ± 4. C) A cartoon showing the location of proteins whose absence keeps the synthetic kinetochore from biorienting (shown in red). Proteins that are dispensable for the synthetic kinetochore are shown in blue, and proteins listed in black were not tested. Components of the DNA-binding complex are outlined in green. Components of the microtubule-binding complex are outlined in purple.