Figure 1. Reconstitution of a kinetochore from individually purified parts and an optical trap-based assay to test for self-assembly of functional chains of kinetochore subcomplexes.

(A) Schematic of the protein complexes of the budding yeast kinetochore. (B) Coomassie-stained SDS-PAGE gel of heterologously expressed budding yeast kinetochore proteins. The asterisk indicates a contaminating E. coli protein or degradation product. (C) Schematic of the optical trap assay used to test for assembly and microtubule attachment prior to quantification of load-bearing ability. (D) Representative force vs. time traces for ruptures in the force-ramp assay.

Figure 1—figure supplement 1. Schematic diagram, drawn approximately to scale, showing two possible bead-microtubule configurations.

Our rupture force assay quantifies the strength of end-on attachments. In this configuration, reconstituted kinetochores on only ~ 3.0% of the bead surface are capable of simultaneously binding to the microtubule. Assuming that the ~ 2900 protein complexes on each bead are evenly distributed,~86 would be capable of binding the microtubule surface. Lateral attachments likely predominate in our self-assembly and microtubule-binding assay. In this configuration, the bead rests against the side of a filament whose tip extends well past the point of contact, maximizing the amount of bead surface in close proximity to the microtubule. Thus, it provides an upper limit for the fraction of bead surface within 84 nm of the microtubule. Reconstituted kinetochores on ~ 9.4% of the bead surface,~270 protein complexes, would be capable of simultaneously binding to the microtubule in this lateral configuration.