Individual kinetochores assembled de novo onto centromeric DNAs capture microtubules. (A) Schematic of the in vitro kinetochore assembly assay. Individual Atto565-labeled centromeric DNAs were tethered sparsely onto a PEG passivated coverslip surface through biotin-avidin linkages. The surface-tethered DNAs were then incubated for 60 min with yeast whole-cell lysate derived from strains with GFP-tagged kinetochore components (Ndc10, Cse4, Ctf19, or Ndc80). Kinetochores assembled spontaneously onto the centromeric DNAs and were then imaged with TIRF microscopy after washing out the extract. (B) Kinetochore subcomplexes colocalized with wild type centromeric DNAs. Locations of Atto565-labeled centromeric DNAs (yellow circles) were mapped onto images of GFP-tagged kinetochore subcomplexes (white spots). Scale bar, 2 µm. (C) Percentages of centromeric DNAs that colocalized with a GFP signal from indicated kinetochore proteins. Bars show average colocalization ± SEM calculated from N > 3,400 DNAs for each kinetochore component from at least nine fields of view recorded across three independent experiments. (D) Assembled Ndc80-GFP kinetochores (cyan) readily captured Alexa647 microtubules (magenta) by their tips (top row of images), and sometimes by their sides (bottom row). Tip-captured and side-captured microtubules were easily distinguished by the relative locations of the kinetochore GFP spots and by the Brownian movement of the filaments. The distal ends of tip-captured microtubules swiveled freely in three dimensions (3D), exploring a hemispherical space above the coverslip. Side-captured microtubules mainly rotated in a two-dimensional (2D) plane parallel to the coverslip in a propeller-like fashion.