In Vivo Protein Architecture of the Eukaryotic Kinetochore with Nanometer Scale Accuracy

Abstract

Summary The kinetochore is a macromolecular protein machine [1] that links centromeric chromatin to the plus-ends of one or more microtubules (MT), and segregates chromosomes during cell division. Its core structure consists of eight multi-component protein complexes, most of which are conserved in all eukaryotes. We use an in vivo two-color fluorescence microscopy technique to determine, for the first time, the location of these proteins along the budding yeast kinetochore axis at nanometer resolution. Together with kinetochore protein counts [2, 3], these localizations predict the 3-D protein architecture of a kinetochore-microtubule attachment, and provide new functional insights. We also find that the kinetochore becomes much shorter in anaphase as metaphase tension is lost. Shortening is mainly due to a decrease in the length of the Ndc80 complex, which may result either from intra-molecular bending of the Ndc80 complex at the kink within the stalk region of the Ndc80/Nuf2 dimer [4, 5] or from a change in its orientation relative to the microtubule axis. Conformational changes within the Ndc80 and Mtw1 complexes may serve as mechanical cues for tension-dependent regulation of MT attachment and the spindle assembly checkpoint. The geometry of the core structure of the budding yeast kinetochore reported here is remarkably similar to that found in mammalian kinetochores, indicating that kinetochore structure is conserved in eukaryotes with either point or regional centromeres.

Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

“…The Ndc80 complex hinges about a flexible loop region, and in vitro and in vivo evidence supports the existence of both a folded conformation and an elongated state (8,11). Here, we show that a temperature-sensitive NDC80 mutant (ndc80-121) that has been previously suggested to affect the stability of a folded complex at 37°C exhibits increased microtubule affinity at 37°C compared with the WT complex.…”

“…Deleting the loop domain of Ndc80 causes lethality, suggesting that flexibility of the complex is necessary in vivo (12). Additional in vivo evidence suggests that a folded Ndc80 complex is important during early mitosis whereas an elongated conformation exists at metaphase (8)(9)(10). We propose that the Ndc80 complex adopts different conformations to modulate the strength of its microtubule attachment.…”

“…Additionally, substantial evidence suggests that the outer kinetochore Ndc80 complex undergoes conformational changes throughout mitosis (8)(9)(10). The Ndc80 complex hinges about a flexible "loop" region and exists in both a folded conformation and an elongated state (8,11). In vivo evidence supports the physiological significance of these conformational changes (10,12), underscoring the importance of different conformations of the Ndc80 complex.…”

“…Additionally, substantial evidence suggests that the outer kinetochore Ndc80 complex undergoes conformational changes throughout mitosis (8)(9)(10). The Ndc80 complex hinges about a flexible "loop" region and exists in both a folded conformation and an elongated state (8,11).…”

Stepwise unfolding supports a subunit model for vertebrate kinetochores

“…Although numerous immuno-electron microscopy (16)(17)(18) and superresolution microscopy (19)(20)(21) studies have mapped the locations of CCAN components relative to one another, the packing of the chromatin fiber in centrochromatin remains unknown. Early studies of stretched chromosomes suggested a repeating "subunit" structure for the kinetochore (1,22).…”