Abstract
Regulation of the outer kinetochore complex Ndc80 is essential to ensure correct kinetochore-microtubule attachments during mitosis. Here, we present a novel mechanism of regulation that is intrinsic to its structure; tight bending of the Ndc80 complex inhibits its microtubule binding. Using single molecule Förster resonance energy transfer (FRET), we show that the Saccharomyces cerevisiae Ndc80 complex can fluctuate between straight and bent forms, and that binding of the complex to microtubules selects for straightened forms. The loop region of the complex enables its bent conformation, as deletion of the loop promotes straightening. In addition, the kinetochore complex MIND enhances microtubule binding by opposing the tightly bent, auto-inhibited conformation of the Ndc80 complex. We suggest that prior to its assembly at the kinetochore, the Ndc80 complex interchanges between bent (auto-inhibited) and open conformations. Once assembled, its association with MIND stabilizes the Ndc80 complex in a straightened form for higher affinity microtubule binding.
Highlights
The highly conserved kinetochore complex Ndc80 (Lampert et al, 2013; Tien et al, 2013; Umbreit et al, 2012; McCleland et al, 2003) is a hub of function within the kinetochore
A yeast Broccoli construct with GFP fused C-terminally to Nuf2 was purified (Broccoli-GFP; Figure 1—figure supplement 1A) and its binding to taxol-stabilized microtubules was observed using single molecule total internal reflection fluorescence (TIRF) microscopy
We have directly examined the functional significance of conformational changes of the Ndc80 complex in vitro
Summary
The highly conserved kinetochore complex Ndc (Lampert et al, 2013; Tien et al, 2013; Umbreit et al, 2012; McCleland et al, 2003) is a hub of function within the kinetochore. Even in instances of Ndc overexpression in yeast or mammalian cells, excess Ndc is not found along the microtubule lattice in the mitotic spindle (Tang and Toda, 2015; Diaz-Rodrıguez et al, 2008) This specific localization to the tips of microtubules mirrors the localization of +TIPs, which often require multiple modes of regulation to ensure accurate targeting to the microtubule plus-end as well as appropriate function (Gireesh et al, 2018). Several studies have suggested that microtubule binding by the Ndc complex is auto-inhibited (Cheeseman et al, 2006; Kudalkar et al, 2015) It remains unclear whether the underlying mechanism of auto-inhibition depends on conformational changes of the complex, or how such regulation might influence Ndc complex function in vivo. Our work reveals a previously unstudied mode of inherent regulation at the kinetochore
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