Assembling the Protein Architecture of the Budding Yeast Kinetochore-Microtubule Attachment using FRET

Abstract

Background The kinetochore is a multiprotein machine that couples chromosome movement to microtubule (MT) polymerization and depolymerization. It uses numerous copies of at least three MT-binding proteins to generate bidirectional movement. The nanoscale organization of these proteins within the kinetochore plays an important role in shaping the mechanisms that drive persistent, bidirectional movement of the kinetochore. Results We used Förster Resonance Energy Transfer (FRET) between genetically encoded fluorescent proteins fused to kinetochore subunits to reconstruct the nanoscale organization of the budding yeast kinetochore. We performed > 60 FRET and high resolution colocalization measurements involving the essential MT-binding kinetochore components: Ndc80, Dam1, Spc105, and Stu2. These measurements reveal that neighboring Ndc80 complexes within the kinetochore are narrowly distributed along the length of the MT. Dam1 complex molecules are concentrated near the MT-binding domains of Ndc80. Stu2 localizes in high abundance within a narrowly defined territory within the kinetochore centered ~ 20 nm on the centromeric side of the Dam1 complex. Conclusions Our data show that the microtubule attachment site of the budding yeast kinetochore is well-organized. Ndc80, Dam1 and Stu2 are all narrowly distributed about their average positions along the kinetochore-MT axis. The relative organization of these components, their narrow distributions, and their known MT-binding properties together elucidate how their combined actions generate persistent, bidirectional kinetochore movement coupled to MT polymerization and depolymerization.

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

“…The N-terminal tail of Ndc80 cross-linked to multiple regions of the complex (Figure S2,C and D). This is consistent with the extended length of the tail (at least 15 nm, Aravamudhan et al 2014) and its predicted disordered nature (Wei et al 2005(Wei et al , 2007Ciferri et al 2008;Alushin et al 2010), allowing it to reach multiple parts of the complex in a cross-linking reaction.…”

“…It was previously proposed that the loop acts as a hinge, conferring the flexibility observed for recombinant Ndc80 complexes on negative-stain electron micrographs (Wang et al 2008). Super-resolution microscopy experiments further suggested that different conformations of the Ndc80 complex exist in vivo; the distance between the two ends of the Ndc80 complex decreases from $40 nm to $20 nm during the metaphaseto-anaphase transition in S. cerevisiae (Aravamudhan et al 2014). Altogether, these results predict that the Ndc80 complex undergoes a conformational change through bending at the loop region.…”

“…The coiled-coil domains are interrupted by a disordered "loop" region that is thought to enable flexion of the complex, as observed for recombinant Ndc80 complexes on negative-stain electron micrographs (Wang et al 2008). The Ndc80 complex adopts elongated and bent conformations in vivo during metaphase and anaphase, respectively (Joglekar et al 2009;Aravamudhan et al 2014). However, it remains to be determined if these conformations arise from the flexibility of the complex at the loop observed in vitro.…”

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