Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation

Abstract

Kinetochores assemble on centromeric DNA and present arrays of proteins that attach directly to the dynamic ends of microtubules. Kinetochore proteins coordinate at the microtubule interface through oligomerization, but how oligomerization contributes to kinetochore function has remained unclear. Here, using a combination of biophysical assays and live-cell imaging, we find that oligomerization of the Dam1 kinetochore complex is required for its ability to form microtubule attachments that are robust against tension in vitro and in vivo. An oligomerization-deficient Dam1 complex that retains wild-type microtubule binding activity is primarily defective in coupling to disassembling microtubule ends under mechanical loads applied by a laser trap in vitro. In cells, the oligomerization-deficient Dam1 complex is unable to support stable bipolar alignment of sister chromatids, indicating failure of kinetochore-microtubule attachments under tension. We propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis.

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

“…Anti-His 5 polystyrene beads (11 pM) were incubated with 40 nM His 6 -tagged Ndc80 or MIND complex as described (40)(41)(42), such that each bead was decorated with ∼1,800 protein complexes. Protein-coated beads were introduced into the flow chambers in BB80 with 1 mM GTP, 1.4 mg·mL −1 tubulin, 200 μg·mL −1 glucose oxidase, 35 μg·mL −1 catalase, 25 mM glucose, and 1 mM DTT.…”

How Kinetochore Architecture Shapes the Mechanisms of Its Function

“…In budding yeast, the Dam1 complex, which is recruited by Ndc80, is essential for force generation [38, 65]. Dam1 molecules likely assemble in the form of an oligomeric ring encircling the microtubule [7, 33, 66]. The Dam1 ring mechanically opposes the outward curling of tubulin protofilaments during depolymerization, and thus, experiences a poleward force [67, 68].…”

Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

“…The contribution of Dam1c to tipcoupling is highest when it is flexibly tethered [155] and when free Dam1c is also present in solution [152,162], presumably because these conditions facilitate oligomerization of Dam1c into a microtubule-encircling ring. A partial Dam1 sub-complex that is specifically deficient in oligomerization forms tip attachments that are far less stable than those formed by the full, wild-type complex [162]. However, direct evidence that the enhancements in tip-coupling afforded by Dam1c oligomers depend on curling protofilaments is lacking.…”

“…Evidence supporting this model is compelling but not definitive [159]. Oligomeric Dam1c rings seem to be ideal structures for harnessing protofilament curls [117,118,160,161], and Dam1c does indeed make a major contribution to the stability and strength of kinetochore-microtubule coupling in vitro [43,162], acting as a processivity factor to enhance Ndc80c-based coupling [154,163]. The contribution of Dam1c to tipcoupling is highest when it is flexibly tethered [155] and when free Dam1c is also present in solution [152,162], presumably because these conditions facilitate oligomerization of Dam1c into a microtubule-encircling ring.…”