The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit

Abstract

The Ndc80 kinetochore complex is essential for robust kinetochore-microtubule (k-MT) attachments during mitosis. Ndc80 has been shown to recruit the Ska1 complex to kinetochores, where Ska1 is thought to aid in k-MT coupling by Ndc80. Our previous work has shown that Cdt1, a DNA replication licensing factor, is a novel mitotic spindle-associated protein that is also recruited to kinetochores via Ndc80 and is required for stabilizing k-MT attachments. In this study, we developed auxin-induced degron (AID)-tagging to validate the previously demonstrated mitotic role of Cdt1. We demonstrate a direct interaction between Cdt1 and Ska1 that is essential for proper recruitment of Cdt1 to kinetochores and spindle microtubules. We find that Cdk1-dependent phosphorylation of Cdt1 during mitosis is critical for Ska1-binding, consequently regulating the stabilization of metaphase k-MT attachments and normal mitotic progression. Total internal reflection fluorescence microscopy (TIR-FM) experiments reveal that Cdt1 synergizes with the Ndc80 and the Ska1 complexes for microtubule-binding. Further, we show that single Cdt1 molecules form diffusive tripartite complexes with Ndc80 and Ska1 that can processively track the ends of dynamic microtubules in vitro. Taken together our data identifies a minimal molecular unit responsible for bidirectional processive tip tracking of kinetochores.

Nuclear lamin A-associated proteins are required for centromere assembly

“…Experiments were performed with a single-channel BLItz system by forte BIO as described in [36]. Anti-Penta-HIS (HIS1K, FORTEBIO, #185121) Biosensors were first hydrated in a black 96-well plate containing HEPES buffer (20 mM HEPES, 150 mM NaCl, and 0.1% Tween 20, pH 7.4) for 10 mins.…”

Coordination between the Ndc80 complex and dynein is essential for microtubule plus-end capture by kinetochores during early mitosis

“…For the titration experiment, dynein was labeled with alexa488 following established protocol ( 77 ) and full-length Ndc80 protein [purification described elsewhere ( 89 )] was labeled with Alexa647 dye using NHS-Alexa 647 as described before ( 90 ). The proteins were immobilized as explained before using 100 nM dynein and varying concentrations of Ndc80.…”

“…Mean intensity of each pixel is then quantified using Fiji, and gliding velocities for each condition were quantified as above. For single-molecule colocalization assay, we used nonfluorescent Taxol stabilized-biotin labeled microtubule, and microtubules were immobilized on the surface as described elsewhere ( 90 ). A solution of premixed dynein (1 nM) and Ndc80 (1 nM) solution in image buffer was flown into the chamber following visualization in appropriate channel.…”