Purification and characterisation of the fission yeast Ndc80 complex

Abstract

The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1.

ZWINT: A potential therapeutic biomarker in patients with glioblastoma correlates with cell proliferation and invasion

“…The mitotic regulator NDC80 is highly expressed in various human malignancies, including hepatocellular carcinoma, colon cancer and osteosarcoma (33)(34)(35). NDC80 is also called Hec1, and its complexes together with NUF2 component of NDC80 kinetochore complex (NUF2), SPC24 component of NDC80 kinetochore complex (SPC24) and SPC25 component of NDC80 kinetochore complex (SPC25) participate in the spindle assembly checkpoint and regulation of mitosis and chromosome segregation (36)(37)(38)(39). Lin et al showed that Hec1 sequentially recruits Zwint-1 and ZW10 to kinetochores during the mitotic phase of the cell cycle, and interruption of the centromeric recruitment led to chromosomal missegregation, incomplete activation of spindle examination points, and ultimately cell death (31).…”

Inositol pyrophosphate-controlled kinetochore architecture and mitotic entry in S. pombe

“…As shown diagrammatically in Figure 3A, plasmid-borne expression of asp1 1-364 increases intracellular IP 8 , while asp1 365-920 expression reduces (but does not eliminate) IP 8 levels [36]. S. pombe kinetochores complexes consist of the same core modules found in human and S. cerevisiae such as KMN(human)/NMS( S. pombe ) and CCAN(human)/Ctf19( S. cerevisiae )/Mis6-Mal2-Sim4( S. pombe ) [84-88]. Expression of either asp1 1-364 or asp1 365-920 from a plasmid had no or only a moderate effect on the growth of 4 temperature sensitive S. pombe KMN mutant strains tested (Figure S2A).…”

AtNUF2 modulates spindle microtubule organization and chromosome segregation during mitosis

“…Biochemical assays and transmission electron microscopy analysis of the NDC80 complex has shown a conserved ‘head‐rod‐head’ architecture in Saccharomyces cerevisiae and humans (Ciferri et al ., 2005; Wei et al ., 2005, 2006). In Schizosaccharomyces pombe , the NDC80 complex may co‐precipitate with polymerized MTs, and this binding is sensitive to ionic strength (Matsuo et al ., 2017; Powers et al ., 2009). In humans, the C‐termini of SPC24 and SPC25 anchor the NDC80 complex to the kinetochore protein, while the N‐terminal domains of NUF2 and HEC1/NDC80 bind to the positive ends of spindle MTs (DeLuca et al ., 2006; Sundin et al ., 2011; Wan et al ., 2009).…”