A Kinesin-5, Cin8, Recruits Protein Phosphatase 1 to Kinetochores and Regulates Chromosome Segregation

Abstract

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8's kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.

Keywords: Cin8; Dam1; Kinesin-5; Ndc80; PP1; biosensor; cell division; kinetochore; mitosis.

Figure 1.. Cin8 localizes to kinetochores in cell cycle-dependent manner.

(A) Representative images of Cin8-GFP and Ndc80-mCherry throughout the cell cycle (left). The plot of normalized GFP intensity at kinetochores throughout the cell cycle (right). The signal intensity was normalized for the mean values of metaphase. (B) Dsn1-His-Flag was immunoprecipitated from asynchronously growing yeast cells expressing Dsn1-His-Flag, Cin8-3V5, or Dsn1-His-Flag/Cin8-3V5 (left) or from Dsn1-His-Flag/Cin8-3V5 cells that were synchronized in G1 (using α-factor), released and harvested after 0, 40, 70, and 101 min (right). The levels of Cin8 that associate with kinetochores was analyzed by immunoblotting. See also Figure S1. (C) Representative images of Cin8-GFP and Ndc80-mCherry in control, nocodazole (Noc), or low dose benomyl (Low Ben) treated cells (left). The plot of normalized GFP intensity (right). The signal intensity was normalized for the mean values of control for either Cin8 or tubulin (Tub1). All values were mean ± SD. n > 80 individual kinetochores for each experiment. * p < 0.05 (t-test). Scale bars were 5 μm. (D) Kinetochores were isolated by immunoprecipitation of Dsn1-His-Flag from asynchronously growing yeast cells expressing Cin8-Snap-3V5 that were treated with or without nocodazole (noc) for 15 min. The levels of Cin8 that associate with kinetochores were detected by immunoblotting.

Figure 2.. The Ndc80 and Dam1 complexes are required for Cin8 kinetochore localization.

(A) Representative images of Cin8-GFP and Ndc80-mCherry at metaphase (left). The separation between sister Cin8, Ndc80, and Spc29 clusters was obtained from cells expressing Spc29-RFP and Cin8-GFP, or Spc29-RFP and Ndc80-GFP. The separation was normalized for the separation values of Spc29-Spc29 (right). Scale bars are 2.5 μm. (B) Representative images of Cin8-GFP at metaphase in control, Ndc80 depleted cells, and Dam1 depleted cells (left). The plot of normalized GFP intensity (right). The signal intensity was normalized for the mean values of each control. All values are mean ± SD. n > 80 individual kinetochores for each experiment. * p < 0.05 (t-test). Scale bars are 5 μm. (C) Kinetochores were purified via immunoprecipitation of Dsnl-His-Flag from WT, ndc80-1 or dad1-1 cells that had been shifted to 37 °C for 2 hours. The levels of Cin8-3V5 that co-purify with kinetochores were analyzed by immunoblotting (left) and quantified by normalizing the intensity relative to the levels of the core kinetochore protein Mtwl (right). (D) Immunoprecipitation with anti-V5 antibody from solution containing purified Cin8-3V5 and Dam1 complex, or Cin8-3V5 and Ndc80 complex (left, See Methods). The plot of Cin8 bound to Ndc80 or Dam1 versus input Cin8 (right).

Figure 3.. Cin8 is essential for producing proper tension at Ndc80.

(A) Representative time-lapse images of control and Cin8 deletion cells (top). The mean duration between prometaphase (PM) and Anaphase onset (Ana onset), and between Ana onset and late anaphase (Late Ana) (bottom). Mitotic stages are determined by sister kinetochore separations (K-K) and cellular bud neck [4]. K-K < 700 nm (Prometaphase), 700 nm < K-K < 1000 nm (Metaphase), K-K > 1000 nm (Anaphase), and Late anaphase is just before Telophase. n = 45 (control) and 24 (Cin8 deletion). (B) Representative FRET images of control and Cin8 deletion cells at prometaphase and metaphase (left). The mean FRET emission ratio in each condition (right). All values are mean ± SD. n > 100 individual kinetochores for each experiment. * p < 0.05 (t-test). Scale bars are 5 μm. See also Figure S2.

Figure 4.. Cin8 binds to PP1 to promote mitotic progression and tension at Ndc80.

(A) Cin8 was immunoprecipitated from cells expressing Cin8-3V5, Glc7-3GFP, or Cin8-3V5/Glc7-3GFP and the levels of associated Glc7 were analyzed by immunoblotting. (B) Schematic of the Cin8 protein, which has a motor domain (black) and four coiled coil (CC) domains (orange). Cin8 has a PP1 binding motif between CC3 and CC4 domains, and two mutants designed to abolish PP1 binding are indicated. The motor mutant, cin8-F467A [46], is also indicated. Note that the authors in [46] used the original annotated Cin8, which started 38 amino-acids upstream of the actual start codon. (C) Cin8, cin8-KAAKA or cin8-F467A were immunoprecipitated from cells that were also expressing Glc7-3GFP and the levels of co-purifying Glc7 were analyzed by immunoblotting. (D) Kinetochores were purified via Dsn1-His-Flag from cells expressing Cin8-3V5, cin8-KAAKA-3V5, or cin8-F467A-4V5, and the levels of associated Glc7 were analyzed by immunoblotting. (E) Percentage of prometaphase (PM) and metaphase (M) cells in control, cin8Δ cells, and cin8-RAKA mutant cells. (F) Representative FRET images of control and cin8-RAKA mutant cells (left). The mean FRET emission ratio in condition left (right). All values were mean ± SD. n > 100 individual kinetochores for each experiment. * p < 0.05 (t-test). Scale bars were 5 μm. See also Figure S3. (G) Schematic models of how yeast kinesin-5 regulates proper force production at the microtubule binding domains of Ndc80. In wild type cells, Cin8 recruits PP1 to kinetochores during early mitosis in manner that requires microtubules, Ndc80 and Dam1 complexes. This leads to PP1 dephosphorylation of the Ndc80 N-terminus to strengthen its attachment to microtubules and to produce force for proper mitotic progression. In Cin8 depleted cells, kinetochore-microtubule attachments are weak because the N-terminal tail of Ndc80 remains phosphorylated due to the loss of PP1.