Arabidopsis KNL1 recruits type one protein phosphatase to kinetochores to silence the spindle assembly checkpoint

Abstract

Proper chromosome segregation during cell division is essential for genomic integrity and organismal development. This process is monitored by the spindle assembly checkpoint (SAC), which delays anaphase onset until all chromosomes are properly attached to the mitotic spindle. The kinetochore protein KNL1 plays a critical role in recruiting SAC proteins. Here, we reveal that Arabidopsis KNL1 regulates SAC silencing through the direct recruitment of type one protein phosphatase (TOPP) to kinetochores. We show that KNL1 interacts with all nine TOPPs via a conserved RVSF motif in its N terminus, and this interaction is required for the proper localization of TOPPs to kinetochores during mitosis. Disrupting KNL1-TOPP interaction leads to persistent SAC activation, resulting in a severe metaphase arrest and defects in plant growth and development. Our findings highlight the evolutionary conservation of KNL1 in coordinating kinetochore-localized phosphatase to ensure timely SAC silencing and faithful chromosome segregation in Arabidopsis.

Fig. 1.. KNL1 interacts with TOPP through the RVSF motif.

(A) Y2H assay to examine interactions between the nine TOPP paralogs (TOPP1-TOPP9) and KNL1 or the KNL1^4A mutant. The empty vector was used as a negative control (NA). Yeast cultures were spotted on vector-selective (-L/-W, left) and interaction-selective (-L/-W/-H/-A, right) media and imaged after 2 days of incubation at 30°C. (B) BIFC assay to visualize the interactions between KNL1 variants (fused with the N-terminal YFP fragment) and the TOPP proteins (fused with the C-terminal YFP fragment) in Nicotiana benthamiana leaves. Scale bars, 10 μm. (C) In vitro pull-down assays using recombinant GST-tagged KNL1 variants and MBP-tagged TOPP4 and TOPP8 proteins. The MBP-TOPP4/TOPP8 fusion proteins were immobilized on MBP-trap beads and incubated with the GST-KNL1 variants. (D) Coimmunoprecipitation analysis of the interaction between KNL1 and TOPP4/8. Total proteins were extracted from 3-day-old transgenic seedlings expressing pTOPP4/8::TOPP4/8-GFP and pKNL1::KNL1-FLAG and were incubated with anti-GFP beads. Both the input and immunoprecipitated samples were subjected to immunoblotting using anti-FLAG or anti-GFP antibodies. These experiments were repeated three times with similar results.

Fig. 2.. KNL1 recruits TOPP to kinetochores.

(A and B) Triple-label immunolocalization of the TOPP paralogs, microtubules, and DNA in wild-type (WT) root meristematic cells undergoing mitosis. TOPP4-GFP (A) and TOPP8-GFP (B) are pseudocolored in green, microtubules are in red, and DNA is in blue. (C) Summary of the localization patterns of all nine TOPPs in Arabidopsis cells. (D) Colocalization of TOPP4-GFP or TOPP8-GFP with KNL1-FLAG in mitotic cells. The merged image has TOPP4 or TOPP8 pseudocolored in green, KNL1 in red, and DNA in blue. (E and F) Localization of TOPP4-GFP (E) and TOPP8-GFP (F) in the knl1 mutant background. The merged image has TOPP4 and TOPP8 pseudocolored in green, microtubules in red, and DNA in blue. (G) Live-cell imaging of wild-type seedlings expressing TOPP4-GFP, with representative snapshot images acquired from movie S1. (H) Assessment of the dynamic changes in TOPP4-GFP fluorescence intensity over time, as observed in the live-cell imaging experiment shown in (G). Scale bars, 5 μm.

Fig. 3.. Expressing KNL1^4A cannot rescue knl1 plants.

(A) Growth phenotypes of 4-week-old Arabidopsis plants, including WT, knl1, and the knl1 mutant expressing either unmodified KNL1 or KNL1^4A. (B to D) Live-cell imaging of WT plant cells (B), knl1 mutant cells (C), and knl1 mutant cells expressing the KNL1^4A construct (D), along with GFP-TUB6 and histone-RFP. Representative snapshot images are acquired from movies S3 to S5. Scale bars, 5 μm.

Fig. 4.. KNL1-TOPP interaction is required for timely SAC silencing.

(A) Growth phenotypes of 7-week-old Arabidopsis plants, including WT plants, WT plants expressing KNL1^4A, the bmf3 mutant, bmf3 mutant plants expressing KNL1^4A, the mad1 mutant, and mad1 mutant plants expressing KNL1^4A. (B) Live-cell imaging of mitotic cells expressing GFP-TUB6 and histone-RFP in WT and WT/KNL1^4A backgrounds. Representative snapshot images are acquired from movies S6 and S7. (C) Live-cell imaging of mitotic cells expressing GFP-TUB6 and histone-RFP in bmf3 and bmf3/KNL1^4A backgrounds. Representative snapshot images are acquired from movies S8 and S9. (D) Live-cell imaging of mitotic cells expressing GFP-TUB6 and histone-RFP in mad1 and mad1/KNL1^4A backgrounds. Representative snapshot images are acquired from movies S10 and S11. (E) Triple-label immunolocalization of BMF3 (green), microtubules (red), and DNA (blue) in control and KNL1^4A-expressing cells. (F) Triple-label immunolocalization of MAD1 (green), microtubules (red), and DNA (blue) in control and KNL1^4A-expressing cells. (G) Quantification of the duration from NEB to anaphase onset in the different genotypes. Scale bars, 5 μm.

Fig. 5.. Inhibiting TOPP results in delayed SAC silencing.

(A) Growth phenotypes of 6-week-old WT plants and WT plants expressing TOPP4^T246M. (B) Growth phenotypes of 6-week-old bmf3 mutant and bmf3 mutant plants expressing TOPP4^T246M. (C and D) Live-cell imaging of mitotic cells expressing GFP-TUB6 and histone-RFP in WT (C) and WT/TOPP4^T246M (D) backgrounds. Representative snapshot images are acquired from movies S12 and S13. (E) Quantification of the duration from NEB to anaphase onset in different seedlings. (F and G) Triple-label immunolocalization of MAD1 (green), microtubules (red), and DNA (blue) in control (F) and TOPP4^T246M-expressing (G) cells. (H and I) Live-cell imaging of mitotic cells expressing GFP-TUB6 and histone-RFP in bmf3 (H) and bmf3/TOPP4^T246M (I) backgrounds. Representative snapshot images are acquired from movies S16 and S17. Scale bars, 5 μm.