Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore

Abstract

Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.

Figure 1.

Generation of KNL-1 microtubule-binding mutants and development of an in vivo system to analyze KNL-1 functions. (A) Primary sequence features of KNL-1. A conserved basic patch is highlighted. Mutations engineered to neutralize the basic patch (4A) or delete it (Δ9) are indicated. (B) Purification of WT and mutant KNL-1^1–505 proteins from bacteria using the indicated steps. The final purified proteins were analyzed by SDS-PAGE and Coomassie staining. (C) Microtubule-bundling analysis of recombinant KNL-1^1–505 proteins. 1 µM taxol-stabilized rhodamine microtubules (MTs) was imaged either alone or in the presence of the indicated 2-µM KNL-1^1–505 variants. Bar, 10 µm. (D) Analysis of recombinant KNL-1^1–505 proteins by gel filtration chromatography. Fractions were analyzed by SDS-PAGE followed by immunoblotting with an anti–KNL-1 antibody. Ipt, input. (E) Bead assay to analyze microtubule-binding activity of KNL-1. 20 fields were photographed, and the number of bound beads was quantified. (F) A schematic of the KNL-1 transgene (KNL-1^RR::mCherry) targeted to a single Mos transposon insertion on chromosome II (Chr II). The transgene has the endogenous knl-1 promoter and 3′ untranslated region (UTR), mCherry fused to the C terminus, and exon 4 modified to preserve coding information but alter nucleotide sequence, thereby enabling RNAi-mediated depletion of endogenous KNL-1. (G) dsRNA targeted to the recoded region selectively depletes >95% of endogenous KNL-1. α-Tubulin serves as a loading control. (H–K) Single-copy transgene insertion–encoded KNL-1^RR::mCherry is fully functional. Kinetochore localization (H), embryonic viability (I; n = number of embryos scored), chromosome segregation phenotype (J), and kinetic analysis of spindle pole separation (K) are shown. (H and J) GFP::H2b and GFP::γ-tubulin were crossed into the KNL-1^RR::mCherry transgenic strain to visualize chromosomes (arrow) and spindle poles (arrowheads), respectively. Bars, 5 µm. (J) Frames from time-lapse sequences aligned relative to NEBD (t = 0); the mCherry signal is not shown. (K) Pole–pole distance measured at 10-s intervals, aligned relative to NEBD, averaged for the indicated number (n) of embryos, and plotted versus time. Inset shows the time of anaphase onset in control (nontransgenic) and transgenic endogenous KNL-1–depleted one-cell embryos. Error bars represent the SEM with a 95% confidence interval.

Figure 2.

Microtubule-binding mutants of KNL-1 do not affect formation of load-bearing attachments or chromosome segregation. (A) An image of a metaphase plate in living one-cell embryos from strains harboring the indicated KNL-1^RR::mCherry transgenes and depleted of endogenous KNL-1. Bar, 2 µm. (B) A schematic of analysis performed after crossing in GFP::histone H2b and GFP::γ-tubulin and depleting endogenous KNL-1. (C) Frames from time-lapse sequences of the first embryonic division for the indicated KNL-1 mutants. Bar, 3 µm. (D) Spindle pole separation kinetics for the indicated conditions. Error bars represent the SEM with a 95% confidence interval. The no transgene knl-1(RNAi) trace is reproduced from Fig. 1 K. (E) Timing of anaphase onset in the indicated conditions. The first visible sign of sister chromatid separation (based on the GFP::H2b signal) was scored as anaphase onset. The gray dashed line indicates the NEBD–anaphase interval for the WT transgene (reproduced from the inset in Fig. 1 K). Error bars represent the SEM with a 95% confidence interval. For comprehensive statistical analysis, see Table S1. (F) Embryonic viability analysis of KNL-1 microtubule-binding mutants. L4-stage worms were injected with dsRNA-targeting endogenous knl-1, and the embryos laid by the injected worms were collected 21–41 h after injection and scored for hatching to form larvae.

Figure 3.

Microtubule-binding mutants of KNL-1 significantly extend the spindle checkpoint–mediated cell cycle delay induced by monopolar spindles. (A) A schematic of the monopolar spindle–based checkpoint signaling assay in C. elegans embryos. Depletion of the centrosome duplication kinase ZYG-1 generates monopolar spindles in the second division, which trigger a spindle checkpoint–dependent cell cycle delay. (B) Mean time from NEBD to chromosome decondensation in the AB cell for the indicated conditions. Error bars represent the SEM with a 95% confidence interval. The gray dashed line marks the duration of AB cell mitosis for the WT transgene, and the red dashed line marks the duration of AB cell mitosis induced by monopolar spindles in the same strain. For comprehensive statistical analysis, see Table S2. (C) Stills from time-lapse sequences of the AB cell monopolar division in worm strains coexpressing GFP::Mad2^MDF-2 and the indicated transgenes. Mad2^MDF-2 accumulation on unattached kinetochores is marked with open arrowheads. Bar, 3 µm.

Figure 4.

KNL-1 microtubule-binding mutants do not extend the cell cycle in the absence of microtubules. (A) A schematic of the experimental approach used to analyze mitotic duration in the AB cell after microtubule (MT) depolymerization. (B) Mean time from NEBD to the onset of cortical contractility for the indicated conditions. Error bars represent the SEM with a 95% confidence interval.

Figure 5.

Microtubule-binding mutants of KNL-1 do not affect the interaction with PP1 and vice versa. (A) The conserved PP1 (PP1c)-docking motif SILK-RRVSF is highlighted, and mutations generated in the docking motif are indicated. (B) KNL-1 interacts with the C. elegans PP1 homologs GSP-1 and -2 but not GSP-3 and -4 in yeast two-hybrid assays (see also Fig. S3, A and B). (C) KNL-1 interaction with GSP-1/2 requires the RRVSF motif (see also Figs. 6 D and S3 C). (D) The 4A and Δ9 mutants do not perturb interaction of KNL-1 with GSP-1 and -2 in yeast two-hybrid analysis. KBP-5, a KNL-1–binding protein that interacts with the C-terminal half of KNL-1, serves as a positive control. (E) Biochemical analysis of KNL-1 interaction with GSP-2. Indicated variants of KNL-1^1–505–6xHis immobilized on nickel agarose beads were incubated with GST–GSP-2, and bead-bound proteins were analyzed by SDS-PAGE. Molecular mass is indicated in kilodaltons. (F) PP1-docking mutant of KNL-1 interacts normally with microtubules. Assays using purified KNL-1^1–505 variants were performed as described in Fig. 1 (C and E). The WT data are reproduced from Fig. 1 E. Error bars represent the SEM with a 95% confidence interval. Bar, 10 µm.

Figure 6.

PP1-docking mutants of KNL-1 exhibit kinetic defects in load-bearing attachment formation and are synthetically lethal with checkpoint inhibition. (A) An image of a metaphase plate in living one-cell embryos from strains harboring the indicated KNL-1^RR::mCherry transgenes and depleted of endogenous KNL-1; the control image is reproduced from Fig. 2 A. Bar, 2 µm. (B) Spindle pole separation kinetics for the indicated conditions. Error bars represent the SEM with a 95% confidence interval. The WT trace is reproduced from Fig. 2 D. (C) Frames from time-lapse sequences of the first embryonic division for the indicated KNL-1 mutants. Bar, 3 µm. (D) Biochemical analysis of KNL-1 interaction with GSP-2 performed as in Fig. 5 E. Molecular mass is indicated in kilodaltons. (E) NEBD–anaphase onset interval in one-cell stage embryos for the indicated conditions. See Table S1 for statistical analysis. (F) Spindle pole separation kinetics for the indicated conditions. Error bars represent the SEM with a 95% confidence interval. The WT trace is reproduced from Fig. 2 D. (G) Embryonic viability analysis for the indicated conditions. Lethality was measured during two intervals (21–41 and 42–64 h) after endogenous KNL-1 depletion. In the earlier time point, embryos are depleted of maternal load but potentially inherit some dsRNA that affects zygotic KNL-1 expression; at the later time point, the maternal load is depleted, but zygotic expression is likely unaffected (see the legend of Fig. S2). (H) NEBD–chromosome decondensation interval in AB cells with bipolar or monopolar spindles for the indicated conditions. The red dashed line marks the duration of AB cell mitosis induced by monopolar spindles in the same strain. See Table S2 for statistical analysis. (E and H) The gray dashed lines mark the duration of AB cell mitosis for the WT transgene. Error bars represent the SEM with a 95% confidence interval.

Figure 7.

Disrupting both microtubule-binding and PP1-docking activities of KNL-1 has an additive phenotype. (A) A still image of a living one-cell metaphase embryo from a strain expressing a 4A;RRASA KNL-1 mutant; endogenous KNL-1 was depleted. Bar, 2 µm. (B and C) Spindle pole separation kinetics for the indicated conditions. WT and single mutant traces (4A and RRASA) are reproduced from Figs. 2 D and 6 B. Error bars represent the SEM with a 95% confidence interval. Anaphase onset times are marked on the x axis. (D) Timing of anaphase onset in the first embryonic division for the indicated conditions. The gray dashed line is drawn as in Fig. 2 E. For statistical analysis, see Table S1. WT and single-mutant timing data are reproduced from Figs. 2 E and 6 E. Error bars represent the SEM with a 95% confidence interval. (E) Mean duration of AB cell mitosis for the indicated conditions. Dashed lines are drawn as in Fig. 3 B; WT and single-mutant data are from Figs. 3 B and 6 H. For statistical analysis, see Table S2. Error bars represent the SEM with a 95% confidence interval. (F) A schematic summarizing the conclusion that the microtubule-binding activity located in the N terminus of KNL-1 participates in checkpoint silencing.