Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells

Abstract

The spindle checkpoint generates a "wait anaphase" signal at unattached kinetochores to prevent premature anaphase onset. Kinetochore-localized dynein is thought to silence the checkpoint by transporting checkpoint proteins from microtubule-attached kinetochores to spindle poles. Throughout metazoans, dynein recruitment to kinetochores requires the protein Spindly. Here, we identify a conserved motif in Spindly that is essential for kinetochore targeting of dynein. Spindly motif mutants, expressed following depletion of endogenous Spindly, target normally to kinetochores but prevent dynein recruitment. Spindly depletion and Spindly motif mutants, despite their similar effects on kinetochore dynein, have opposite consequences on chromosome alignment and checkpoint silencing. Spindly depletion delays chromosome alignment, but Spindly motif mutants ameliorate this defect, indicating that Spindly has a dynein recruitment-independent role in alignment. In Spindly depletions, the checkpoint is silenced following delayed alignment by a kinetochore dynein-independent mechanism. In contrast, Spindly motif mutants are retained on microtubule-attached kinetochores along with checkpoint proteins, resulting in persistent checkpoint signaling. Thus, dynein-mediated removal of Spindly from microtubule-attached kinetochores, rather than poleward transport per se, is the critical reaction in checkpoint silencing. In the absence of Spindly, a second mechanism silences the checkpoint; this mechanism is likely evolutionarily ancient, as fungi and higher plants lack kinetochore dynein.

Figure 1.

Single amino acid changes in Spindly uncouple its kinetochore localization from dynein/dynactin recruitment. (A) Mitotic HeLa cells fixed and immunostained for Spindly and centromere antigens (ACA). In addition to kinetochores, Spindly is also visible at spindle poles (arrows) during chromosome alignment. Spindly is absent from kinetochores at the metaphase plate, but is detectable at an unaligned kinetochore pair (arrowhead). (B) Immunoblot and immunofluorescence 48 h after transfection of HeLa cells with control and Spindly siRNA. α-Tubulin was used as a loading control for the immunoblot. Cells were treated with nocodazole for 4 h prior to fixation and immunostaining. (C) Localization of dynein intermediate chains and the dynactin subunit p150^Glued at unattached kinetochores in control and Spindly siRNA-treated cells. Cells were incubated in nocodazole for 4 h to accumulate dynein/dynactin at kinetochores (see also Supplemental Fig. S1D). (D) Sequence alignment of the highly conserved motif in the Spindly protein family. The conserved serine and phenylalanine (S256 and F258 in human Spindly) that were individually mutated to alanine are indicated. (E) Immunoblot monitoring endogenous and RNAi-resistant (RR) transgenic Spindly expression. Cells were treated with control or Spindly siRNA for 22 h followed by induction with 0.2 μg/mL tetracycline for 8 h. α-Tubulin is used as a loading control. (F,G) Cell lines expressing GFP:^RRSpindly^WT or the point mutants depicted in D immunostained for GFP, centromere antigens (ACA), and either the dynactin subunit p150^Glued (F) or dynein intermediate chains using the monoclonal antibody 70.1 (G). Cells were treated with Spindly siRNA for 24 h, transgene expression was induced with tetracycline for 16 h, and nocodazole was added for 4 h prior to fixation. Bars, 5 μm; inset in A, 1 μm.

Figure 2.

Spindly and its ability to target dynein/dynactin to kinetochores is required for poleward transport of checkpoint proteins. (A) Schematic of the ATP reduction assay used to analyze kinetochore dynein-mediated transport of checkpoint proteins (Howell et al. 2000). (B) Immunostaining of Spindly and centromere antigens (ACA) in the indicated states. (C) Immunostaining of Mad1 and Mad2 in control and Spindly siRNA-treated cells with normal or reduced ATP levels. Similar results were obtained for Zwilch and CENP-E (Supplemental Fig. S2A,B; Supplemental Movie S1). (D) The ATP reduction assay performed in cells expressing GFP:^RRSpindly^WT or GFP:^RRSpindly^F258A. Cells were treated with Spindly siRNA and tetracycline as described for Figure 1F and immunostained for GFP, α-tubulin, and centromere antigens (ACA). (E) Cells were treated as in D and immunostained for GFP, α-tubulin, and Mad1. For similar analysis of Mad2, see Supplemental Figure S2C. Arrowheads denote accumulation of the indicated proteins at spindle poles. Bars, 5 μm.

Figure 3.

Checkpoint proteins are not retained at bioriented kinetochores in Spindly-depleted cells. (A–E) Control or Spindly siRNA-treated cells immunostained for the checkpoint proteins Mad1 (A), Mad2 (B), BubR1 (C), Zwilch (D), and CENP-E (E). For each protein, staining is shown in an early prometaphase (PM) cell with no kinetochore–microtubule attachments, and in a cell where all (metaphase [M] in control siRNA) or most (“Late PM” in Spindly siRNA) chromosomes have congressed. Arrows and arrowheads point to examples of aligned (A) and unaligned (U) kinetochores, respectively, used for quantitation of checkpoint protein signals. Kinetochore intensity measurements for each protein were normalized relative to prometaphase of control siRNA-treated cells. Error bars represent the SEM with a 95% confidence interval. Bars, 5 μm; inset in E, 1 μm.

Figure 4.

Spindly motif mutants are retained together with Mad1 and Mad2 on kinetochores of aligned chromosomes. (A) Images from a time-lapse imaging sequence of cells expressing histone H2b:mRFP and either GFP:^RRSpindly^WT or GFP:^RRSpindly^F258A. Cells were treated with siRNAs for 32 h, and expression of the Spindly transgenes was induced for 16 h before filming. Bar, 5 μm. (B,C) Immunofluorescence images of cells with congressed chromosomes expressing GFP:^RRSpindly^WT or GFP:^RRSpindly^F258A stained for GFP and Mad1 (B) or BubR1 (C). Bars, 5 μm. (D) Quantitation of checkpoint protein levels at kinetochores of aligned chromosomes relative to unaligned kinetochores in early prometaphase control cells for the indicated cell lines, siRNA treatments, and transgenes.

Figure 5.

Aligned kinetochores retaining Spindly motif mutants have achieved stable, bioriented microtubule attachments. (A) Projection of three optical sections from an immunofluorescence Z-stack of a cell depleted of endogenous Spindly expressing GFP:^RRSpindly^F258A stained for GFP and Mad1. Cells were treated with siRNAs for 32 h, and expression of the Spindly transgenes was induced for 16 h before fixation. Bar, 5 μm; inset, 1 μm. (B) Distance between the ACA signal of sister kinetochores at metaphase in the indicated states. Error bars represent the SEM with a 95% confidence interval. (C) Cold-stable kinetochore fibers visualized by immunofluorescence in cells depleted of endogenous Spindly expressing GFP:^RRSpindly^WT or GFP:^RRSpindly^F258A. Blowups of individual kinetochore fibers represent projections of selected sections of the image Z-stack. Bar, 5 μm; blowups, 2 μm. (D) Distance (δ) between the inner kinetochore component CENP-I and the outer kinetochore component Ndc80/Hec1, measured at bioriented metaphase kinetochores in the indicated states. The value for unperturbed metaphase kinetochores in HeLa cells was previously determined to be 62 ± 9 nm (Wan et al. 2009). Values are given as the mean ± standard deviation. (E) Kymographs of aligned sister kinetochore pairs marked by GFP:Mis12 (Kline et al. 2006) or GFP:^RRSpindly^F258A after the indicated treatments. Bar, 2 μm; kymograph panels, 1 μm.

Figure 6.

Differential effects of Spindly motif mutants and Spindly depletions on chromosome alignment and spindle checkpoint silencing. (A) Selected images from a time-lapse series of cells expressing histone H2b:mRFP with or without Spindly transgenes (see also Supplemental Movie S3). The experimental protocol prior to the start of filming was identical to that for the immunoblot in Figure 1E. A blowup shows the onset of the scattering phenotype (see also the Supplemental Material; Supplemental Fig. S5; Supplemental Movies S4–S6). Bar, 5 μm; blowup, 2 μm. (B,C) Quantitative analysis of mitotic intervals for the experimental conditions shown in A. Interval averages are marked by horizontal bars (see also Supplemental Table S1). Two independent experiments were performed for each condition, and the number (n) of cells scored is indicated.

Figure 7.

Model explaining the different consequences of Spindly depletion and Spindly motif mutants. Spindly depletion and replacement of endogenous Spindly with Spindly motif mutants abrogate recruitment of dynein/dynactin to kinetochores, but have differential effects on chromosome alignment and checkpoint silencing. See the text for details.