Janczyk PŁ et al. (2023), Aurora A phosphorylates Ndel1 to reduce the lev...

XB-ART-59457

Mol Biol Cell 2023 Jan 01;341:br1. doi: 10.1091/mbc.E21-09-0438.

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Aurora A phosphorylates Ndel1 to reduce the levels of Mad1 and NuMA at spindle poles.

Janczyk PŁ , Żyłkiewicz E , De Hoyos H , West T , Matson DR , Choi WC , Young HMR , Derewenda ZS , Stukenberg PT .

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Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally absent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters in Xenopus extracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.

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Species referenced: Xenopus laevis
Genes referenced: Mad1l1 ndel1 numa1 ran

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	FIGURE 4: Phosphorylation of Ndel1 on Ser285 also limits the localization of NuMA to spindle poles and negatively regulates dynein-dependent aster formation in Xenopus egg extracts. (A, B) A nonphosphorylatable mutant of Ndel1 allows for increased recruitment of NuMA to the centrosomal region. (A) Representative images depicting localization of NuMA (red) in HeLa T-Rex cells expressing Ndel1 WT or Ndel1 S285A mutant in mitotic cells; bar = 5 Î¼m. (B) Quantification of experiment depicted in panel A (n = 20 centrosomal regions; Studentâs t test, with **, p < 0.0005). (C, D) A nonphosphorylatable mutant of Ndel1 generates Ran asters at lower concentrations than WT Ndel1. Xenopus egg extracts were depleted of Ndel1 and rescued with the indicated concentrations of recombinant mNdel1. Note that Ndel1 S285A mutant rescued aster formation at 10 nM (10-fold less than endogenous Ndel1 level), whereas the WT protein mNdel18â310 required higher concentrations to generate Ran asters. Representative microtubule structures are shown in panel C, bar = 10 Î¼m, and the quantification is shown in panel D (n > 100 microtubule structures; N = 3; Studentâs t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with , p < 0.005). (E, F) A phosphomimetic mutant of Ser285 is deficient in Ran aster formation in Xenopus egg extracts. Experiment was performed as in panels C and D, except the Ndel1 S285D mutant was used. Note that at endogenous concentrations of 100 nM the S285D mutant did not rescue aster formation. Representative microtubule structures are shown in panel E (bar = 10 Î¼m) and the quantification is shown in panel F (n > 100 microtubule structures; N = 3; Studentâs t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with **, p < 0.0005).
	FIGURE 1:. The Mad1 SAC protein relocalizes from kinetochores to spindle poles after inhibition of Aurora A kinase activity in prometaphase cells. (A, B) Representative images depicting the localization of Mad1 (red) in HeLa T-Rex cells treated with increasing concentrations of Aurora A inhibitor MLN8054, with tubulin (cyan) and centromere (green) costaining. Note: Aurora A inhibition by MLN had an unexpected effect on anti-centromere antigen (ACA) staining so we altered the levels of ACA in each image so that centromeres are clearly visible. The ACA staining has not been adjusted in Supplemental Figure S1. Bar = 5 µm; N = 3 experimental repeats. (C, D) Quantification of Mad1 fluorescent intensity at the kinetochores (C) and at the spindle poles (D) in the experiment depicted in panels A and B. We have not ratioed the Mad1 staining by the ACA signal; however, we confirmed that the results were similar if this was done (Supplemental Figure S1). (Box, 25th–75th percentile; whisker, 5th–95th percentile; bar in middle, median. One-way ANOVA, with , p < 0.05; , p < 0.0005). (E) Representative images depicting localization of Mad1 (red) in HeLa T-Rex cells expressing GFP (−CC1) or GFP-CC1 (+CC1) in mitotic cells treated with 1.5 μM MLN8054; bar = 5 μm. (F) Quantification of cells containing visible Mad1 signal at centrosomes from experiment depicted in panel C (n > 100 cells; N = 3; *, p < 0.005; Student’s t test, unpaired).
	FIGURE 2:. Aurora kinases phosphorylate Ndel1 near a C-terminal helix I Ser285 at spindle poles and kinetochores. (A) Domain structure of Ndel1. Crystal structure of the N-terminal coiled-coil domain (PDB: 2V71) with critical amino acids required for dynein intermediate chain (DIC) binding (red ball and stick) and Lis1 (green ball and stick) highlighted. The C terminus of Ndel1 is mostly unstructured but contains two protein binding sites and a number of previously identified phosphorylation sites (purple), including previously reported Aurora site Ser251 (red). Here we describe a new Aurora phosphorylation site Ser285 (red with asterisks). Note that two Aurora sites are conserved in frogs, rodents, and humans and flank an alpha helical domain (box) that is required for DISC1 binding. (B) The C terminus of Ndel1 is phosphorylated by Aurora kinases. The indicated mNdel1 fragments were phosphorylated in vitro by either recombinant Aurora A or Aurora B kinase and analyzed on the SDS–PAGE and autoradiography. (C) Ser285 is the dominant Aurora phosphorylation site on Ndel1. The serines that fit the known Aurora consensus in the C terminus of Ndel1 were mutated to alanines and tested in in vitro kinase assays with Aurora A or Aurora B kinase. (D) Ndel1 (red) signal colocalizes with Ndc80 (blue) at most kinetochores and centrosomes in both prometaphase and metaphase in Xenopus laevis S3 cells; bar = 10 μm. (E) Ndel1 pS285 (red) signal colocalizes with Ndc80 (blue) at kinetochores and centrosomes in mitotic Xenopus laevis S3 cells; bar = 10 μm.
	FIGURE 3:. Phosphorylation of Ndel1 at S285 regulates dynein stripping and spindle checkpoint function. (A) Representative images depicting localization of Mad1 (red) in HeLa T-Rex cells stably expressing Ndel1 WT or S285A after removal of endogenous Ndel1 by siRNA; bar = 5 μm. Insets represent maximum projection of one to two Z-slices of the centrosomal region. (B) Quantification of Mad1 fluorescent intensity at centrosomes (left graph, n > 16 centrosomal regions) or kinetochores (right graph, n > 300 kinetochores) from the experiment depicted in panel A. (N = 3; , p < 0.05; n.s., p > 0.05; Student’s t test, unpaired). (C) A nonphosphorylatable mutant of Ndel1 shows increased proliferation rates in the presence of 20 nM nocodazole. Graph depicts confluency normalized to day 0 of HeLa T-Rex cells expressing either Ndel1 WT or Ndel1 S285A mutant grown in the presence of 20 nM nocodazole (n = 4; N = 3; Student’s t test, with , p < 0.05; **, p < 0.005). (D) HeLa T-Rex cells expressing either Ndel1 WT or Ndel1 S285A mutant were imaged in the presence of 20 nM nocodazole and 100 nM SiR-DNA for 24 h. Graph represents cumulative frequency of time spent from NEBD until mitotic exit through anaphase or mitotic catastrophy (n > 130 cells; N = 2; Kolmogorov-Smirnov test).
	FIGURE 4:. Phosphorylation of Ndel1 on Ser285 also limits the localization of NuMA to spindle poles and negatively regulates dynein-dependent aster formation in Xenopus egg extracts. (A, B) A nonphosphorylatable mutant of Ndel1 allows for increased recruitment of NuMA to the centrosomal region. (A) Representative images depicting localization of NuMA (red) in HeLa T-Rex cells expressing Ndel1 WT or Ndel1 S285A mutant in mitotic cells; bar = 5 μm. (B) Quantification of experiment depicted in panel A (n = 20 centrosomal regions; Student’s t test, with **, p < 0.0005). (C, D) A nonphosphorylatable mutant of Ndel1 generates Ran asters at lower concentrations than WT Ndel1. Xenopus egg extracts were depleted of Ndel1 and rescued with the indicated concentrations of recombinant mNdel1. Note that Ndel1 S285A mutant rescued aster formation at 10 nM (10-fold less than endogenous Ndel1 level), whereas the WT protein mNdel18–310 required higher concentrations to generate Ran asters. Representative microtubule structures are shown in panel C, bar = 10 μm, and the quantification is shown in panel D (n > 100 microtubule structures; N = 3; Student’s t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with , p < 0.005). (E, F) A phosphomimetic mutant of Ser285 is deficient in Ran aster formation in Xenopus egg extracts. Experiment was performed as in panels C and D, except the Ndel1 S285D mutant was used. Note that at endogenous concentrations of 100 nM the S285D mutant did not rescue aster formation. Representative microtubule structures are shown in panel E (bar = 10 μm) and the quantification is shown in panel F (n > 100 microtubule structures; N = 3; Student’s t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with **, p < 0.0005).

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	FIGURE 4: Phosphorylation of Ndel1 on Ser285 also limits the localization of NuMA to spindle poles and negatively regulates dynein-dependent aster formation in Xenopus egg extracts. (A, B) A nonphosphorylatable mutant of Ndel1 allows for increased recruitment of NuMA to the centrosomal region. (A) Representative images depicting localization of NuMA (red) in HeLa T-Rex cells expressing Ndel1 WT or Ndel1 S285A mutant in mitotic cells; bar = 5 Î¼m. (B) Quantification of experiment depicted in panel A (n = 20 centrosomal regions; Studentâs t test, with **, p < 0.0005). (C, D) A nonphosphorylatable mutant of Ndel1 generates Ran asters at lower concentrations than WT Ndel1. Xenopus egg extracts were depleted of Ndel1 and rescued with the indicated concentrations of recombinant mNdel1. Note that Ndel1 S285A mutant rescued aster formation at 10 nM (10-fold less than endogenous Ndel1 level), whereas the WT protein mNdel18â310 required higher concentrations to generate Ran asters. Representative microtubule structures are shown in panel C, bar = 10 Î¼m, and the quantification is shown in panel D (n > 100 microtubule structures; N = 3; Studentâs t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with , p < 0.005). (E, F) A phosphomimetic mutant of Ser285 is deficient in Ran aster formation in Xenopus egg extracts. Experiment was performed as in panels C and D, except the Ndel1 S285D mutant was used. Note that at endogenous concentrations of 100 nM the S285D mutant did not rescue aster formation. Representative microtubule structures are shown in panel E (bar = 10 Î¼m) and the quantification is shown in panel F (n > 100 microtubule structures; N = 3; Studentâs t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with **, p < 0.0005).
	FIGURE 1:. The Mad1 SAC protein relocalizes from kinetochores to spindle poles after inhibition of Aurora A kinase activity in prometaphase cells. (A, B) Representative images depicting the localization of Mad1 (red) in HeLa T-Rex cells treated with increasing concentrations of Aurora A inhibitor MLN8054, with tubulin (cyan) and centromere (green) costaining. Note: Aurora A inhibition by MLN had an unexpected effect on anti-centromere antigen (ACA) staining so we altered the levels of ACA in each image so that centromeres are clearly visible. The ACA staining has not been adjusted in Supplemental Figure S1. Bar = 5 µm; N = 3 experimental repeats. (C, D) Quantification of Mad1 fluorescent intensity at the kinetochores (C) and at the spindle poles (D) in the experiment depicted in panels A and B. We have not ratioed the Mad1 staining by the ACA signal; however, we confirmed that the results were similar if this was done (Supplemental Figure S1). (Box, 25th–75th percentile; whisker, 5th–95th percentile; bar in middle, median. One-way ANOVA, with , p < 0.05; , p < 0.0005). (E) Representative images depicting localization of Mad1 (red) in HeLa T-Rex cells expressing GFP (−CC1) or GFP-CC1 (+CC1) in mitotic cells treated with 1.5 μM MLN8054; bar = 5 μm. (F) Quantification of cells containing visible Mad1 signal at centrosomes from experiment depicted in panel C (n > 100 cells; N = 3; *, p < 0.005; Student’s t test, unpaired).
	FIGURE 2:. Aurora kinases phosphorylate Ndel1 near a C-terminal helix I Ser285 at spindle poles and kinetochores. (A) Domain structure of Ndel1. Crystal structure of the N-terminal coiled-coil domain (PDB: 2V71) with critical amino acids required for dynein intermediate chain (DIC) binding (red ball and stick) and Lis1 (green ball and stick) highlighted. The C terminus of Ndel1 is mostly unstructured but contains two protein binding sites and a number of previously identified phosphorylation sites (purple), including previously reported Aurora site Ser251 (red). Here we describe a new Aurora phosphorylation site Ser285 (red with asterisks). Note that two Aurora sites are conserved in frogs, rodents, and humans and flank an alpha helical domain (box) that is required for DISC1 binding. (B) The C terminus of Ndel1 is phosphorylated by Aurora kinases. The indicated mNdel1 fragments were phosphorylated in vitro by either recombinant Aurora A or Aurora B kinase and analyzed on the SDS–PAGE and autoradiography. (C) Ser285 is the dominant Aurora phosphorylation site on Ndel1. The serines that fit the known Aurora consensus in the C terminus of Ndel1 were mutated to alanines and tested in in vitro kinase assays with Aurora A or Aurora B kinase. (D) Ndel1 (red) signal colocalizes with Ndc80 (blue) at most kinetochores and centrosomes in both prometaphase and metaphase in Xenopus laevis S3 cells; bar = 10 μm. (E) Ndel1 pS285 (red) signal colocalizes with Ndc80 (blue) at kinetochores and centrosomes in mitotic Xenopus laevis S3 cells; bar = 10 μm.
	FIGURE 3:. Phosphorylation of Ndel1 at S285 regulates dynein stripping and spindle checkpoint function. (A) Representative images depicting localization of Mad1 (red) in HeLa T-Rex cells stably expressing Ndel1 WT or S285A after removal of endogenous Ndel1 by siRNA; bar = 5 μm. Insets represent maximum projection of one to two Z-slices of the centrosomal region. (B) Quantification of Mad1 fluorescent intensity at centrosomes (left graph, n > 16 centrosomal regions) or kinetochores (right graph, n > 300 kinetochores) from the experiment depicted in panel A. (N = 3; , p < 0.05; n.s., p > 0.05; Student’s t test, unpaired). (C) A nonphosphorylatable mutant of Ndel1 shows increased proliferation rates in the presence of 20 nM nocodazole. Graph depicts confluency normalized to day 0 of HeLa T-Rex cells expressing either Ndel1 WT or Ndel1 S285A mutant grown in the presence of 20 nM nocodazole (n = 4; N = 3; Student’s t test, with , p < 0.05; **, p < 0.005). (D) HeLa T-Rex cells expressing either Ndel1 WT or Ndel1 S285A mutant were imaged in the presence of 20 nM nocodazole and 100 nM SiR-DNA for 24 h. Graph represents cumulative frequency of time spent from NEBD until mitotic exit through anaphase or mitotic catastrophy (n > 130 cells; N = 2; Kolmogorov-Smirnov test).
	FIGURE 4:. Phosphorylation of Ndel1 on Ser285 also limits the localization of NuMA to spindle poles and negatively regulates dynein-dependent aster formation in Xenopus egg extracts. (A, B) A nonphosphorylatable mutant of Ndel1 allows for increased recruitment of NuMA to the centrosomal region. (A) Representative images depicting localization of NuMA (red) in HeLa T-Rex cells expressing Ndel1 WT or Ndel1 S285A mutant in mitotic cells; bar = 5 μm. (B) Quantification of experiment depicted in panel A (n = 20 centrosomal regions; Student’s t test, with **, p < 0.0005). (C, D) A nonphosphorylatable mutant of Ndel1 generates Ran asters at lower concentrations than WT Ndel1. Xenopus egg extracts were depleted of Ndel1 and rescued with the indicated concentrations of recombinant mNdel1. Note that Ndel1 S285A mutant rescued aster formation at 10 nM (10-fold less than endogenous Ndel1 level), whereas the WT protein mNdel18–310 required higher concentrations to generate Ran asters. Representative microtubule structures are shown in panel C, bar = 10 μm, and the quantification is shown in panel D (n > 100 microtubule structures; N = 3; Student’s t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with , p < 0.005). (E, F) A phosphomimetic mutant of Ser285 is deficient in Ran aster formation in Xenopus egg extracts. Experiment was performed as in panels C and D, except the Ndel1 S285D mutant was used. Note that at endogenous concentrations of 100 nM the S285D mutant did not rescue aster formation. Representative microtubule structures are shown in panel E (bar = 10 μm) and the quantification is shown in panel F (n > 100 microtubule structures; N = 3; Student’s t test, with , p < 0.05; , p < 0.005; , p < 0.0005; two-way ANOVA [red], with **, p < 0.0005).