Figure 5.
Ser-240 phosphorylation of NuSAP promotes its interaction with Kif2A and reduces the localization of Kif2A on spindle poles. (A and C) Mitotic HEK293T cells cotransfected with the indicated GFP-tagged proteins and mCherry-tagged NuSAP constructs were immunoprecipitated with GFP-Trap beads. (B and D) The quantification of protein–protein interaction discrepancy. Error bars indicate SD. Three independent replicates were quantified (n = 3). Unpaired two-tailed t test: P = 0.0005 for WT/S240A, P = 0.0135 for S240A/S240D, P = 0.3489 for WT/S240D in B; P = 0.2274 for WT/S240A, P = 0.1531 for S240A/S240D, P = 0.2562 for WT/S240D in D. (E) NuSAP-knockout HeLa cells were transfected with GFP-tagged NuSAP-WT, -S240A, or -S240D (green) followed by immunofluorescence labeling using anti-Kif2A (red) and anti-α-tubulin (magenta) antibodies. DNA was stained with DAPI (blue). (F) Quantification of relative Kif2A intensity on spindle poles in E. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0049 for WT/S240A; P = 0.0051 for S240A/S240D; P = 0.3347 for WT/S240D. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = the number of independent experiments presented. Scale bars, 10 μm. (G) Along with the nuclear envelope broken down during mitotic entry, microtubule asters nucleate around the chromosomes and the centrosomes to form small acentrosomal and large centrosomal asters. These oriented microtubules are bundled and then sorted into the large centrosomal microtubule asters to form a bipolar spindle structure by molecular motors and microtubule-associated proteins till to a well-organized metaphase spindle formation with a dynamically constant length, in which NuSAP plays a crucial role. (H) NuSAP is mainly localized on microtubules near chromosomes and is phosphorylated by the mitotic kinase Aurora A on S240. As a MAP, NuSAP regulates the spindle dynamics to maintain metaphase spindle length through coordinating microtubule depolymerization and microtubule–microtubule sliding. On the one hand, NuSAP interacts with Eg5 on the adjacent microtubules, which allows Eg5 to move along the microtubules and to mediate the sliding of antiparallel interpolar microtubules (shown by the black arrow), which increases spindle length. On the other hand, S240-phosphorylated NuSAP interacts with more Kif2A on the spindle body and then reduces the amount of Kif2A on spindle poles, thus restraining the excess accumulation of Kif2A at the minus ends of microtubules to ensure the proper microtubule depolymerization. At the plus end, NuSAP attenuates MCAK depolymerization activity to stabilize kinetochore microtubules. NuSAP regulates microtubule flux (shown by the orange arrow) and controls metaphase spindle length by coordinating with Eg5, Kif2A, and MCAK. In experimental conditions, NuSAP knockout results in a reduction of Eg5 localization on spindle microtubules, excess accumulation of Kif2A on spindle poles, and high MCAK depolymerization activity. This abnormality leads to faster microtubule flux, kinetochore–microtubule attachment failure, and the formation of a shorter metaphase spindle. Alternatively, overexpression of NuSAP-S240A, a loss-of-function mutant that interacts with less Kif2A on the spindle body, also causes faster microtubule flux and shorter metaphase spindle formation. Source data are available for this figure: SourceData F5.

Ser-240 phosphorylation of NuSAP promotes its interaction with Kif2A and reduces the localization of Kif2A on spindle poles. (A and C) Mitotic HEK293T cells cotransfected with the indicated GFP-tagged proteins and mCherry-tagged NuSAP constructs were immunoprecipitated with GFP-Trap beads. (B and D) The quantification of protein–protein interaction discrepancy. Error bars indicate SD. Three independent replicates were quantified (n = 3). Unpaired two-tailed t test: P = 0.0005 for WT/S240A, P = 0.0135 for S240A/S240D, P = 0.3489 for WT/S240D in B; P = 0.2274 for WT/S240A, P = 0.1531 for S240A/S240D, P = 0.2562 for WT/S240D in D. (E) NuSAP-knockout HeLa cells were transfected with GFP-tagged NuSAP-WT, -S240A, or -S240D (green) followed by immunofluorescence labeling using anti-Kif2A (red) and anti-α-tubulin (magenta) antibodies. DNA was stained with DAPI (blue). (F) Quantification of relative Kif2A intensity on spindle poles in E. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0049 for WT/S240A; P = 0.0051 for S240A/S240D; P = 0.3347 for WT/S240D. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = the number of independent experiments presented. Scale bars, 10 μm. (G) Along with the nuclear envelope broken down during mitotic entry, microtubule asters nucleate around the chromosomes and the centrosomes to form small acentrosomal and large centrosomal asters. These oriented microtubules are bundled and then sorted into the large centrosomal microtubule asters to form a bipolar spindle structure by molecular motors and microtubule-associated proteins till to a well-organized metaphase spindle formation with a dynamically constant length, in which NuSAP plays a crucial role. (H) NuSAP is mainly localized on microtubules near chromosomes and is phosphorylated by the mitotic kinase Aurora A on S240. As a MAP, NuSAP regulates the spindle dynamics to maintain metaphase spindle length through coordinating microtubule depolymerization and microtubule–microtubule sliding. On the one hand, NuSAP interacts with Eg5 on the adjacent microtubules, which allows Eg5 to move along the microtubules and to mediate the sliding of antiparallel interpolar microtubules (shown by the black arrow), which increases spindle length. On the other hand, S240-phosphorylated NuSAP interacts with more Kif2A on the spindle body and then reduces the amount of Kif2A on spindle poles, thus restraining the excess accumulation of Kif2A at the minus ends of microtubules to ensure the proper microtubule depolymerization. At the plus end, NuSAP attenuates MCAK depolymerization activity to stabilize kinetochore microtubules. NuSAP regulates microtubule flux (shown by the orange arrow) and controls metaphase spindle length by coordinating with Eg5, Kif2A, and MCAK. In experimental conditions, NuSAP knockout results in a reduction of Eg5 localization on spindle microtubules, excess accumulation of Kif2A on spindle poles, and high MCAK depolymerization activity. This abnormality leads to faster microtubule flux, kinetochore–microtubule attachment failure, and the formation of a shorter metaphase spindle. Alternatively, overexpression of NuSAP-S240A, a loss-of-function mutant that interacts with less Kif2A on the spindle body, also causes faster microtubule flux and shorter metaphase spindle formation. Source data are available for this figure: SourceData F5.

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