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A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends

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ABSTRACT

The Astrin/SKAP complex regulates mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, Kern et al. demonstrate that a previously unappreciated short SKAP isoform mediates mitotic spindle positioning at astral microtubule plus ends.

No MeSH data available.


Related in: MedlinePlus

SKAP ΔEB mutant cells display a spindle mispositioning defect. (A, left) IF images showing microtubules in the SKAP ΔEB mutant spindle. Images represent maximum-intensity projections. The dashed line indicates the cell boundary. (right) Graph showing difference in the distances between each spindle pole and the closest position on the cell cortex (see diagram at bottom right of the figure). A value of 0 represents spindle positioning in the cell center, with equivalent distances to each cell cortex. n = 100 total cells per condition collected from two (ΔEB without depletion) or three independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. *, difference with P = 0.0153. (B, left) Images showing maximum-intensity projections for microtubule staining to show for spindle mispositioning phenotype and its suppression by LGN depletion. (right) Graph showing the pole–cortex difference for LGN experiment (plotted as in A). n = 100 cells per condition collected from three independent experiments. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (C, left) Maximum-intensity projections for microtubule staining show the spindle mispositioning phenotype and its suppression by 20 nM nocodazole. (right) Graph showing the pole–cortex difference (as in A and B) for the low-dose nocodazole experiment. n = 100 cells per condition collected from two independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (D) Kymographs of videos from cells in which wild-type (WT) SKAP or the SKAP ΔEB mutant replaces endogenous SKAP. Arrows mark the start of the spindle shift in ΔEB cells. Also see Video 4. DIC, differential interference contrast.
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fig5: SKAP ΔEB mutant cells display a spindle mispositioning defect. (A, left) IF images showing microtubules in the SKAP ΔEB mutant spindle. Images represent maximum-intensity projections. The dashed line indicates the cell boundary. (right) Graph showing difference in the distances between each spindle pole and the closest position on the cell cortex (see diagram at bottom right of the figure). A value of 0 represents spindle positioning in the cell center, with equivalent distances to each cell cortex. n = 100 total cells per condition collected from two (ΔEB without depletion) or three independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. *, difference with P = 0.0153. (B, left) Images showing maximum-intensity projections for microtubule staining to show for spindle mispositioning phenotype and its suppression by LGN depletion. (right) Graph showing the pole–cortex difference for LGN experiment (plotted as in A). n = 100 cells per condition collected from three independent experiments. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (C, left) Maximum-intensity projections for microtubule staining show the spindle mispositioning phenotype and its suppression by 20 nM nocodazole. (right) Graph showing the pole–cortex difference (as in A and B) for the low-dose nocodazole experiment. n = 100 cells per condition collected from two independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (D) Kymographs of videos from cells in which wild-type (WT) SKAP or the SKAP ΔEB mutant replaces endogenous SKAP. Arrows mark the start of the spindle shift in ΔEB cells. Also see Video 4. DIC, differential interference contrast.

Mentions: Although the SKAP ΔEB mutant was able to facilitate normal chromosome segregation, during our analysis of chromosome alignment, we unexpectedly observed that many spindles in the SKAP ΔEB mutant were dramatically mispositioned away from the cell midzone, often adjacent to or abutting the cell cortex (Fig. 5 A). In these extreme cases, cells appeared to have centrosomes and their astral microtubules directly in contact with the cortex (Fig. S3 C). In control cells or cells in which wild-type SKAP rescued endogenous SKAP depletion, we found that the spindle was typically positioned symmetrically within the dividing cells with similar distances between each spindle pole and the cell cortex (mean spindle displacement: control, 0.62 µm; rescue, 0.87 µm; Fig. 5 A). In contrast, we found that the spindle was positioned asymmetrically within the cell in SKAP ΔEB mutant cells such that one spindle pole was often much closer to the cell cortex (mean spindle displacement: ΔEB, 2.1 µm; Fig. 5 A), including cells with extremely mispositioned spindles (Fig. 5 A). Expression of the SKAP ΔEB mutant without depletion of the endogenous protein resulted in a modest, but statistically significant, shift in spindle positioning (mean spindle displacement: 1.05 µm; Fig. 5 A), potentially because of the formation of mixed Astrin/SKAP complexes. SKAP-depleted cells display dramatic and pleiotropic defects in chromosome alignment and centrosome stability (Fig. 2), which can influence spindle positioning indirectly (Kiyomitsu and Cheeseman, 2012; Tame et al., 2016), thereby preventing a directed analysis of spindle positioning phenotypes in SKAP-depleted cells with more extreme phenotypes (see Video 1). To test for spindle positioning defects in SKAP-depleted cells without substantial secondary defects, we quantified cells with clearly defined metaphase plates, which likely represent cells with an intermediate SKAP depletion. In these depleted cells, we found substantial spindle mispositioning (mean spindle displacement: 1.62 µm; Fig. 5 A), although slightly less than that observed in the SKAP ΔEB mutant. We conclude that SKAP ΔEB mutant replacement has a potent and specific effect on spindle positioning.


A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends
SKAP ΔEB mutant cells display a spindle mispositioning defect. (A, left) IF images showing microtubules in the SKAP ΔEB mutant spindle. Images represent maximum-intensity projections. The dashed line indicates the cell boundary. (right) Graph showing difference in the distances between each spindle pole and the closest position on the cell cortex (see diagram at bottom right of the figure). A value of 0 represents spindle positioning in the cell center, with equivalent distances to each cell cortex. n = 100 total cells per condition collected from two (ΔEB without depletion) or three independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. *, difference with P = 0.0153. (B, left) Images showing maximum-intensity projections for microtubule staining to show for spindle mispositioning phenotype and its suppression by LGN depletion. (right) Graph showing the pole–cortex difference for LGN experiment (plotted as in A). n = 100 cells per condition collected from three independent experiments. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (C, left) Maximum-intensity projections for microtubule staining show the spindle mispositioning phenotype and its suppression by 20 nM nocodazole. (right) Graph showing the pole–cortex difference (as in A and B) for the low-dose nocodazole experiment. n = 100 cells per condition collected from two independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (D) Kymographs of videos from cells in which wild-type (WT) SKAP or the SKAP ΔEB mutant replaces endogenous SKAP. Arrows mark the start of the spindle shift in ΔEB cells. Also see Video 4. DIC, differential interference contrast.
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fig5: SKAP ΔEB mutant cells display a spindle mispositioning defect. (A, left) IF images showing microtubules in the SKAP ΔEB mutant spindle. Images represent maximum-intensity projections. The dashed line indicates the cell boundary. (right) Graph showing difference in the distances between each spindle pole and the closest position on the cell cortex (see diagram at bottom right of the figure). A value of 0 represents spindle positioning in the cell center, with equivalent distances to each cell cortex. n = 100 total cells per condition collected from two (ΔEB without depletion) or three independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. *, difference with P = 0.0153. (B, left) Images showing maximum-intensity projections for microtubule staining to show for spindle mispositioning phenotype and its suppression by LGN depletion. (right) Graph showing the pole–cortex difference for LGN experiment (plotted as in A). n = 100 cells per condition collected from three independent experiments. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (C, left) Maximum-intensity projections for microtubule staining show the spindle mispositioning phenotype and its suppression by 20 nM nocodazole. (right) Graph showing the pole–cortex difference (as in A and B) for the low-dose nocodazole experiment. n = 100 cells per condition collected from two independent experiments. Mean and SD are plotted. ****, P < 0.0001, significant difference assessed by an unpaired two-tailed t test. (D) Kymographs of videos from cells in which wild-type (WT) SKAP or the SKAP ΔEB mutant replaces endogenous SKAP. Arrows mark the start of the spindle shift in ΔEB cells. Also see Video 4. DIC, differential interference contrast.
Mentions: Although the SKAP ΔEB mutant was able to facilitate normal chromosome segregation, during our analysis of chromosome alignment, we unexpectedly observed that many spindles in the SKAP ΔEB mutant were dramatically mispositioned away from the cell midzone, often adjacent to or abutting the cell cortex (Fig. 5 A). In these extreme cases, cells appeared to have centrosomes and their astral microtubules directly in contact with the cortex (Fig. S3 C). In control cells or cells in which wild-type SKAP rescued endogenous SKAP depletion, we found that the spindle was typically positioned symmetrically within the dividing cells with similar distances between each spindle pole and the cell cortex (mean spindle displacement: control, 0.62 µm; rescue, 0.87 µm; Fig. 5 A). In contrast, we found that the spindle was positioned asymmetrically within the cell in SKAP ΔEB mutant cells such that one spindle pole was often much closer to the cell cortex (mean spindle displacement: ΔEB, 2.1 µm; Fig. 5 A), including cells with extremely mispositioned spindles (Fig. 5 A). Expression of the SKAP ΔEB mutant without depletion of the endogenous protein resulted in a modest, but statistically significant, shift in spindle positioning (mean spindle displacement: 1.05 µm; Fig. 5 A), potentially because of the formation of mixed Astrin/SKAP complexes. SKAP-depleted cells display dramatic and pleiotropic defects in chromosome alignment and centrosome stability (Fig. 2), which can influence spindle positioning indirectly (Kiyomitsu and Cheeseman, 2012; Tame et al., 2016), thereby preventing a directed analysis of spindle positioning phenotypes in SKAP-depleted cells with more extreme phenotypes (see Video 1). To test for spindle positioning defects in SKAP-depleted cells without substantial secondary defects, we quantified cells with clearly defined metaphase plates, which likely represent cells with an intermediate SKAP depletion. In these depleted cells, we found substantial spindle mispositioning (mean spindle displacement: 1.62 µm; Fig. 5 A), although slightly less than that observed in the SKAP ΔEB mutant. We conclude that SKAP ΔEB mutant replacement has a potent and specific effect on spindle positioning.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

The Astrin/SKAP complex regulates mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, Kern et al. demonstrate that a previously unappreciated short SKAP isoform mediates mitotic spindle positioning at astral microtubule plus ends.

No MeSH data available.


Related in: MedlinePlus