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Length-dependent anisotropic scaling of spindle shape.

Young S, Besson S, Welburn JP - Biol Open (2014)

Bottom Line: We demonstrate that spindle shape scaling is independent of the nature of the molecules that regulate dynamic microtubule properties, but is dependent on the steady-state metaphase spindle length.The shape of the spindle scales anisotropically with increasing length.Our results suggest that intrinsic mechanisms control the shape of the spindle to ensure the efficient capture and alignment of chromosomes independently of spindle length.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.

No MeSH data available.


Related in: MedlinePlus

The chromokinesin Kid opposes Clasp1 and Clasp2 during spindle formation.(A) Time-lapse imaging of U2OS cells expressing mCherry-tubulin after a STLC washout. (B–D) Graphs representing the average spindle length, width and aspect ratio and the corresponding SD, during elongation for each condition described in panel B. (E) Time-lapse imaging of U2OS cells expressing mCherry-tubulin and PA-GFP-tubulin before and at 30 s intervals after photoactivation at 405 nm. (F,G) Quantification of microtubule poleward rate + SD and fast turnover + sem in cells treated with Control, Kid, Clasp2 or Clasp2/Kid siRNA. *, ** and *** represent a P<0.5, P<0.01 and P<0.001 respectively. Scale bars: 10 µm.
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f02: The chromokinesin Kid opposes Clasp1 and Clasp2 during spindle formation.(A) Time-lapse imaging of U2OS cells expressing mCherry-tubulin after a STLC washout. (B–D) Graphs representing the average spindle length, width and aspect ratio and the corresponding SD, during elongation for each condition described in panel B. (E) Time-lapse imaging of U2OS cells expressing mCherry-tubulin and PA-GFP-tubulin before and at 30 s intervals after photoactivation at 405 nm. (F,G) Quantification of microtubule poleward rate + SD and fast turnover + sem in cells treated with Control, Kid, Clasp2 or Clasp2/Kid siRNA. *, ** and *** represent a P<0.5, P<0.01 and P<0.001 respectively. Scale bars: 10 µm.

Mentions: Polar ejection forces contribute to chromosome alignment by pushing chromatin away from the spindle poles during prometaphase (Magidson et al., 2011). The kinesin-10 family chromokinesin Kid is the major contributor to polar ejection forces (Bieling et al., 2010; Cane et al., 2013; Stumpff et al., 2012). We hypothesized that polar ejection forces could play a role in spindle shape. Kid depletion alone did not alter spindle length or width significantly by live-cell imaging (12.8 ± 1.7 µm and 8.8 ± 1.2 µm, respectively), in agreement with previous work (Stumpff et al., 2012), although a study measuring spindle length in Kid-depleted cells in fixed cells reported a small decrease in length (Fig. 2A–C) (Tokai-Nishizumi et al., 2005). The final spindle width was however reached later than that of control cells, due to reduced polar ejection forces that position chromosomes during prometaphase (supplementary material Fig. S2E,F) (Magidson et al., 2011). As Kid interacts with CLASP (Patel et al., 2012), we also tested whether Kid acts synergistically with Clasps during bipolar spindle formation. When Kid and Clasp2 were co-depleted, spindle length was restored to 12.7 ± 1.2 µm and spindle width was not significantly modified, averaging 8.5+0.9 µm (Fig. 2A–C; supplementary material Table S1). Under these conditions, Kid and Clasp2 were efficiently co-depleted (supplementary material Fig. S1E). We also observed a similar rescue in spindle length following the co-depletion of Kid and Clasp1 (11.5 ± 1.5 µm; supplementary material Fig. S1F; Table S1). Interestingly, the spindles did not contract after maximal elongation in Clasp1 or Clasp2 and Kid co-depleted cells, leading to the higher aspect ratio of Clasp2/Kid spindles (Fig. 2D). Our data suggest that Kid-mediated polar ejection forces on their own do not alter the diamond-shape spindle geometry, but that Clasp1 and Clasp2 have a functional and antagonistic relationship with Kid for regulating spindle geometry.


Length-dependent anisotropic scaling of spindle shape.

Young S, Besson S, Welburn JP - Biol Open (2014)

The chromokinesin Kid opposes Clasp1 and Clasp2 during spindle formation.(A) Time-lapse imaging of U2OS cells expressing mCherry-tubulin after a STLC washout. (B–D) Graphs representing the average spindle length, width and aspect ratio and the corresponding SD, during elongation for each condition described in panel B. (E) Time-lapse imaging of U2OS cells expressing mCherry-tubulin and PA-GFP-tubulin before and at 30 s intervals after photoactivation at 405 nm. (F,G) Quantification of microtubule poleward rate + SD and fast turnover + sem in cells treated with Control, Kid, Clasp2 or Clasp2/Kid siRNA. *, ** and *** represent a P<0.5, P<0.01 and P<0.001 respectively. Scale bars: 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265759&req=5

f02: The chromokinesin Kid opposes Clasp1 and Clasp2 during spindle formation.(A) Time-lapse imaging of U2OS cells expressing mCherry-tubulin after a STLC washout. (B–D) Graphs representing the average spindle length, width and aspect ratio and the corresponding SD, during elongation for each condition described in panel B. (E) Time-lapse imaging of U2OS cells expressing mCherry-tubulin and PA-GFP-tubulin before and at 30 s intervals after photoactivation at 405 nm. (F,G) Quantification of microtubule poleward rate + SD and fast turnover + sem in cells treated with Control, Kid, Clasp2 or Clasp2/Kid siRNA. *, ** and *** represent a P<0.5, P<0.01 and P<0.001 respectively. Scale bars: 10 µm.
Mentions: Polar ejection forces contribute to chromosome alignment by pushing chromatin away from the spindle poles during prometaphase (Magidson et al., 2011). The kinesin-10 family chromokinesin Kid is the major contributor to polar ejection forces (Bieling et al., 2010; Cane et al., 2013; Stumpff et al., 2012). We hypothesized that polar ejection forces could play a role in spindle shape. Kid depletion alone did not alter spindle length or width significantly by live-cell imaging (12.8 ± 1.7 µm and 8.8 ± 1.2 µm, respectively), in agreement with previous work (Stumpff et al., 2012), although a study measuring spindle length in Kid-depleted cells in fixed cells reported a small decrease in length (Fig. 2A–C) (Tokai-Nishizumi et al., 2005). The final spindle width was however reached later than that of control cells, due to reduced polar ejection forces that position chromosomes during prometaphase (supplementary material Fig. S2E,F) (Magidson et al., 2011). As Kid interacts with CLASP (Patel et al., 2012), we also tested whether Kid acts synergistically with Clasps during bipolar spindle formation. When Kid and Clasp2 were co-depleted, spindle length was restored to 12.7 ± 1.2 µm and spindle width was not significantly modified, averaging 8.5+0.9 µm (Fig. 2A–C; supplementary material Table S1). Under these conditions, Kid and Clasp2 were efficiently co-depleted (supplementary material Fig. S1E). We also observed a similar rescue in spindle length following the co-depletion of Kid and Clasp1 (11.5 ± 1.5 µm; supplementary material Fig. S1F; Table S1). Interestingly, the spindles did not contract after maximal elongation in Clasp1 or Clasp2 and Kid co-depleted cells, leading to the higher aspect ratio of Clasp2/Kid spindles (Fig. 2D). Our data suggest that Kid-mediated polar ejection forces on their own do not alter the diamond-shape spindle geometry, but that Clasp1 and Clasp2 have a functional and antagonistic relationship with Kid for regulating spindle geometry.

Bottom Line: We demonstrate that spindle shape scaling is independent of the nature of the molecules that regulate dynamic microtubule properties, but is dependent on the steady-state metaphase spindle length.The shape of the spindle scales anisotropically with increasing length.Our results suggest that intrinsic mechanisms control the shape of the spindle to ensure the efficient capture and alignment of chromosomes independently of spindle length.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.

No MeSH data available.


Related in: MedlinePlus