Suzuki K et al. (2017), Mechanical properties of spindle poles are symm...

XB-ART-53625

Biophys Physicobiol 2017 Jan 24;14:1-11. doi: 10.2142/biophysico.14.0_1.

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Mechanical properties of spindle poles are symmetrically balanced.

Suzuki K , Itabashi T , Ishiwata S .

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The metaphase spindle is organized for accurate chromosome segregation. One of the fundamental features of the spindle across the species is its symmetrical shape; the spindle consists of two polar arrays of microtubules at both ends. Although it has been suggested that the formation of the bipolar shape requires force balance coordination by molecular motors, i.e., kinesins and dyneins, quantitative analysis for the pole mechanics has not been conducted. Here, we demonstrate that it is not only the shape but also the stiffness and microtubule density of the pairs of pole regions are symmetrically balanced in single spindles self-assembled in Xenopus egg extracts. We found that the inhibition of dynein functions dramatically reduced the stiffness and microtubule density in the pole region. By contrast, the inhibition of one of the kinesins, Eg5, which is the antagonistic motor protein of dynein, increased the value of these parameters. Moreover, the inhibition of both dynein and Eg5 recovered these parameter values to those of non-treated spindle poles. We also found that, when one pole structure was held widened with the use of two glass microneedles, the opposite pole structure spontaneously widened, resulting in the formation of the barrel-like shaped spindle. The values of stiffness and microtubule density in the manipulated pole region decreased, following the spontaneous decrement of those in the paired unmanipulated pole region. These results suggest that the spindle possesses a mechanism to dynamically maintain its symmetry in mechanical properties.

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Species referenced: Xenopus
Genes referenced: abl1 kif11

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	Figure 1. A pair of poles of a spindle is symmetric in shape. (A) The spindle width at 5–50% of spindle length away from the pole (left), and definitions of pole width Wp, pole length Lp and pole area S of the pole region (right). In the left panel, the distance from the pole end is normalized with L (pole-to-pole distance), and each plot includes 28 spindles (error bars, ± S.D.). The orange and blue lines indicate the linear fit (slope: 0.47 and 0.14) for the plots of 5–20% and 20–50% of spindle length away from the pole, respectively. In the right panel, scale bar: 10 μm. (B, C) Comparison of the aspect ratio (B) and γ (C) between pairs of pole regions in single spindles. The aspect ratio and γ of pole regions were calculated as Wp/Lp and S/(Wp×Lp)×2, respectively. The numbers 1 and 2 for the pole region labeled on the abscissa and ordinate represent the left and right pole regions in single spindles, respectively. The mean ± S.D. of the aspect ratio and γ value of pole regions were 0.51 ± 0.08 and 1.31 ± 0.09, respectively (n = 78 poles of 39 spindles). The r indicates the correlation coefficient. Schematic illustrations in (C) represent the spindle shape having the corresponding γ values. If the value of γ is 1.0 or 1.57, the pole shape is analogous to a triangle or a half circle, respectively. If the value of γ is between 1.0 and 1.57, the pole shape is analogous to a half ellipse.
	Figure 2. Stiffness of a pair of pole regions is symmetrical in a spindle. (A) Schematic illustration and (B) microscopic images of the spindle and glass microneedles during measurement of the stiffness of the pole region. In (B), SN and FN in the left image indicate stiff and flexible needles, respectively. The right image is the kymograph of the region within the yellow box in the left image. In the left image, scale bar: 10 μm. In the kymograph, scale bars: 10 μm (vertical) and 4 sec (horizontal). (C) Time courses of the displacement of the stiff needle and the distance between two needles d, obtained from the manipulation experiment shown in (B). In the left panel, red plots indicate the force estimated from the deflection of the flexible needle, and black plots denote the displacement of the stiff needle. (D) The relationship between d and force determined from the deflection of the flexible needle shown in (B). The grey line indicates the linear fit for the plots in the region where d was 0–2 μm. The stiffness of the pole region was calculated from the slope of the d versus force plot in the region where d was 0–2 μm, because the force had a linear correlation with d in this region. (E–H) The dependencies of the stiffness of the pole region on spindle length (E), spindle width (width at the equatorial plane of the spindle) (F), the aspect ratio of the pole region (G) and γ (H) of the pole region. The r indicates the correlation coefficient. We examined n = 39 poles of 39 spindles (one pole of each spindle). (I) Comparison of the stiffness between pairs of pole regions in single spindles. The numbers 1 and 2 for the pole region labeled on the abscissa and ordinate represent the left and right pole regions in single spindles, respectively.
	Figure 3. Dynein and Eg5 are involved in the maintenance of the mechanical strength of spindle poles. In all panels, blue, green and orange colors represent p150-CC1- (dynein inhibitor), monastrol- (Eg5 inhibitor) and p150-CC1 and monastrol-treated spindles, respectively. (A, B) Comparison of the aspect ratio (A) and the γ value (B) between pairs of pole regions in single spindles in the presence of p150-CC1 and monastrol. The numbers 1 and 2 for the pole region labeled on the abscissa and ordinate represent the left and right pole regions in single spindles, respectively. Mean ± S.D. is shown in the upper left corners (the mean ± S.D. with the asterisk means that the value is significantly different from that of nontreated spindles, p < 0.05 by Student’s t-test). Closed circles represent the mean values. (C) The stiffness of the pole region in the presence of inhibitors. Solid lines indicate the mean values. Mean ± S.D.; 0.84 ± 0.40 (non-treated), 0.23 ± 0.14 (p150-CC1), 1.12 ± 0.44 (monastrol) and 0.81 ± 0.41 (both p150-CC1 and monastrol). n = 39 (non-treated), 18 (p150-CC1), 44 (monastrol) and 40 (both p150-CC1 and monastrol) spindles. n.s. (not significant): p > 0.1 by Mann–Whitney U test. (D) Comparison of the stiffness between pairs of pole regions in single spindles. n = 26 (non-treated), 15 (p150-CC1), 15 (monastrol) and 11 (both p150-CC1 and monastrol) spindles. The correlation coefficients are 0.82 (non-treated), 0.60 (p150-CC1), 0.44 (monastrol) and 0.80 (both p150-CC1 and monastrol). The plots represent mean values (error bars, ± S.D.). The r indicates the correlation coefficient.
	Figure 4. Dynein and Eg5 regulate microtubule density in the pole region. In all panels, black, blue, green and orange colors represent non-treated, p150-CC1, monastrol and both of p150-CC1 and monastrol-treated spindles, respectively. (A) Microtubule density D in the pole region: 28 poles of 14 spindles (non-treated), 16 poles of 8 spindles (p150-CC1), 16 poles of 8 spindles (monastrol) and 20 poles of 10 spindles (both p150-CC1 and monastrol). n.s. (not significant): p > 0.1 by Student’s t-test. (B) Comparison of microtubule density D between pairs of pole regions in single spindles. The numbers 1 and 2 for the pole region labeled on the abscissa and ordinate represent the left and right pole regions in single spindles, respectively. n = 19 spindles (non-treated), 8 spindles (p150-CC1), 8 spindles (monastrol) and 10 spindles (both p150-CC1 and monastrol). Error bars, ± S.D. (C) Relationship between microtubule density D and the stiffness of the pole region. The solid curve indicates the best fit by the stiffness (= a×Db where a = 1.34×10−4 and b = 1.91).
	Figure 5. A pair of pole regions in a single spindle is symmetrically balanced in the shape, stiffness and microtubule density. (A) Time-lapse images of the spindle which was held asymmetrically deformed for ~10 min. In the 0 min image, the two orange rods indicate the glass microneedles for the widening, and the blue rod represents the microneedle positioned to avoid incline of the spindle from the focal plane). 0 min: the timing of the start of the widening, Scale bar: 10 μm. (B) Time courses of the γ value (left panel) and the microtubule density D in the pole regions (right panel) in the spindle shown in (A). (C) The relationship between the spindle width (left panel), spindle length (right panel) and the time period from the widening to the timing when the γ value of the unmanipulated pole region became the maximum value during the observation (at this timing, all spindles formed the barrel-like shape as in (A)). n = 15 spindles. (D) The stiffness of the pole region before and after the widening of only one pole structure in a single spindle. The same colors indicate the same spindle (e.g., the black open and closed circles indicate the stiffness of the manipulated and unmanipulated pole regions, respectively, in a single spindle). n = 4 spindles.

References [+] :

Belmont, Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. 1996, Pubmed, Xenbase