Takagi J et al. (2013), Chromosome position at the spindle equator is r...

XB-ART-47476

Sci Rep 2013 Sep 30;3:2808. doi: 10.1038/srep02808.

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Chromosome position at the spindle equator is regulated by chromokinesin and a bipolar microtubule array.

Takagi J , Itabashi T , Suzuki K , Ishiwata S .

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The chromosome alignment is mediated by polar ejection and poleward forces acting on the chromosome arm and kinetochores, respectively. Although components of the motile machinery such as chromokinesin have been characterized, their dynamics within the spindle is poorly understood. Here we show that a quantum dot (Qdot) binding up to four Xenopus chromokinesin (Xkid) molecules behaved like a nanosize chromosome arm in the meiotic spindle, which is self-organized in cytoplasmic egg extracts. Xkid-Qdots travelled long distances along microtubules by changing several tracks, resulting in their accumulation toward and distribution around the metaphase plate. The analysis indicated that the direction of motion and velocity depend on the distribution of microtubule polarity within the spindle. Thus, this mechanism is governed by chromokinesin motors, which is dependent on symmetrical microtubule orientation that may allow chromosomes to maintain their position around the spindle equator until correct microtubule-kinetochore attachment is established.

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Species referenced: Xenopus laevis
Genes referenced: kif22 rps3a

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	Figure 1. Analysis of Xkid-Qdot movement within the meiotic spindle.(a) Localization of Xkid-GFP-FL and Xkid-GFP-FL-T125N in the metaphase spindle in self-organized in Xenopus egg extracts. The Xkid constructs are shown in green. TMR- bovine brain tubulin and 4′,6-diamidino-2-phenylindole (DAPI) were added to visualize microtubules (red) and DNA (blue), respectively. Scale bar represents 10 μm. (b) Localization and motion of Xkid-Qdots (green or yellow, when merged with red) in the spindle. Arrows indicate Xkid-Qdots moving on the spindle microtubules (red). The right panel shows a kymograph of Xkid-Qdots motion along the pole-to-pole axis of the spindle depicted on the left panels. Scale bars represent 10 μm and 100 s. (c) Trajectories of Xkid-Qdots in the spindle. Open circles indicate the initial position of trajectory.
	Figure 2. Xkid-Qdots traverse long distances towards the spindle equator.(a) Schematic of the analysis of Xkid-Qdot motion. Xkid-Qdot motion was categorized into the three modes, Equator (E), Pole (P) and E-P, by the direction of movement along the pole-to-pole axis of spindle, and was analysed by calculating instantaneous velocity, run length, and lifetime in each mode, total run length, and total lifetime in each Qdot (see Methods). (b) Time course of the motion of Xkid-Qdots along the pole-to-pole axis of the spindle (n = 2 Qdots; red, equator mode; blue, pole mode; green, E-P mode). (c) Histograms of total run lengths and lifetimes for Xkid-Qdots (n = 9 spindles, 170 Qdots). The indicated average total run length (L) and average total lifetime (t) were determined by fitting the data to single exponentials excluding the first bin. (d) Histograms of instantaneous velocity for Xkid-Qdots in each mode fitted to a single Gaussian distribution (n = 9 spindles, 170 Qdots). (e) Average instantaneous velocity, run length and lifetime for Xkid-Qdots in each mode obtained by Gaussian or exponential fitting (mean ± s.e.m., data are shown in Supplementary Table S1, see also (d) and Supplementary Fig. S1e, f). Asterisks indicate the result of t- and the Mann-Whitney U tests (p < 0.05).
	Figure 3. The motion and direction of movement of Xkid-Qdots depend on the position in the spindle along the pole-to-pole axis.Spatial distribution of Xkid-Qdots and their motion along the pole-to-pole axis. (a) Histogram of the spatial distribution of Xkid-Qdots at each time point (red, equator mode; blue, pole mode; green, E-P mode) (n = 9 spindles, 170 Qdots, 5,234 time points) and DNA distribution along the pole-to-pole axis (mean ± s.e.m.; blue, n = 7 spindles) as a function of the distance from equator (AU). (b) Spatial distribution of the mode of motion along the pole-to-pole axis (red, equator mode; blue, pole mode; green, E-P mode) shown in (a). Each region represents at least 100 time points. (c) Spatial distribution of the instantaneous velocity along the pole-to-pole axis (mean ± s.e.m.; red, equator mode; blue, pole mode; green, E-P mode). Data with statistical significance (p) are shown in Supplementary Table S2, see also Supplementary Fig. S3a. (d) Spatial distribution of the run length and lifetime along the pole-to-pole axis (mean ± s.e.m.; red, equator mode; blue, pole mode; green, E-P mode). Data are shown in Supplementary Table S2, see also Supplementary Fig. S3b, c.
	Figure 4. Xkid-Qdots accumulate towards the plus ends of microtubules in the spindle-like microtubule structure.The monopolar microtubule structure ((a)–(c), n = 12 spindles, 569 Qdots, 12,362 time points) and the spindle-like bipolar structure ((d)–(f), n = 1 spindle, 518 Qdots, 13,792 time points) were formed in the presence of 200 μM monastrol. (a) Localization and motion of Xkid-Qdots (green) in the monopolar microtubule structure (red). The right panel shows a kymograph of Xkid-Qdot motion in a region depicted in the left panel (white dashed box) along the white arrow. (b) Histogram of spatial distribution of Xkid-Qdots in the monopolar microtubule structure for each time point from the pole to outward (red, outward mode; blue, pole mode; green, O-P mode). (c) Spatial distribution of the mode of motion in the monopolar microtubule structure from the pole to outward (red, outward mode; blue, pole mode; green, O-P mode). Each region represents at least 100 time points. (d) Localization and movement of Xkid-Qdots (green) in the spindle-like bipolar assembly of a pair of monopolar microtubule structures (red). The right panel shows a kymograph of Xkid-Qdots in a region depicted in the left panel (white dashed box) along the white arrow. (e) Histogram of spatial distribution of Xkid-Qdots in the spindle-like bipolar structure at each time point along the axis perpendicular to the blue dashed line in the left panel of (d) (red, outward mode; blue, pole mode; green, O-P mode). (f) Spatial distribution of the mode of motion in the spindle-like bipolar structure along the axis perpendicular to the blue dashed line in the left panel of (d) (red, outward mode; blue, pole mode; green, O-P mode). Each region represents at least 100 time points. (g) Schematic showing Xkid-Qdot motion along the spindle's symmetrically ordered microtubule structure. Xkid-Qdot movement along the spindle is indicated by yellow circles, and their size represents the probability of each directional movement. Dashed and solid lines represent the proportion of polarity and average length of microtubules derived from each spindle pole (red and blue), respectively. Scale bars in (a) and (d) represent 10 μm and 100 s.

References [+] :

Antonio, Xkid, a chromokinesin required for chromosome alignment on the metaphase plate. 2000, Pubmed, Xenbase