Gatlin JC et al. (2010), Directly probing the mechanical properties of t...

XB-ART-41191

J Cell Biol 2010 Feb 22;1884:481-9. doi: 10.1083/jcb.200907110.

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Directly probing the mechanical properties of the spindle and its matrix.

Matov A , Danuser G , Mitchison TJ , Salmon ED .

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Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 numa1 pnp

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	Figure 1. Experimental approach for spindle skewering. (A) The cartoon shows the experimental setup used in all skewering experiments (see Materials and methods for more details). (B) Example of a skewered spindle visualized by the addition of X-rhodamineâlabeled tubulin to the extract. The cross section of the microneedle is seen as a dark annulus. Bar, 25 Âµm.
	Figure 2. Intrinsic spindle forces move impaling microneedles through the spindle. (A) Images shown were selected from three time-lapse series of skewered spindles. Dashed lines indicate the position of the microneedle, which remained essentially stationary during the experimental time course. (B) Kymographs were used to analyze velocities of spindles as they translocated off microneedles. Because skewered spindles often rotated during time-lapse experiments as the result of changing flows within the extract (e.g., spindle #3), each image in the series was rotated using custom software to maintain a fixed orientation of the spindleâs interpolar axis (arrowheads mark the position of the needle). (C) The distance between the nearest pole and the middle of the needle was plotted versus time. Slopes were calculated using linear regressions from these plots of pole to needle distances of >5 Âµm and those equal to 5 Âµm. (D) Two microneedles were used to impale spindles. In each case, the needles were positioned on the same side of the spindle midzone, and the spindle moved off both needles, regardless of the predominant direction of extract flow (indicated by the white arrow). Dashed lines indicate the position of the microneedles used to skewer the spindles. Bars, 25 Âµm.
	Figure 3. Intrinsic forces push but do not pull the spindle off impaling microneedles. (A) The time-lapse series shows the dynamic morphology of a spindle pole, labeled with Alexa Fluor 488 anti-NuMA antibodies (green), as it is split by a microneedle. Spindles continued to move despite a lack of any detectable microtubules (red) on the distal side of the microneedle. (B) Time-lapse images show the behavior of skewered spindles â¼5â10 min after the addition of function-perturbing antibodies against the 70.1-kD DIC (anti-DIC). The distance between the metaphase plate and the needle were measured and plotted as a function of time for multiple spindles in the corresponding graph. (C) Assembled spindles were treated with 1.5 ÂµM AMP-PNP and skewered within 5â10 min after treatment. AMP-PNP at this concentration inhibited flux, in agreement with nearly horizontal plots of needle to spindle midpoint distance versus time (graph). (B and C) Dashed lines indicate the position of the microneedle used to skewer the spindles. Bars: (A) 5 Âµm; (B and C) 25 Âµm.
	Figure 4. Lateral interactions between microtubules are more robust near the spindle poles. (A and C) The cartoons show the initial positions of the two skewering needle tips within the spindle and the direction of needle movement. (A) The two needles were initially positioned one on each side of the metaphase plate and then spread apart along the interpolar axis. The images show a representative time-lapse series of a spindle being longitudinally stretched. Dashed lines represent the position of the spindle poles before the onset of stretching. (B) Changes in spindle length during longitudinal stretching (normalized to the initial length of the spindle) are plotted versus time, shown as black lines, whereas corresponding plots of needle separation versus time are shown in red. Matching markers indicate data taken from the same experiment. Percentages are the ratio of needle separation at the onset of spindle elongation to the initial spindle length (elongation onset was arbitrarily defined as the time point at which the spindle became 0.5% longer than its initial length). (C) Spindles were also stretched in the orthogonal direction, transverse to their interpolar axes. In these experiments, spindle deformation began at the onset of needle separation and continued until the needles were stopped. White arrowheads indicate the position of the interpolar axis after transverse spindle stretching. Bars, 25 Âµm.
	Figure 5. Predicted experimental outcomes of single needle experiments for different types of matrices assuming flux forces can be transmitted to the needle by lateral microtubule cross-links. (AâC) Thin red lines represent the matrix, whereas green lines represent spindle microtubules. Thicker sections along the microtubules depict the movement of a photoactivated mark on the microtubule lattice to illustrate poleward flux over time. The blue shapes within the spindle midzone represent aligned metaphase chromosomes. The dashed line runs through the midpoint of the stationary needle tip, as seen in cross-section (depicted as a white annulus with a filled, black center). See âDoes a spindle matrix play a mechanical role in spindle length regulation?â for explanations of each cartoon.

References [+] :

Begg, Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers. 1979, Pubmed