The distribution of polar ejection forces determines the amplitude of chromosome directional instability - PubMed (original) (raw)
The distribution of polar ejection forces determines the amplitude of chromosome directional instability
Kevin Ke et al. Curr Biol. 2009.
Abstract
Background: Polar ejection forces have often been hypothesized to guide directional instability of mitotic chromosomes, but a direct link has never been established. This has led, in part, to the resurgence of alternative theories. By taking advantage of extremely precise femtosecond pulsed laser microsurgery, we abruptly alter the magnitude of polar ejection forces by severing vertebrate chromosome arms.
Results: Reduction of polar ejection forces increases the amplitude of directional instability without altering other characteristics, thus establishing a direct link between polar ejection forces and the direction of chromosome movements. We find that polar ejection forces limit the range of chromosome oscillations by increasing the probability that motors at a leading kinetochore abruptly disengage or turn off, leading to a direction reversal.
Conclusions: From the relation between the change in oscillation amplitude and the amount a chromosome arm is shortened, we are able to map the distribution of polar ejection forces across the spindle, which is surprisingly different from previously assumed distributions. These results allow us to differentiate between the mechanisms proposed to underlie the directional instability of chromosomes.
Figures
Figure 1. Forces driving chromosome movement
The known forces acting on a chromosome are the poleward force on kinetochore microtubules (kMT), and PEFs. The PEFs that push the arms away from a nearby pole are developed by chromokinesin motors (green and black) that move toward the plus ends of microtubules, located distal to the spindle pole. Polymerizing microtubules impinging on the arms may also contribute to PEFs. PEFs from each pole are oppositely directed, with the PEF from the nearer (left) pole is expected to be greater due to the higher microtubule density. The depicted chromosome will move toward the left pole if the poleward left kinetochore force exceeds the net PEF. Drawing is not to scale, and for clarity the number of polar and kinetochore microtubules is greatly underrepresented. Upper right inset models PEFs analogous to a potential well and illustrates how this predicts a change in PEFs affects chromosome movements. Confined by PEFs pushing the chromosome toward the spindle equator, the oscillation amplitude depends on the force required to cause reversal of the leading kinetochores. Upon severing the chromosome arm, the PEF is decreased, leading to increased oscillation amplitude.
Figure 2. Laser microsurgery setup
An ultrafast pulsed laser is focused to a diffraction limited spot slightly exceeding the critical intensity for breakdown of biological material. Chromosomes are visualized by phase contrast, and targeted with a nanopositioning stage. A portion of an arm is severed by scanning the chromosome across the laser focus (e.g. within broken line). Circles show the locations of the spindle poles.
Figure 3. Chromosome movement before and after severing an arm
In the images and corresponding traces, blue indicates the severed arm, and green the kinetochore-containing region. Positions are relative to the spindle equator. In the images below the trace, the initially intact chromosome oscillates at average 1.4 μm peak-to-peak amplitude. The arm is severed immediately before the second frame. The region containing the kinetochores continues to exhibit directional instability but at higher amplitude (average 2.7 μm). The severed arm (blue) initially follows the rest of the chromosome, but loses synchronization by 1000 seconds. By 2063s, the chromosome and the severed arm have moved to opposite sides of the spindle equator. The oscillation amplitude of the control chromosomes does not increase.
Figure 4. Shortening a chromosome arm does not change speed
Antipoleward (toward spindle equator) and poleward are compared before and after severing chromosome arms. The average speed was about 31 nm/s, consistent with the work of Skibbens et al. [32], and was not changed by severing chromosome arms.
Figure 5. Chromosome oscillations before and after the arms are shortened
Each panel shows a different chromosome's movements before and after surgery (indicated by the vertical line on the traces). Positions are relative to the spindle equator. The arrow in the micrograph to the left of each trace indicates where each chromosome was severed, and the spindle poles are circled. The solid lines (Figure A & B) are linear regression fits used to determine oscillation speeds and amplitudes. The numbers in the ovals compare the oscillation amplitude before and after the ablation. A-I show significant increase in average amplitude (p<0.05, black oval). J-L show significant decrease in amplitude (p<0.05, dotted oval). M-P show no significant change (p>0.05, gray oval).
Figure 6. Estimation of the polar ejection force distribution
A) The relative change in the average amplitude of directional instability after severing a chromosome arm as a function of the average amplitude before severing the arm (squares). Black diamonds are control chromosomes located in the same cell as an experimental chromosome: in these cases the before and after amplitudes correspond to before and after severing the arm of the experimental chromosome. The average oscillation amplitude of the control chromosomes is 2.9 ± 1.6 μm (dashed line), and the dotted line is 2 standard deviations from the average. B) Estimation of the PEF distribution from the equation _F_=KAxn (see text). The two circled outlying data points were excluded from the line fit. The slope of the line, n, is 0.57 ± 0.11. The line fit does not pass through zero because of the decrease in oscillation amplitude as the mitosis progresses toward anaphase (see supplemental information), presumably due to increasing spindle microtubule density (e.g. [17]). C) From the line fit the PEF distribution is estimated to be proportional to _x_0.57 (black line), where x is the distance from the spindle equator. The straight dotted segments indicate regions outside the range of data where reversals occur. This differs substantially from the inverse square distribution (gray line), where the PEF is proportional to 1/(Λ-x)2, where Λ is half of the spindle length. D) Interpolar microtubule density at metaphase, and early anaphase of PtK1 cells, calculated from the data of Mastronarde et al. [17]. The polarity adjusted (net) density (squares) – the amount that the number of microtubules from one pole exceeds the other – is calculated by subtracting the right pole values (circles) from the left pole values (diamonds), and is similar to our estimation of PEF distribution (C, black line) for newt lung cells: steepest near the equator, and flattening toward the poles. The microtubule density then drops off very near the poles.
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