Mol Biol Cell
January 1, 2021;
Spatial Variation of Microtubule Depolymerization in Large Asters.
plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg
extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule
assembly is a MAP that inhibits depolymerization, and that egg
asters are tuned to low microtubule
density. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
Mol Biol Cell
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FIGURE 1:. EB1 tracking-based measurements of microtubule plus-end polymerization rate and catastrophe rate during aster growth. (A) Time-lapse movies of EB1-GFP comets are analyzed by particle tracking. Region in the white box is magnified on the right. Purple spots show the location of individual EB1 comets in this frame, while the trailing lines show the trajectory of the corresponding plus end for the preceding 10 s. The velocity of EB1 comets report microtubule polymerization rate. (B) Polymerization rate as a function radial distance from the center of the aster. N = 9348 EB1 tracks are represented as blue dots. Filled black circles and the shaded region indicate the mean and the SD of the polymerization rate for each spatial bin. The estimated mean difference in the periphery (distance > 300 µm) vs. interior (50 µm < distance < 280 µm) for t = 1.5 min was 3.7 µm/min (95% CI = [2.4, 4.9]). (C) EB1 comet density as a function radial distance. (D) Polymerization rate vs. EB1 comet density. Filled black circles and error bars indicate the mean and the SD of polymerization rate for each EB1 density bin. The different colors correspond to the same time points as in the previous panel. The estimated mean difference in the dense (0.066 1/µm2) vs. sparse (0.022 1/µm2) was 3.8 µm/min (95% CI = [2.6, 5.0]). (E) Catastrophe rate is calculated from the duration of EB1 tracks and plotted over radial distance. Filled black circles and the shaded region indicate the mean and the corresponding 95% confidence interval for the catastrophe rate for each spatial bin. Original data used for this analysis is Supplemental Movie 1. Scale bars, 100 µm and 10 µm.
FIGURE 2:. Intensity difference–based measurement of polymerization and depolymerization rates in the interior and the periphery of microtubule asters. (A) TIRF microscopy movies of fluorescent tubulin were subjected to intensity difference analysis, which revealed a spatial map of polymerization and depolymerization for the interior (<100 µm away from the MTOC) and peripheral (>200 µm from the MTOC with few microtubules) region of microtubule asters. These images correspond to Supplemental Movies 2 and 3. (B) The distributions of polymerization and depolymerization rates measured from N = 6 interior movies and N = 6 peripheral movies. The polymerization rate (median 32.0 vs. 32.0 µm/min) showed no statistical difference in the interior vs. periphery (p-value = 0.819, t test). The depolymerization rate (median 37.6 vs. 26.7 µm/min) was higher in the interior (p-value = 4.44e-16, two sample Kolmogorov-Smirnov tests) with estimated difference of mean and median depolymerization rates of 7.1 µm/min (95% CI = [5.9, 8.3]) and 11.0 µm/min (95% CI = [8.4, 12.4]). Vertical lines indicate the mean for each distribution. See Supplemental Figure 1 for analysis of individual movies. Scale bar, 20 µm.
FIGURE 3:. Measurement of depolymerization rates following laser ablation. (A) An interphase aster was subjected to a circular laser cut with radius 56 µm (left), which induced a wave of microtubule depolymerization (Supplemental Movie 4). The resulting movie was used to construct a differential intensity movie (right and Supplemental Movie 5). Dotted wedge region indicates the region excluded from image analysis. (B) Radial sum of differential intensities at different time points (from dark to light blue) of the same laser cut experiment. The area under each curve equals the mass of microtubules depolymerized per time interval of 5 s. Vertical dotted line indicates the location of the laser cut. (C) For each cut, the peak position of the differential intensity travels at constant speed. (D) Depolymerization rates measured from N = 16 laser cuts positioned at 15–56 µm from the center of the aster. (E) The area under differential intensity curves decreases as the depolymerization wave travels inward. This example corresponds to the cut depicted in panels A–C. Vertical dotted line indicates the location of the laser cut.
FIGURE 4:. Fluorescent intensity profiles of tubulin and EB1 indicate spatial variation of these species within growing interphase asters. (A) Wide-field images of a growing aster visualized with Alexa 647–labeled tubulin and EB1-GFP. For both images, the contrast is adjusted to emphasize the zone of low fluorescence intensity. (B) Corresponding quantification of the fluorescence intensity profiles averaged over the quadrant and normalized to the intensity outside the aster (top) and EB1 comet density profile (bottom). We define the aster as the region that has EB1 density higher than the background level + SD. Scale bar, 100 µm.
FIGURE 5:. Proposed model for the regulation of depolymerization rate by a MAP species as the limiting component. (A) We consider the equilibrium of a single MAP species that associates with the microtubule lattice with dissociation constant . We hypothesize that the degree at which a microtubule is bound by this MAP species slows down the depolymerization rate. This effect is greater in the aster periphery, where the microtubule density is lower. (B) Fraction of free MAP and occupied microtubule lattice as a function of microtubule concentration for a hypothetical MAP present at 0.2 µM.
Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression.