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FIGURE 1: Kif4A localization at aster interaction zones in zygotes. Xenopus eggs were fixed between first mitosis and first cleavage (70â100 min postfertilization), triple stained for Kif4A, AurkB (a subunit of the CPC), and tubulin, and imaged by laser scanning confocal microscopy in a clearing solvent. (A) Anaphaseâtelophase. Kif4A is enriched at anti-parallel bundles between asters. At this stage, it is still present on mitotic chromosomes (Chr). (B) Shortly before the asters reach the cortex. Kif4A is enriched at the interaction zone between asters, where it colocalizes with the CPC. (C) Similar stage to B, different embryo. Higher magnification of the interaction zone between asters illustrating anti-parallel microtubule bundles with Kif4A and CPC enriched at the midline.
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FIGURE 2: Localization of Prc1E and Kif4A in interphase egg extracts. (a) Spinning disk confocal time-lapse sequence of asters nucleated from AurkA beads in interphase extract. Probes: microtubules (Alexa 647-tubulin), mCherry-Prc1E, and Kif4A-GFP (Supplemental Video 1). (b) Widefield sequence using GFP-DasraA subunit to visualize the CPC (Supplemental Video 2). (aâ², bâ²) Kymograph analysis along a 30-μm-wide line (cyan box in a and b). Note that the CPC is more focused in interaction zones than Prc1E or Kif4A.
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FIGURE 3: Visualization of microtubule dynamics between asters. Panels aâh show analysis of EB1-GFP trajectories imaged by spinning disk confocal microscopy at interaction zones between two asters. (a) EB1 comet trajectories colored by mean direction. (b) EB1 comet trajectories colored by instantaneous velocity. (c) EB1 comet initiations colored by velocity for 5 s after initiation. (d) EB1 comet termination colored by velocity 5 s before termination. (e) Spatial distributions EB1 comet trajectories classified by direction (gray dots: fraction of EB1 comets moving from left to right; blue curve: sigmoidal fit to grey dots; red curve: sigmoidal fit to fraction of EB1 comets moving in opposite direction). (f) Mean instantaneous growth rates (mean ± SEM). (g) EB1 comet density. (h) Fractions of EB1 comets that are growth initiation/terminations (mean ± SD), n = 12 neighboring ROIs (see Materials and Methods for data analysis). (i) Tubulin-A647 image sequence of a field of asters growing and interacting; 20à widefield. Interaction zones between asters were established by 24 min in this example. Note that the microtubule density between asters remained approximately constant for a further 40 min, showing that there is no increase in the density of anti-parallel bundles over time. Images were linearly rescaled to 8 bits to correct for mild photobleaching over the long image sequence.
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FIGURE 4: Immunodepletion of Prc1E and Kif4A. (a) Low-mag widefield tubulin images of asters depleted as shown or treated with 100 μM ZM-447439 (AurkB inhibitor). Prc1E and Kif4A depletion had no discernalbe effect on nucleation of microtubule density. AurkB inhibition caused descrease of MT density over time. (bâe) High-mag TIRF images showing microtubules (Alexa 647-tubulin), mCherry-Prc1E, and Kif4A-GFP under the depletion/inhibition above. Zoom-ups of boxed areas are shown on the right. Note that Kif4A localization to bundles depended on Prc1E. Prc1E localization did not depend on Kif4A, but bundles were more spread out in its absence. Both proteins were recruited to bundles when AurkB was inhibited, but the bundles are much more spread out.
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FIGURE 5: Prc1E and Kif4A are required for the block to interpenetration between asters. (aâf) Analyses of microtubule growth directions by EB1 tracking of spinning disk confocal images sequences as per Figure 2a. Depletions and add-backs as noted. (g) The D60 parameter, a metric for the degree of interpenetration, was defined as the difference between the interpolated distances where the red and blue curves crossed 60% (see panel a). Plot of mean D60 values (± SD) measured for each treatment (n ⥠3 interaction zones each). The last two bars (control and AurkB inhibition with 100 μM ZM-447439) were replotted from Nguyen et al. (2014) to provide a comparison. Asterisks indicate significant of different from control IgG depletion based on unpaired t test analyses, *p < 0.01, **p < 0.001.
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FIGURE 6: Prc1E and Kif4A enforce radial order in isolated asters. (aâe) Plus-end growth trajectories within isolated asters colored by mean direction. Imaging and EB1 tracking methods similar to Figure 2a. Asterisks indicate position of the nucleating center, Immunodepletion/add back conditions as labeled. (aâ²âeâ²) Radial order heat maps showing the local radial order quantified by the R parameter; each square cell measures 5 à 5 μm2 (see Materials and Methods). Redder colors represent higher radial order. White pixels contained too few comet tracks to measure radial order. (f) Radial order parameter R as a function of distance from the aster center for the five asters shown above. Error bars are standard deviations (see Materials and Methods for analysis). Data are truncated at <20 μm, where microtubule growth components in the z-axis complicate analysis.
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FIGURE 7: Examples of anti-parallel overlaps in growing asters being eliminated. Examples of anti-parallel microtubule overlap formation and elimination at the edge of growing asters imaged by spinning disk confocal microscopy (Supplemental Videos 7 and 8). Proteins visualized: (a) Alexa 647-tubulin and Kif4A-GFP, Alexa 647-tubulin; (b) Kif4A-GFP, and mCherry-Prc1E. Arrowheads indicate the following: likely plus ends (red), likely minus ends (cyan), microtubule growing out radially from aster (empty), and microtubule growing in opposite direction (full). (aâ²,bâ²) Kymographs of examples above along the radially growing microtubule. Events indicated: likely catastrophe (c), likely stabilization (st), and likely rescue (r).
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FIGURE 8: Model for function of Prc1E and Kif4A within and between asters. Left box, general function of Prc1E and Kif4A in anti-parallel bundles. Middle box, block to interpenetration at the interaction zone between asters. Right box, enforcement of radial order within single asters by pruning of anti-parallel overlaps. Left box, general function of Prc1E and Kif4A in anti-parallel bundles. Middle box, Block to interpenetration at the interaction zone between asters. Right box, enforcement of radial order within single asters by pruning of anti-parallel overlaps.
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FIGURE 1:. Kif4A localization at aster interaction zones in zygotes. Xenopus eggs were fixed between first mitosis and first cleavage (70–100 min postfertilization), triple stained for Kif4A, AurkB (a subunit of the CPC), and tubulin, and imaged by laser scanning confocal microscopy in a clearing solvent. (A) Anaphase–telophase. Kif4A is enriched at anti-parallel bundles between asters. At this stage, it is still present on mitotic chromosomes (Chr). (B) Shortly before the asters reach the cortex. Kif4A is enriched at the interaction zone between asters, where it colocalizes with the CPC. (C) Similar stage to B, different embryo. Higher magnification of the interaction zone between asters illustrating anti-parallel microtubule bundles with Kif4A and CPC enriched at the midline.
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FIGURE 2:. Localization of Prc1E and Kif4A in interphase egg extracts. (a) Spinning disk confocal time-lapse sequence of asters nucleated from AurkA beads in interphase extract. Probes: microtubules (Alexa 647-tubulin), mCherry-Prc1E, and Kif4A-GFP (Supplemental Video 1). (b) Widefield sequence using GFP-DasraA subunit to visualize the CPC (Supplemental Video 2). (a′, b′) Kymograph analysis along a 30-μm-wide line (cyan box in a and b). Note that the CPC is more focused in interaction zones than Prc1E or Kif4A.
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FIGURE 3:. Visualization of microtubule dynamics between asters. Panels a–h show analysis of EB1-GFP trajectories imaged by spinning disk confocal microscopy at interaction zones between two asters. (a) EB1 comet trajectories colored by mean direction. (b) EB1 comet trajectories colored by instantaneous velocity. (c) EB1 comet initiations colored by velocity for 5 s after initiation. (d) EB1 comet termination colored by velocity 5 s before termination. (e) Spatial distributions EB1 comet trajectories classified by direction (gray dots: fraction of EB1 comets moving from left to right; blue curve: sigmoidal fit to grey dots; red curve: sigmoidal fit to fraction of EB1 comets moving in opposite direction). (f) Mean instantaneous growth rates (mean ± SEM). (g) EB1 comet density. (h) Fractions of EB1 comets that are growth initiation/terminations (mean ± SD), n = 12 neighboring ROIs (see Materials and Methods for data analysis). (i) Tubulin-A647 image sequence of a field of asters growing and interacting; 20× widefield. Interaction zones between asters were established by 24 min in this example. Note that the microtubule density between asters remained approximately constant for a further 40 min, showing that there is no increase in the density of anti-parallel bundles over time. Images were linearly rescaled to 8 bits to correct for mild photobleaching over the long image sequence.
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FIGURE 4:. Immunodepletion of Prc1E and Kif4A. (a) Low-mag widefield tubulin images of asters depleted as shown or treated with 100 μM ZM-447439 (AurkB inhibitor). Prc1E and Kif4A depletion had no discernalbe effect on nucleation of microtubule density. AurkB inhibition caused descrease of MT density over time. (b–e) High-mag TIRF images showing microtubules (Alexa 647-tubulin), mCherry-Prc1E, and Kif4A-GFP under the depletion/inhibition above. Zoom-ups of boxed areas are shown on the right. Note that Kif4A localization to bundles depended on Prc1E. Prc1E localization did not depend on Kif4A, but bundles were more spread out in its absence. Both proteins were recruited to bundles when AurkB was inhibited, but the bundles are much more spread out.
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FIGURE 5:. Prc1E and Kif4A are required for the block to interpenetration between asters. (a–f) Analyses of microtubule growth directions by EB1 tracking of spinning disk confocal images sequences as per Figure 2a. Depletions and add-backs as noted. (g) The D60 parameter, a metric for the degree of interpenetration, was defined as the difference between the interpolated distances where the red and blue curves crossed 60% (see panel a). Plot of mean D60 values (± SD) measured for each treatment (n ≥ 3 interaction zones each). The last two bars (control and AurkB inhibition with 100 μM ZM-447439) were replotted from Nguyen et al. (2014) to provide a comparison. Asterisks indicate significant of different from control IgG depletion based on unpaired t test analyses, *p < 0.01, **p < 0.001.
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FIGURE 6:. Prc1E and Kif4A enforce radial order in isolated asters. (a–e) Plus-end growth trajectories within isolated asters colored by mean direction. Imaging and EB1 tracking methods similar to Figure 2a. Asterisks indicate position of the nucleating center, Immunodepletion/add back conditions as labeled. (a′–e′) Radial order heat maps showing the local radial order quantified by the R parameter; each square cell measures 5 × 5 μm2 (see Materials and Methods). Redder colors represent higher radial order. White pixels contained too few comet tracks to measure radial order. (f) Radial order parameter R as a function of distance from the aster center for the five asters shown above. Error bars are standard deviations (see Materials and Methods for analysis). Data are truncated at <20 μm, where microtubule growth components in the z-axis complicate analysis.
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