Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation - PubMed (original) (raw)

Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation

Daniel Hayward et al. Dev Cell. 2014.

Abstract

The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. We have tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. Our work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules.

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Figures

Graphical abstract

Figure 1

Cold Treatment of Drosophila Embryos Reveals MT Nucleation from Chromatin during Mitosis (A and B) Stills from movies of spindle formation in embryos expressing α-Tubulin-GFP (green) and Histone-RFP (red) to visualize MTs and chromatin, respectively (A), or the MT growing plus-end marker EB1-GFP (B). Mitotic spindles unambiguously form in an “outwards-in” manner. (C and D) Composite kymographs of Tubulin-GFP (C) and EB1-GFP (D) with heat-map representations of MT intensity. (E) Schematic diagram of cold-treatment protocol (see Results). (F and G) Stills from movies of spindle reformation in syncytial embryos expressing Tubulin-GFP; Histone-RFP (F) or EB1-GFP (G) following cold treatment. MT regrowth is apparent both at centrosomes and around chromatin. (H and I) Composite kymographs of Tubulin-GFP (H) and EB1-GFP (I) in embryos following cold-treatment recovery, with heat-map representations of MT intensity. (J) Graph showing the initial random directionality of EB1 comets emanating from chromatin on cold-treatment recovery. Scale bars, 5 μm. See also Movies S1 and S2.

Figure 2

D-HURP and D-TPX2 Are Mitotic MAPs that Dynamically Associate with MTs during Drosophila Syncytial Mitoses (A) Schematic representation comparing D-TPX2/Mei-38 and D-HURP/Mars to human TPX2 and HURP. GKAP, guanylate kinase-associated protein; NLS, nuclear localization signal; NES, nuclear export signal. (B and C) Stills from movies of GFP-D-HURP (B) and GFP-TPX2 (C) during embryonic divisions. GFP-D-HURP is nuclear in interphase before accumulating on specifically on spindle, but not astral, MTs following NEB; GFP-TPX2 is nuclear in interphase and localizes weakly to the area of the spindle and to the centrosomes (arrow) during mitosis. Scale bars, 5 μm. See also Movie S3.

Figure 3

MT Nucleation from Chromatin Is Mediated by D-HURP (A and B) Stills from movies of spindle formation in d-tpx2 (mei-38 1 ) (A) and d-hurp (mars p ) (B) embryos expressing EB1-GFP. Note the presence of abnormal spindles. (C) Bar chart of control (WT), d-tpx2, and d-hurp spindle length across mitotic cycles 10, 11, and 12. Error bars indicate SEM. (D and E) Composite kymographs of MT nucleation (EB1-GFP) during mitosis in d-tpx2 (D) and d-hurp (E) cycling embryos. (F and G) Stills from movies of spindle reformation in syncytial d-tpx2 (F) and d-hurp (G) mutant embryos expressing EB1-GFP. (H and I) Composite kymographs of MT nucleation (EB1-GFP) during mitosis in d-tpx2 (H) and d-hurp (I) embryos following cold treatment, demonstrating the requirement of D-HURP in chromatin-mediated MT generation. (J) Stills from movies of GFP-D-HURP localization following cold treatment; the protein is now initially present on mitotic chromatin, gradually relocalizing to spindle MTs as they form. Scale bars, 5 μm. See also Movies S4 and S5.

Figure 4

Augmin Is Required for Maintenance of Mitotic Spindle Integrity (A and B) Stills from movies of spindle formation in embryos expressing Tubulin-GFP; Histone-RFP (A) or EB1-GFP (B) injected with interfering antibodies generated against the Augmin subunit, Dgt6. Astral MT and spindle formation are delayed, progressing to weak elongated spindles which arrest. (C) Stills from movies of spindle formation in msd1 ex51 embryos expressing Tubulin-GFP. (D) Stills from movies of Msd1-GFP-expressing embryos. Msd1-GFP localizes to MTs in interphase (i) and metaphase (ii). Following injection of anti-Dgt6 antibodies, Msd1-GFP dissipates from all MTs close to the site of injection during interphase (iii) and mitosis (iv). Weak MT localization remains in areas distant (∼50 μm) from the site of injection (v). (E and F) Stills from movies of spindle formation in embryos expressing Rod-GFP (E). Rod localizes to assembling kinetochores in prophase and streams poleward on MT-kinetochore attachment, gradually decreasing with time (30”–120”). In (F), upon injection of anti-Dgt6 antibodies, Rod-GFP streaming is delayed but occurs, signifying the presence of K-fibers. Localization of Rod-GFP to kinetochores and kMTs persists throughout the observation period. Scale bars, 5 μm. See also Movies S6 and S7.

Figure 5

Augmin Is Essential for MT Generation around Chromatin (A and B) Stills from movies of spindle reformation in embryos expressing Tubulin-GFP Histone-RFP (A) or EB1-GFP (B) injected with anti-Dgt6 antibodies. (C) Stills from movies of D-HURP-GFP localization following cold treatment in an anti-Dgt6-injected embryo; localization of D-HURP-GFP is not dependent on Augmin. (D) Composite kymographs of MT nucleation (EB1-GFP) during mitosis in control (WT) and anti-Dgt6-injected embryos following cold treatment; chromatin-dependent MT generation is completely absent upon Augmin disruption. See also Movie S8. Scale bars, 5 μm.

Figure 6

Centrosome/PCM Disruption Leads to Spindle Formation via Augmin-Dependent aMTOCs (A) Stills from movies of spindle formation in an EB1-GFP-expressing cnn mutant embryo. (B and C) Stills from movies of spindle formation in EB1-GFP expressing (B) and Tubulin-GFP; Histone-RFP embryos (C) injected with a high concentration of anti-DSpd-antibody. In all cases, spindles form predominantly from cytoplasmic, acentriolar MTOCs. (D) Stills from movies of spindle formation in an EB1-GFP-expressing cnn embryo injected with anti-Dgt6 antibodies. MTs are present in interphase, but mitotic aMTOCs do not form. Scale bars, 5 μm. See also Movie S9.

Figure 7

MT-Generating Pathways Work Synergistically to Promote Robust Mitotic Spindle Formation (A) Stills from movies of spindle formation in an EB1-GFP-expressing embryo injected with low levels of anti-DSpd-2, to dampen astral input. (B) Stills from movies of spindle reformation following cold treatment in an EB1-GFP-expressing embryo injected with low levels of anti-DSpd-2. (C) Line graph showing the fluorescence intensity over time in the region of mitotic chromatin following cold treatment in control (WT) embryos and embryos injected with anti-DSpd-2 antibodies. Reducing astral input results in increased generation of MTs around chromatin. Error bars represent SEM. n ≥ 20 spindles from at least three embryos. (D) EB1-GFP comets in control (WT) embryos, control embryos immediately following cold treatment, and embryos injected with anti-Dgt6 antibodies immediately following cold treatment. (E) Histogram of mitotic EB1-GFP comet length in embryos under different conditions. Curves to the right represent the histogram trends for comparison of data sets; the table below shows mean comet length and statistical significance between samples of interest. (F) Stills from time-lapse movies of spindle formation in an EB1-GFP-expressing embryo injected with both anti-Dgt6 and low levels of anti-DSpd-2. Mitotic spindles initially form from limited astral input, allowing measurement of EB1-GFP comet length, but intensity reduces and spindles eventually collapse. (G) Histogram of EB1-GFP comet velocity in embryos under different conditions. Curves to the right represent the histogram trends for comparison of data sets; the table below shows mean comet velocities of the samples of interest. Scale bars, 5 μm. See also Movie S10.

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Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation - PubMed (original) (raw)