The <i>Drosophila</i> Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

<div><p>Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of <i>Drosophila</i> embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle <i>in vivo</i>. Both <i>in vivo</i> and <i>in vitro</i> data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.</p></div

Overexpression of GFP-C Mars causes embryonic lethality and impairs the assembly of the mitotic spindle.

<p>(A, B) Subcellular localization of GFP-C-Mars at interphase (A) and metaphase (B). (C–F) Defects caused by overexpression of GFP-C-Mars in embryos. (C) Overview of GFP-C-Mars overexpressing embryo showing abnormal pattern of early mitoses. (D) Giant nuclei. (E) Poorly organized mitotic spindles. (F) Excessive formation of astral microtubules. Numbers to the right of panels (D–F) indicate the frequency of the respective phenotypes. Numbers add up to more than 100% since some spindles show a combination of two or more abnormalities. 200 mitotic spindles from 10 embryos were scored. (G) Spindles from control embryo overexpressing GFP-Mars show normal morphology. Embryos were fixed and stained with tubulin antibody (red), GFP antibody (green) and DAPI (turquoise). Scale bars are 40 µm in panel (C) and 5 µm in panels (A, B, D–G). (H) Still images from live imaging of GFP-C-Mars showing fusion of two nuclei (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060596#pone.0060596.s005" target="_blank">Movie S4</a>). Scale bar is 20 µm.</p

Rescue of <i>mars⁹¹</i> mutant phenotypes by GFP-N-Mars and GFP-C-Mars.

<p>(A) Typical mitotic spindles in wild type and <i>mars⁹¹</i> mutant embryos upon expression of GFP-Mars, GFP-N-Mars or GFP-C-Mars. Embryos were fixed and stained with tubulin antibody (red), GFP antibody (green) and DAPI (turquoise). Scale bar is 5 µm. (B) Quantification of larval hatching of wild type embryos, <i>mars⁹¹</i> mutant embryos and <i>mars⁹¹</i> mutant embryos rescued by the respective transgenes. (C) Illustration of spindle parameters quantified in (D–F). (D–F) Quantification of mitotic spindle parameters from wild type embryos, <i>mars⁹¹</i> mutant embryos and <i>mars⁹¹</i> mutant embryos rescued by GFP-Mars, GFP-N-Mars or GFP-C-Mars. Embryos analyzed in (C–F) were fixed and stained by tubulin antibody (red). Scale bar in (C) is 5 µm. t-test was performed by Prism 5 (GraphPad Software).</p

Overexpression of GFP-N Mars causes embryonic lethality and mitotic spindle defects.

<p>(A–E) Subcellular localization of GFP-N-Mars in transgenic fly embryos. Embryos were fixed and stained by tubulin antibody (red), GFP antibody (green) and DAPI (turquoise). Scale bar is 5 µm. (F–K) Overexpression of GFP-N-Mars causes embryonic lethality and defects in mitotic spindle morphology. (F) Overview of GFP-N-Mars overexpressing embryo showing abnormal pattern of early mitoses. (G) Acentrosomal spindle with pointed spindle poles. (H) Chromosome lagging at spindle pole. (I) Attached multipolar spindles. (J) Tripolar spindle. (K) Chromosome segregation failure with chromosomal bridges. Numbers to the right of panels (G–K) indicate the frequency of the respective phenotypes. Numbers add up to more than 100% since some spindles show a combination of two or more abnormalities. 300 mitotic spindles from 15 embryos were scored. (L) Spindles from control embryo overexpressing GFP-Mars show normal morphology. Embryos were fixed and stained by antibodies described in (A–E). Scale bars are 40 µm for panel (F) and 5 µm for panels (G–L). (M) Still images from live imaging of GFP-N-Mars showing fusion of two mitotic spindles (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060596#pone.0060596.s003" target="_blank">Movie S2</a>). Scale bar is 10 µm.</p

Overexpression of GFP-N-Mars causes reduced localization of endogenous Mars in the nucleus and on the mitotic spindle.

<p>(A) Wild type embryos and embryos overexpressing GFP-N-Mars were fixed and stained by GFP antibody, Mars antibody raised against the C-terminus (red) and DAPI (turquoise). (B) Wild type embryos and embryos overexpressing GFP-C-Mars were fixed and stained by GFP antibody, Mars antibody raised against the N-terminus (red) and DAPI (turquoise). GFP channels not shown. Scale bar is 5 µm.</p

MBP-N Mars binds and stabilizes microtubules in vitro.

<p>(A) Scheme of recombinant proteins used in this study. (B) MBP-N-Mars binds to MTs <i>in vitro</i>. 1 µg of purified MBP-N-Mars was incubated with taxol-stabilized MTs at different concentrations before ultracentrifugation through a glycerol cushion. (C) Plot of bound fraction of MBP-N-Mars against MT concentration. The calculated Kd value is given above the curve. (D) MBP-N-Mars stimulates the assembly of MTs. The indicated amounts of MBP protein, MBP-N-Mars and MBP-C-Mars were incubated with tubulin solution. Taxol was used as a positive control. (E) Quantification of the tubulin in the pellet after sedimentation of the samples according to the same procedure as in (D). (F) MT dilution assay in the absence or presence of MBP-N-Mars. Top: Coomassie Brilliant Blue staining of the pellet samples separated by SDS-PAGE. Bottom: Quantification of the Coomassie staining results by LI-COR ODYSSEY SA system. (G, H) Microtubule bundling assay. The same amount of MBP (G) and MBP-N-Mars (H) was incubated with tubulin solution in the presence of a low concentration of taxol. Tubulin structures were fixed by formaldehyde, stained with tubulin-FITC antibody and imaged by fluorescence microscopy. Scale bar is 5 µm.</p

Mars stabilizes the mitotic spindle <i>in vivo</i>.

<p>(A, B) Wild type (A) and <i>mars⁹¹</i> mutant embryos (B) were mixed and treated with 0.15 µM demecolcine to destabilize mitotic spindles. Embryos were fixed and stained by tubulin antibody (green), Mars antibody (red) and DAPI (turquoise). (C) Quantification of spindle phenotypes for the genotypes shown in (A, B). (D, E) Wild type embryos (D) and embryos overexpressing GFP-Mars driven by daughterless>Gal4 (E) were mixed and treated like in (A, B). Embryos were fixed and stained by tubulin antibody (red), GFP antibody (green) and DAPI (turquoise). (F) Quantification of spindle phenotypes for the genotypes shown in (D, E). For the quantifications in (C) and (F), 200 spindles from 10 embryos were scored for each genotype. Scale bar is 5 µm.</p

Comprehensive Identification of SUMO2/3 Targets and Their Dynamics during Mitosis

<div><p>During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.</p></div

Identification of mitotic SUMOylation targets using quantitative proteomics.

<p>A) Schematic outline of the protocol for growth and synchronization of cells in roller bottles. B) HeLa FRT TRex SUMO2 and HeLa FRT TRex SUMO2ΔGG cells were arrested in S phase by thymidine or synchronized in mitosis with thymidine and taxol, followed by provoking mitotic exit by the addition of ZM447439 for the indicated times. Cell lysates were analyzed by western blotting using antibodies against SUMO2/3, Cyclin B1 and Aurora A as markers for mitotic progression, and Vinculin. (^★) indicates un-conjugated SUMO2 fusion proteins, (^⧫) indicates endogenous SUMO2/3. C) Schematic outline of the quantitative proteomics strategy. Cells are grown in large-scale and synchronized in roller bottles as shown in A. SUMO2ΔGG cells were isotopically labelled with light “L”/R0K0 amino acids and arrested in prometaphase (grey), cells expressing SUMO2 were labelled with medium “M”/R6K4 amino acids and represented the mitotic exit stage one hour after ZM447439 addition (green), and cells expressing SUMO2 were labelled with heavy “H”/R10K8 amino acids and arrested in prometaphase (red). Equal amounts of cell lysate from the labelled populations were mixed and SUMO2 conjugated proteins were purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100692#pone-0100692-g001" target="_blank">Figure 1</a>. The SUMO2 conjugate enriched sample was separated by SDS-PAGE, digested with trypsin and analyzed by mass spectrometry (MS). Lysates from the different experimental conditions were analyzed by western blotting antibodies against FLAG, Cyclin B1, Aurora A and Vinculin to confirm conjugation state and mitotic stage. (^★) indicates un-conjugated SUMO2 fusion proteins. D) Scatter plot of the entire data set from screen I and screen II. The plot is showing the value of log₂(M/L) and log₂(H/L) SILAC ratios that are used to identify SUMOylation targets. The red dashed line at log₂(M/L) = 1 and log₂(H/L) = 1 represents the cut-off ratio of ≥2 for the respective SILAC pairs. Each point represents a single identified protein, proteins identified in screen I are illustrated in blue and proteins identified in screen II are in purple. Identified proteins that are classified as SUMOylation hits are above the dashed cut-off line and are darker colored, whereas identified proteins classified as background is below the cut-off and lighter. E) Diagram showing the number of identified targets (hits) of SUMO2/3 modification in screen I (blue), screen II (purple) and the overlap (dark blue) of hits that are identified in both. F) Scatter plot with the correlation between the screen I and screen II log₂(H/M) ratios of identified SUMO2/3 target proteins. Each point represents a SUMO2/3 target. The pearson correlation, R, is shown.</p

RhoGDIα is specifically modified by SUMO in prometaphase.

<p>A) Identification of RhoGDIα as a SUMO2/3 target. SILAC ratios and MS data from screen I and II. B) Schematic representation of RhoGDIα with the amino acid sequence around lysine residues K138 and K141. The consensus modification motifs are indicated and the lysines are highlighted in red. C) Purification of Venus-RhoGDIα or Venus-RhoGDIα K138R/K141R from taxol arrested cells using GFP-trap beads. SUMO conjugation pathway components, Ubc9 and PIAS1-4, were depleted by RNAi, expression of Venus-RhoGDIα fusion proteins in the generated stable HeLa FRT Trex cell lines was induced with doxycycline and ZM447439 was added for one hour as indicated. The SUMOylation states of purified RhoGDIα and RhoGDIα K138R/K141R from the different experimental conditions were analyzed by western blotting with antibodies specific for SUMO2/3 and RhoGDIα. D) Parental HeLa FRT and HeLa FRT TRex SUMO2 cells were arrested in S phase by thymidine or synchronized in mitosis with thymidine and taxol, followed by mitotic checkpoint override and progression by the addition of ZM447439 for the indicated times. Cell lysates were analyzed by western blotting using antibodies against Cyclin B1, RhoGDIα, RhoA and α-tubulin, and shows that RhoGDIα itself is stable in mitosis. E) Using 3 different siRNA oligos for each target, normal HeLa cells were depleted for RhoGDIα or RhoGDIβ using RNAi. Lysates were analyzed for depletion efficiency and RhoA stabilization by western blot with RhoGDIα and RhoA specific antibodies. F) Stable HeLa FRT TRex FLAG-RhoGDIα or FLAG-RhoGDIα K138R/K141R cells were depleted for endogenous RhoGDIα and arrested in mitosis with taxol. Rescue with exogenous RhoGDIα fusion proteins were titrated in with increasing concentrations of doxycycline (ng/ml) as indicated. Lysates were analyzed for depletion efficiency, expression level of exogenous RhoGDIα and RhoA stabilization by western blot with RhoGDIα and RhoA specific antibodies. G) Representative still images from time-lapse movies of stable HeLa cell lines expressing Venus-RhoGDIα and Venus-RhoGDIα K138R/K141R as they progress through an unperturbed mitosis. The DIC and Venus channels are shown and the time of nuclear envelope breakdown (NEBD), metaphase and anaphase is indicated. H) As F) but using Venus-RhoGDIα.</p