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

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 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