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. 2011 Oct;22(20):3779-90.
doi: 10.1091/mbc.E11-06-0569. Epub 2011 Aug 24.

Role of Survivin in cytokinesis revealed by a separation-of-function allele

Edith Szafer-Glusman  1 , Margaret T Fuller, Maria Grazia Giansanti
Affiliations

Role of Survivin in cytokinesis revealed by a separation-of-function allele

Edith Szafer-Glusman et al. Mol Biol Cell. 2011 Oct.

Abstract

The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. Here we identify a missense allele of Drosophila Survivin that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this "separation-of-function" allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems.

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Figures

FIGURE 1:
FIGURE 1:
Drosophila Survivin protein localization in dividing cells in a pattern typical of chromosomal passenger proteins. Immunofluorescence images of wild-type (A) larval neuroblasts undergoing mitosis and (B) primary spermatocytes undergoing meiosis, stained for Survivin (red), tubulin (green), and DNA (blue). Left, Survivin channel only, in grayscale. (C) Selected snapshots from Supplemental Movies S1 and S2 showing the dynamic behavior of wild-type Drosophila Survivin-GFP protein in a dividing spermatocyte. Top, GFP; bottom, phase-contrast image. Same cell viewed throughout. Scale bar, 10 μm.
FIGURE 2:
FIGURE 2:
The scpo mutation separates action of survivin in cytokinesis from earlier functions. (A–C) Onion-stage spermatids from (A) scpoz2775 mutant males raised at 18°C showing normal spermatids, each containing a mitochondrial derivative (arrow) associated with a nucleus of similar size (arrowhead); (B) scpoz2775 males raised at 25°C; and (C) scpoz2775/Df(3L)Exel5780 males raised at 25°C. In B and C most spermatids contained a large mitochondrial derivative (arrow) associated with two or four nuclei of similar size. (D) Mitotic parameters in larval brain preparations from Drosophila Oregon-R and scpoz2775/Df(3R)Exel5780 mutants stained for tubulin and DNA. OF, optic field: the circular area defined by a phase-contrast Neofluar 100X Zeiss objective, using 10× oculars and the Optovar set at 1.25. MF, Number of mitotic cells scored. MI, mitotic index: average number of mitotic figures per optic field. (E) Alignment of Drosophila Deterin/Survivin with human Survivin. Arrowheads indicate amino acids mutated in the scapolo male sterile allele: open arrowhead, permissive substitution of glutamine for glutamic acid in position 83, also present in the background chromosome; solid arrowhead, substitution of Ser for wild-type proline at position 86 that underlies the scpo phenotype. Asterisks, acidic patch near the Pro substitution; #, residues involved in Zn coordination. (F) Ribbon representation of dSurvivin (amino acids 20–106 of the 152 amino acid–long protein) modeled with Swiss Model and visualized with PyMOL. Blue sticks, Pro-86; magenta ribbon, acidic patch; yellow ribbon, amino acids in the Zn coordination loop: Cys-70 and -73 and His-90 and -93 (Jeyaprakash et al., 2007). (G) Immunofluorescence images of scpoz2775/Df(3R)Exel5780 larval neuroblasts stained for Survivin (red), tubulin (green), and DNA (blue). Scale bar, 10 μm.
FIGURE 3:
FIGURE 3:
Aurora B complex components reflect the localization of survivin in scpo mutant cells. (A–C) Wild-type and scpoz2775/Df(3R)Exel5780 (A, B) spermatocytes or (C) neuroblasts stained for tubulin (green), DNA (light blue), and either (A) INCENP (red) or (B, C) Aurora B (red). Aurora B and INCENP were enriched at metaphase centromeres of both wild-type and scpoz2775/Df(3R)Exel5780 spermatocytes and neuroblasts but failed to accumulate at the spindle midzone of mutant ana/telophases. (D) Wild-type and scpoz2775/Df(3R)Exel5780 mutant testes stained for tubulin (green), phosphohistone H3 Ser-10 (red), and DNA (blue). Scale bar, 10 μm.
FIGURE 4:
FIGURE 4:
Pole-to-equator microtubules initially contact the cortex but fail to form a stable central spindle in scpo mutant primary spermatocytes undergoing meiosis. Time-lapse imaging of central spindle formation in wild-type and scpoz2775/Df(3R)Exel5780 mutant primary spermatocytes expressing EGFP–β-tubulin imaged every 60 s. (A) Wild type (selected frames from Supplemental Movie S3): a robust central spindle formed in anaphase (frame 4.59) and constricted during telophase (frames 8.59, 15.00, and 25.00). (B) scpoz2775/Df(3R)Exel5780 mutant (selected frames from Supplemental Movie S4): microtubules bundled only transiently (see dots in frame 11), and a robust central spindle failed to form. Scale bar, 10 μm.
FIGURE 5:
FIGURE 5:
Loss of Survivin function during anaphase affects localization of the centralspindlin component Pav but not the microtubule-bundling protein Feo in scpo mutant spermatocytes. Wild -type and scpoz2775/Df(3R)Exel5780 mutant spermatocytes stained for tubulin (green), DNA (blue), and either (A) Feo (red) or (B) Pav (red). Scale bar, 10 μm.
FIGURE 6:
FIGURE 6:
Loss of Survivin function disrupts localization of the centralspindlin component Pav but not accumulation of the microtubule-bundling protein Feo in scpoz2775/Df(3R)Exel5780 neuroblasts. Localization of Fascetto (Feo) and Pavarotti (Pav) in wild-type and scpoz2775/Df(3R)Exel5780 mutant neuroblasts stained for tubulin (green), DNA (blue), and either (A) Feo (red) or (B) Pav (red). Arrows in A point to telophase nuclei. Scale bar, 10 μm.
FIGURE 7:
FIGURE 7:
Recruitment of Polo to the midzone is impaired in spermatocytes but not in neuroblasts from scpoz2775/Df(3R)Exel5780 mutants. Wild-type and scpoz2775/Df(3R)Exel5780 (A) spermatocytes or (B) neuroblasts stained for DNA (blue) and Polo (green). Polo is present at the midzone of anaphase wild-type and mutant neuroblasts but fails to form a tight band in telophase of scpo mutant neuroblast (arrows in B). Scale bar, 10 μm.
FIGURE 8:
FIGURE 8:
Rho1 and Sqh-GFP localization to the midzone is disrupted in spermatocytes from scpoz2775/Df(3R)Exel5780 mutants. Wild-type and scpoz2775/Df(3R)Exel5780 spermatocytes stained for DNA (blue), centrosomin (red), and Rho1 (green) (A) and for DNA (blue), microtubules (red), and Sqh-GFP (green) (B). Arrows in B point to the cells' equator. Scale bar, 10 μm.
FIGURE 9:
FIGURE 9:
Loss of Survivin function does not impair the recruitment of Rho1 and Myosin II to the cortex of scpoz2775/Df(3R)5780 mutant neuroblasts but affects the formation of a compact, fully constricted ring. Localization of Rho1 (A) and myosin II (B) in wild-type and scpoz2775/Df(3R)Exel5780 mutant neuroblasts stained for Rho1 (green in A), centrosomin (red), tubulin (green in B), DNA (blue), and myosin II (red in B). Scale bar, 10 μm.
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