InteractiveFly: GeneBrief

Deterin/Survivin: Biological Overview | References

BIOLOGICAL OVERVIEW

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. This study identified a missense allele of Drosophila Survivin (FlyBase name: Deterin) 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 (Szafer-Glusman, 2011).

The chromosomal passenger complex (CPC), composed of the Ser-Thr kinase Aurora B and three partner proteins, plays several key roles in mitosis and meiosis, including regulation of attachment of kinetochores to microtubules, the spindle checkpoint that delays anaphase onset until all chromosomes are under tension on the spindle, regulation of sister chromatid cohesion, and cytokinesis. To accomplish these different tasks, the Aurora B kinase must be exquisitely localized in space and regulated in time. During mitosis, Aurora B associates with the microtubule-binding protein Inner Centromere Protein (INCENP), Borealin/DASRA/CSC-1, and the small, multifunctional BIR-motif protein Survivin to form the CPC (Szafer-Glusman, 2011).

Dissecting the role of individual CPC components has been hampered by the extraordinary interdependence of the four subunits; depletion of any single CPC protein by RNA interference knockdown in human cells affected the structural unit, localization, and function of the CPC. The structural basis of this interdependence is evident in the crystal structure of the Survivin-Borealin-INCENP core of the CPC complex, in which Borealin and INCENP associate with the C-terminal helical domain of Survivin to form a tight three-helix bundle (Jeyaprakash, 2007; Szafer-Glusman, 2011 and references therein).

Strict localization of Aurora B by the CPC ensures that this kinase, which has multiple substrates, phosphorylates the correct targets at the proper points in cell cycle progression. Concentrated on chromosomes from G2, then at inner centromeres from prometaphase until the metaphase-to-anaphase transition, the CPC is required to regulate chromosome condensation, spindle formation and dynamics, kinetochore maturation, kinetochore-microtubule interaction, correct chromosome alignment, and control of the spindle checkpoint. At anaphase onset the CPC then translocates to the central spindle (CS) midzone and equatorial cortex and is involved in CS formation. At the CS Aurora B phosphorylates the centralspindlin components, Pav/mitotic kinesin-like protein 1 (MKLP1)/Zen-4 and the RacGAP50/MgcRacGAP/Cyc-4. However, the mechanisms that target the CPC to the spindle midzone and equatorial cortex after onset of anaphase and the mechanisms by which the CPC regulates central spindle formation and cytokinesis are not understood. In addition, the requirements for CPC function for critical events in metaphase and at the metaphase-anaphase transition have complicated analysis of how the CPC is localized and functions at later stages for cytokinesis (Szafer-Glusman, 2011).

This study characterized the role of Drosophila Survivin (dSurvivin), previously termed deterin and analyzed in its antiapoptotic activity (Jones, 2000), as a regulator of cell division, identifying a missense mutation (scapolo) in the Drosophila Survivin BIR domain that allows recruitment and function of the CPC in metaphase but disrupts CPC localization and function in anaphase and telophase. The findings reveal that Survivin plays a role in targeting the CPC and centralspindlin to the central spindle and the equatorial cell cortex during anaphase. In spermatocytes, Survivin function is also required to localize Polo and localize the small GTPase RhoA to set up the contractile ring machinery at the onset of cytokinesis (Giansanti, 2004; this study). In larval neuroblasts undergoing mitotic division, however, the scapolo mutant did not block initial accumulation of Rho to a band at the equatorial cortex, although it did cause failure of cytokinesis. The different requirements for Survivin function for equatorial accumulation of Rho in spermatocytes versus neuroblasts may reflect a fundamental difference in the series of steps that lead to formation of the contractile ring in these two cell types (Szafer-Glusman, 2011).

A missense mutation leading to substitution of serine for the wild-type Pro-86 of Drosophila Survivin uncouples the function of Survivin in metaphase from function during anaphase and telophase, indicating a direct requirement for Survivin and the chromosomal passenger complex in orchestrating the profound reorganization of the cortical cytoskeleton at the cell equator at the onset of cytokinesis. This 'separation-of-function' allele allowed analysis of Survivin and CPC function during cytokinesis, which is normally obscured by the better-known roles of the CPC at centromeres during metaphase, when it facilitates alignment of chromosomes to the spindle equator and mediates the spindle checkpoint. The finding that a point mutation in the BIR domain disrupts activity of Survivin during cytokinesis challenges the model that the C-terminal domain of Survivin is sufficient for cytokinesis function (Lens, 2006) and indicates that residues in the BIR domain are important for localization and activity of Survivin at the central spindle (Szafer-Glusman, 2011).

Survivin associates with kinetochores and the central spindle with different dynamics, being highly mobile in prometaphase and metaphase and strongly immobile at the anaphase central spindle (Beardmore, 2004; Delacour-Larose, 2004). This change in dynamics may underlie the largely normal localization and function of scapolo (scpo) (a missense allele of the Drosophila Survivin) at metaphase but the fully penetrant effect on assembly of the F-actin contractile ring and cytokinesis observed in scpo mutants (Giansanti, 2004; Szafer-Glusman, 2011).

Cytokinesis depends on the assembly of an equatorial actomyosin ring regulated by local activation of the small GTPase RhoA at the cortex, in turn catalyzed by the RhoGEF Ect2/Pebble. It has been proposed that association of RhoGEF/Pebble with centralspindlin promotes local RhoA activation at the cortex. In addition, the kinase polo (PLK1) has been implicated in RhoGEF localization and Rho activation, at least in part by phosphorylation of the centralspindlin component MgcRacGAP. The current observations that the Drosophila RhoA homologue, Rho1, failed to accumulate at the equatorial cortex in scpo mutant spermatocytes implicate Survivin and the CPC in the mechanism(s) that localize and activate RhoA at the equatorial cortex in these cells. This requirement may in part act through effects on Polo kinase. Failure to localize Polo to the central spindle in scpo mutant spermatocytes could prevent localization of RhoGEF by the centralspindlin complex and the consequent activation of Rho at the cortex. In this model, failure to localize Polo may contribute to the failure to form an equatorial ring of localized Rho1 and, in consequence, the inability to form a localized ring of myosin regulatory light chain and F-actin in scpo mutant male germ cells undergoing meiotic division. This mechanism may also explain the failure to maintain pole-to-equatorial microtubules observed in scpo mutant spermatocytes. It is likely that Rho-mediated activation of the Formin Dia helps stabilize microtubule arrays at the equatorial cortex of dividing cells, as active Rho and Formin (mDia) have been shown to regulate stabilization of microtubule arrays at the cortex of migrating fibroblasts. Consistent with this model, this study found that microtubules reached the plasma membrane at the equator of scpo dividing spermatocytes, but the bundles are transient and fail to form stable arrays at the cortex (Szafer-Glusman, 2011).

A striking finding of this work is the difference in requirement for Survivin function for localization of the Polo kinase and RhoA in anaphase neuroblasts versus spermatocytes. This difference raises two possibilities: either Survivin is not part of a universal signaling mechanism that directs cytokinesis, or different semiredundant mechanisms can drive cytokinesis, similar to redundancy between astral pulling and sliding of central spindle microtubules for anaphase B, and different cell types rely more strongly on one mechanism or the other. Indeed, consistent with the latter possibility, spermatocytes and neuroblasts display different cytoskeletal architectures during cytokinesis (Giansanti, 2006). In neuroblasts, actomyosin initially accumulates in a broad cortical band, presumably because this is the region of the cell cortex that escapes repression of Rho associated with the plus ends of astral microtubule. This initial wide band gradually narrows into a tight equatorial ring as the cell progresses into telophase (Giansanti, 2006). Thus assembly of the contractile apparatus in neuroblasts proceeds, as proposed by Lewellyn (2011) for Caenorhabditis elegans embryos, in 'two genetically separable steps' in which localization of contractile machinery is initially independent of the central spindle. In support of this model, this study found that Rho1 accumulates in a broad cortical band in scpo mutant neuroblasts, suggesting that the first stage can occur independent of Survivin and CPC localization to the central spindle (Szafer-Glusman, 2011).

Spermatocytes, in contrast, do not form an initial wide equatorial band of contractile ring components. Instead, from their first appearance in early anaphase, the actomyosin rings in spermatocytes are tightly focused at the cell equator (Giansanti, 2006). It is speculated that this restricted initial localization of contractile ring components and the apparent lack of a preceding wide equatorial band may be a consequence of a more stringent global block to Rho1 activation at the cortex in spermatocytes than in neuroblasts. It is proposed that this global block is eventually overridden by positive regulation of Rho1 by local concentration of RhoGEF, in turn facilitated by CPC-dependent events associated with and/or localized by central spindle microtubules. Rho1 activation would then occur within a narrow peak exactly at the site where pole-to-equator microtubules interact to maximize RhoGEF deposition/concentration at the cortex. Indeed, F-actin ring assembly occurs locally and cytokinesis initiates immediately after the pole-to-equator microtubules contact the cortex in Drosophila spermatocytes. It is proposed that, according to this model, the defects in Survivin lead to lack of CPC activity and abnormal centralspindlin, resulting in absence of Rho1 and Polo kinase from the equator of scpo mutant spermatocytes (Szafer-Glusman, 2011).

In neuroblasts, where a more permissive cortex allows a broad belt of Rho1 activation at the cell equator, Survivin and CPC appear to promote gradual convergence of the initial broad band into a narrow ring centered at the maximum of RhoGEF activity at the cortex. In scpo mutants, which display irregular anaphase central spindles devoid of Pav, the broad Rho1 cortical band fails to narrow, the cells fail to form a focused, narrow ring of myosin, and cell division proceeds with inefficient and incomplete constriction (Szafer-Glusman, 2011).

A key difference between neuroblasts and spermatocytes that may account, at least in part, for the differences in behavior of Rho1 and myosin complex proteins is in the relationship between Polo kinase and the CPC observed in scpo mutant mitotic versus male meiotic cells. In spermatocytes, Polo and the CPC are interdependent and Polo colocalizes with the CPC along its full journey from metaphase through anaphase and telophase. In neuroblasts, in contrast, Polo localization during cytokinesis appears to be independent of the CPC and centralspindlin, at least at early stages of cell division, but Polo appears to colocalize with Feo, the Drosophila homolog of PRC1, that required for central-spindle formation and cytokinesis. A second difference between neuroblasts and spermatocytes may be the recently described, spindle-independent backup system that can localize myosin to a broad band at the cell cortex near the future cleavage plane under control of the neuroblast cell polarity system. The broad localization of myosin to the cell cortex observed in ana/telophase neuroblasts in scpo mutants may be in part due to these redundant mechanisms (Szafer-Glusman, 2011).

Structure of a Survivin-Borealin-INCENP core complex reveals how chromosomal passengers travel together

The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connected to its localization. The complex first localizes to centromeres and later associates with the central spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. The 1.4 A resolution crystal structure of the regulatory core of the CPC was determined, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. siRNA rescue experiments were used with structure-based mutants to explore the requirements for CPC localization. The intertwined structural interactions of the core components were shown to lead to functional interdependence. Association of the core 'passenger' proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for central spindle and midbody localization (Jeyaprakash, 2007; full online article).

Participation of Survivin in mitotic and apoptotic activities of normal and tumor-derived cells

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, containing a single baculovirus IAP repeat (BIR) and no RING finger, that is expressed in many human cancers. Although it has been proposed to be involved in mitotic and cytokinetic processes, its functional subcellular distribution in the cytoplasm and nucleus, and its binding to centrosomes, spindle fibers, and centromeres in relation to these processes, is not fully resolved. The localization of Survivin was analyzed in normal (Detroit 551, IMR-90) and tumor-derived (HeLa, Saos-2) cell lines, and it was found to colocalize with centrosomes in the cytoplasm during interphase, then moves to centromeres during mitosis, and finally localizes to the midbody spindle fibers during telophase. However, Taxol, a popular microtubule stabilizing agent that is frequently used in the study of these processes, severely disrupted the localization of Survivin. Taxol treatment of cells promoted extensive relocalization of Survivin with alpha-tubulin on microtubules during either interphase or mitosis. Survivin antisense oligonucleotide markedly sensitized HeLa cells to cell death induced by agents acting at the level of cell surface receptor (Fas pathway) or at the level of mitochondria (etoposide). HeLa cell death induced by Survivin antisense oligonucleotide could be partially complemented by Deterin, the Drosophila homolog of Survivin. Reciprocally, a chimera of the Deterin BIR domain and Survivin C-terminus could rescue Drosophila Kc cells from death induced by transfection of a human caspase-7-expressing plasmid. These results indicate common components of Survivin and Deterin antiapoptotic action in the vertebrate and invertebrate phyla (Jiang, 2001).

Deterin, a new inhibitor of apoptosis from Drosophila melanogaster

Deterin, a new apoptosis inhibitor from Drosophila melanogaster, possesses an unusual structure of only a single baculovirus inhibitor of apoptosis (IAP)-type repeat and no RING finger motif. The biochemical actions of deterin are demonstrated in SF9 and S2 cell transfection assays, in which the expressed protein acts in the cytoplasm to inhibit or deter cells from apoptosis otherwise induced by the caspase-dependent apoptosis activator reaper or by cytotoxicants. A loss of function phenotype for deterin of cell death was indicated by transfections with either a dominant negative deterin mutant or with inhibitory RNA (RNAi) for deterin. The dominant negative C-terminal fragment that antagonized antiapoptotic activity of deterin did not affect antiapoptotic activity of DIAP1 or p35. Both the baculovirus IAP-type repeat (BIR) domain and the alpha-helical C-terminal domain are necessary in both SF9 and S2 cells for deterin to manifest its activity to prevent cell death. The approximately 650-base deterin transcript is present in embryos, third instar larvae, and late stage nurse cells of adult females. The deterin transcript is distributed throughout early stage embryos, whereas in later stage embryos it becomes progressively restricted to the central nervous system and gonads. Whereas the nematode survivin-type IAP has thus far been implicated only as a mitotic regulator, Drosophila deterin constitutes the first invertebrate member of the survivin-type IAP group to exhibit apoptosis-inhibitory activity (Jones, 2000).

Search PubMed for articles about Drosophila Survivin

Beardmore, V. A., Ahonen, L. J., Gorbsky, G. J. and Kallio, M. J. (2004). Survivin dynamics increases at centromeres during G2/M phase transition and is regulated by microtubule-attachment and Aurora B kinase activity. J Cell Sci 117: 4033-4042. PubMed ID: 15280424

Delacour-Larose, M., Molla, A., Skoufias, D. A., Margolis, R. L. and Dimitrov, S. (2004). Distinct dynamics of Aurora B and Survivin during mitosis. Cell Cycle 3: 1418-1426. PubMed ID: 15483398

Giansanti, M. G., Farkas, R. M., Bonaccorsi, S., Lindsley, D. L., Wakimoto, B. T., Fuller, M. T. and Gatti, M. (2004). Genetic dissection of meiotic cytokinesis in Drosophila males. Mol Biol Cell 15: 2509-2522. PubMed ID: 15004238

Giansanti, M. G., Bonaccorsi, S., Kurek, R., Farkas, R. M., Dimitri, P., Fuller, M. T. and Gatti, M. (2006). The class I PITP giotto is required for Drosophila cytokinesis. Curr Biol 16: 195-201. PubMed ID: 16431372

Jeyaprakash, A. A., Klein, U. R., Lindner, D., Ebert, J., Nigg, E. A. and Conti, E. (2007). Structure of a Survivin-Borealin-INCENP core complex reveals how chromosomal passengers travel together. Cell 131: 271-285. PubMed ID: 17956729

Jiang, X., Wilford, C., Duensing, S., Munger, K., Jones, G. and Jones, D. (2001). Participation of Survivin in mitotic and apoptotic activities of normal and tumor-derived cells. J Cell Biochem 83: 342-354. PubMed ID: 11573250

Jones, G., Jones, D., Zhou, L., Steller, H. and Chu, Y. (2000). Deterin, a new inhibitor of apoptosis from Drosophila melanogaster. J Biol Chem 275: 22157-22165. PubMed ID: 10764741

Lens, S. M., Wolthuis, R. M., Klompmaker, R., Kauw, J., Agami, R., Brummelkamp, T., Kops, G. and Medema, R. H. (2003). Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension. EMBO J 22: 2934-2947. PubMed ID: 12805209

Lewellyn, L., Carvalho, A., Desai, A., Maddox, A. S. and Oegema, K. (2011). The chromosomal passenger complex and centralspindlin independently contribute to contractile ring assembly. J Cell Biol 193: 155-169. PubMed ID: 21464231lewell

Szafer-Glusman, E., Fuller, M. T. and Giansanti, M. G. (2011). Role of Survivin in cytokinesis revealed by a separation-of-function allele. Mol Biol Cell 22: 3779-3790. PubMed ID: 21865602

date revised: 6 May, 2013

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