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. 2000 Dec 25;151(7):1575-82.
doi: 10.1083/jcb.151.7.1575.

Human survivin is a kinetochore-associated passenger protein

D A Skoufias  1 , C Mollinari, F B Lacroix, R L Margolis
Affiliations

V体育平台登录 - Human survivin is a kinetochore-associated passenger protein

D A Skoufias et al. J Cell Biol. .

Abstract

Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle VSports手机版. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Delta106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage. .

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Figures

Figure 1
Figure 1
Transient overexpression of human survivin reveals kinetochore and cleavage furrow association. (A) Immunoblot analysis of HeLa cell extracts using antibodies specific to HA and to survivin from cells transiently expressing HA-tagged survivin. (B) Immunofluorescence microscopy of transfected cells stained with anti–HA (green) and propidium iodide (red) for chromatin. In different interphase cells, survivin localizes predominantly either in the cytoplasm (a) or the nucleus (b). In prophase through metaphase (c–e), survivin localizes in distinct spots on the chromatin. In anaphase, leaves the chromatin, associates with the spindle midzone (f), extending to the cortex (g), and remains localized in telophase (h) and the midbody (i). (C) Survivin (green) colocalizes with kinetochores stained with a human CREST serum (red) during mitosis until metaphase (a and b), and then dissociates from kinetochores and accumulates in the cleavage furrow and midbody (c–e). (D) Survivin (green) colocalizes with the kinetochore passenger protein TD-60 stained with a human autoimmune serum (red) through all the stages of mitosis: (a) prometaphase, (b) metaphase, (c) anaphase, (d) telophase, and (e) late telophase. Bar: 10 μm.
Figure 2
Figure 2
Cell-cycle distribution of survivin in permanently overexpressing HA-tagged survivin NIH3T3 cells. Immunofluorescence microscopy of expressing cells stained with an anti–HA (green) and propidium iodide (red). (A) During mitosis, survivin localizes to kinetochores in late G2/prophase (a), prometaphase (b), and metaphase (c). In late mitosis, survivin localizes to the spindle midzone (d), and then the cleavage furrow (e) and midbody (f). (B) Survivin retains kinetochore localization after disruption of the mitotic spindle by nocodazole (0.1 μg/ml, a) or taxol (20 μM, b). (C) Low-magnification fields of random cycling cells and cells blocked for 16 h in G2 with the topoisomerase II inhibitor VP16 (2 μg/ml). In untreated cells, survivin exhibits a cytoplasmic staining, nuclear punctate staining (arrow) and midbody staining (arrowhead). In 90% of G2 arrested cells, (VP16) survivin has a nuclear punctate distribution (right; see also B, c). Bar: 10 μm.
Figure 3
Figure 3
A BIR-motif C84A mutation and a Δ106 COOH-terminal truncation on survivin abolish kinetochore and cleavage furrow association. (A) Schematic representation of survivin and the introduced C84A point mutation (red arrowhead) and Δ106 deletion (black arrowhead). The locations of the introduced mutations are also depicted on the survivin dimer crystal structure (Chantalat et al. 2000). (B) Immunofluorescence microscopy of HeLa cells transiently overexpressing wild-type (WT) HA-survivin, HA-C84A, and HA-Δ106 survivin 48 h after transfection. Cells stained with anti–HA (green) and propidium iodide (red). As opposed to wild-type survivin, neither mutant protein localizes to kinetochores (left), the cleavage furrow (middle), or midbody (right). Bar: 10 μm.
Figure 4
Figure 4
The BIR-motif C84A mutation and the Δ106 COOH-terminal truncation on survivin do not alter the distribution of the kinetochore passenger protein TD-60. Double immunofluorescence of HeLa cells transiently transfected with wild type (A), C84A (B), and Δ106 (C) survivin stained with anti–HA (SRV) and with a human autoimmune serum recognizing TD-60. Both mutants of survivin do not alter the TD-60 localization to kinetochores (left) and neither mutant alters the distribution of TD-60 to the cleavage furrow (middle) or to midbody (left). Bar: 10 μm.
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