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Human survivin is a kinetochore-associated passenger protein.

Skoufias DA, Mollinari C, Lacroix FB, Margolis RL - J. Cell Biol. (2000)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat á l'Energie Atomique-Centre National de la Recherche Scientifique), 38027 Grenoble Cedex 1, France.

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. 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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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.
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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.

Mentions: We have transiently transfected HeLa cells with plasmid expressing human HA-tagged survivin by either electroporation or with exgen, and have examined the protein's localization through the cell cycle. With either method the results are equivalent. The HA antibody recognized one strong band in Western blots of whole cell extracts, and reacted with no protein in control cells (Fig. 1 A). Western blots with a survivin antibody confirmed the expression of HA-tagged survivin at levels higher than the endogenous protein (Fig. 1 A). The HA tag has been introduced into the NH2 terminus of the protein, where structural analysis had previously shown modification did not interfere with native folding of the protein dimer (Chantalat et al. 2000).


Human survivin is a kinetochore-associated passenger protein.

Skoufias DA, Mollinari C, Lacroix FB, Margolis RL - J. Cell Biol. (2000)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150675&req=5

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.
Mentions: We have transiently transfected HeLa cells with plasmid expressing human HA-tagged survivin by either electroporation or with exgen, and have examined the protein's localization through the cell cycle. With either method the results are equivalent. The HA antibody recognized one strong band in Western blots of whole cell extracts, and reacted with no protein in control cells (Fig. 1 A). Western blots with a survivin antibody confirmed the expression of HA-tagged survivin at levels higher than the endogenous protein (Fig. 1 A). The HA tag has been introduced into the NH2 terminus of the protein, where structural analysis had previously shown modification did not interfere with native folding of the protein dimer (Chantalat et al. 2000).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat á l'Energie Atomique-Centre National de la Recherche Scientifique), 38027 Grenoble Cedex 1, France.

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. 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.

Show MeSH
Related in: MedlinePlus