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CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Zeitlin SG, Shelby RD, Sullivan KF - J. Cell Biol. (2001)

Bottom Line: The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1.Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell.These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

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Related in: MedlinePlus

Live cell analysis: Flemming body lifetime and midbody length. (a–c) Cell division was visualized by live cell phase contrast microscopy at a magnification of 20×. Representative cells are shown for each stage of cell division (TTA cells closely resembled C4; unpublished data). For videos, see online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200108125/DC1. Cells were followed from interphase through initial rounding up, cell division, and reflattening until they ultimately separated. Midbodies were easily detectable in S7A and S7E cells but were smaller, and sometimes not visible, in C4. Arrows indicate cells of interest, midbodies, and newly separated cells. Times shown are hours:minutes, with the time of Flemming body appearance set to zero. (d) CENP-A mutant cell lines exhibit significantly longer midbody lifetimes than wild-type cells filmed under identical conditions of substrate attachment and cell seeding density. Images were taken once every 2 min. Midbody lifetimes were measured beginning when a visible Flemming body became visible between the two daughter cells, and ending when the midbody split, at which time the Flemming body was engulfed by one of the daughter cells and cell separation was considered complete. Standard error is shown for each cell line (bars). (e) Midbody lengths are shown in μm (± standard error) from videos of live cells (black) compared with lengths from fixed cells stained with antitubulin (gray). For live analysis, the length of the intercellular bridge was measured at each time point of 2 min, from when it first became visible to when the two cells completed separation and the Flemming body was engulfed by one of the daughter cells. These values were then averaged for each cell over time, and averaged again across the total number of cells filmed in this way (n = 7, 12, and 7 for C4, S7A, and S7E, respectively). Midbodies were observed to oscillate in a stretching motion before breaking at their maximum length. For fixed analysis, asynchronous cells were stained with antitubulin and a minimum of 150 cells were counted for each cell line. Midbody lengths for both mutants are significantly different from wild-type (P = 0) based on Chi-squared analysis. (f and g) Midbody morphology. The brightly labeled tubulin bundle (red) does not exactly match the length of the intercellular bridge (DIC). (f) In early cytokinesis, the tubulin bundle is longer than the intercellular bridge, defined as the distance between the two cell bodies. (g) In late cytokinesis, the tubulin bundle is shorter than the intercellular bridge, thus leaving an unstained gap between the end of the tubulin bundle and the edge of the cell body. Bars, 10 μm.
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fig4: Live cell analysis: Flemming body lifetime and midbody length. (a–c) Cell division was visualized by live cell phase contrast microscopy at a magnification of 20×. Representative cells are shown for each stage of cell division (TTA cells closely resembled C4; unpublished data). For videos, see online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200108125/DC1. Cells were followed from interphase through initial rounding up, cell division, and reflattening until they ultimately separated. Midbodies were easily detectable in S7A and S7E cells but were smaller, and sometimes not visible, in C4. Arrows indicate cells of interest, midbodies, and newly separated cells. Times shown are hours:minutes, with the time of Flemming body appearance set to zero. (d) CENP-A mutant cell lines exhibit significantly longer midbody lifetimes than wild-type cells filmed under identical conditions of substrate attachment and cell seeding density. Images were taken once every 2 min. Midbody lifetimes were measured beginning when a visible Flemming body became visible between the two daughter cells, and ending when the midbody split, at which time the Flemming body was engulfed by one of the daughter cells and cell separation was considered complete. Standard error is shown for each cell line (bars). (e) Midbody lengths are shown in μm (± standard error) from videos of live cells (black) compared with lengths from fixed cells stained with antitubulin (gray). For live analysis, the length of the intercellular bridge was measured at each time point of 2 min, from when it first became visible to when the two cells completed separation and the Flemming body was engulfed by one of the daughter cells. These values were then averaged for each cell over time, and averaged again across the total number of cells filmed in this way (n = 7, 12, and 7 for C4, S7A, and S7E, respectively). Midbodies were observed to oscillate in a stretching motion before breaking at their maximum length. For fixed analysis, asynchronous cells were stained with antitubulin and a minimum of 150 cells were counted for each cell line. Midbody lengths for both mutants are significantly different from wild-type (P = 0) based on Chi-squared analysis. (f and g) Midbody morphology. The brightly labeled tubulin bundle (red) does not exactly match the length of the intercellular bridge (DIC). (f) In early cytokinesis, the tubulin bundle is longer than the intercellular bridge, defined as the distance between the two cell bodies. (g) In late cytokinesis, the tubulin bundle is shorter than the intercellular bridge, thus leaving an unstained gap between the end of the tubulin bundle and the edge of the cell body. Bars, 10 μm.

Mentions: We directly examined the execution of mitosis in mutant cell lines by time-lapse phase-contrast microscopy (representative images are shown in Fig. 4, a–c). One phase image was taken every 2 min overnight on a heated stage. HeLa cells characteristically round up when they enter mitosis, allowing approximate staging of cell division. The time from the onset to completion of cell rounding was similar for all cell lines. Although this analysis cannot rule out subtle differences in mitotic progression, cells in all lines appeared to progress into anaphase B (oval-shape, with visibly separated chromatin masses) and telophase with similar kinetics. However, the kinetics of the final stage of cytokinesis, scored from the first appearance of an intercellular bridge to the disappearance of the Flemming body, were quite different in CENP-A Ser7 mutant cells (Fig. 4, a–c). Wild-type or C4 cells complete this process within 30–40 min (Fig. 4 d). S7E cells exhibit a dramatic delay, requiring ∼150 min on average, whereas S7A cells took an average of ∼70 min (Fig. 4 d). The midbody structure is very dynamic during this process, oscillating in length before finally breaking (see videos available at http://www.jcb.org/cgi/content/full/jcb.200108125/DC1).


CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Zeitlin SG, Shelby RD, Sullivan KF - J. Cell Biol. (2001)

Live cell analysis: Flemming body lifetime and midbody length. (a–c) Cell division was visualized by live cell phase contrast microscopy at a magnification of 20×. Representative cells are shown for each stage of cell division (TTA cells closely resembled C4; unpublished data). For videos, see online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200108125/DC1. Cells were followed from interphase through initial rounding up, cell division, and reflattening until they ultimately separated. Midbodies were easily detectable in S7A and S7E cells but were smaller, and sometimes not visible, in C4. Arrows indicate cells of interest, midbodies, and newly separated cells. Times shown are hours:minutes, with the time of Flemming body appearance set to zero. (d) CENP-A mutant cell lines exhibit significantly longer midbody lifetimes than wild-type cells filmed under identical conditions of substrate attachment and cell seeding density. Images were taken once every 2 min. Midbody lifetimes were measured beginning when a visible Flemming body became visible between the two daughter cells, and ending when the midbody split, at which time the Flemming body was engulfed by one of the daughter cells and cell separation was considered complete. Standard error is shown for each cell line (bars). (e) Midbody lengths are shown in μm (± standard error) from videos of live cells (black) compared with lengths from fixed cells stained with antitubulin (gray). For live analysis, the length of the intercellular bridge was measured at each time point of 2 min, from when it first became visible to when the two cells completed separation and the Flemming body was engulfed by one of the daughter cells. These values were then averaged for each cell over time, and averaged again across the total number of cells filmed in this way (n = 7, 12, and 7 for C4, S7A, and S7E, respectively). Midbodies were observed to oscillate in a stretching motion before breaking at their maximum length. For fixed analysis, asynchronous cells were stained with antitubulin and a minimum of 150 cells were counted for each cell line. Midbody lengths for both mutants are significantly different from wild-type (P = 0) based on Chi-squared analysis. (f and g) Midbody morphology. The brightly labeled tubulin bundle (red) does not exactly match the length of the intercellular bridge (DIC). (f) In early cytokinesis, the tubulin bundle is longer than the intercellular bridge, defined as the distance between the two cell bodies. (g) In late cytokinesis, the tubulin bundle is shorter than the intercellular bridge, thus leaving an unstained gap between the end of the tubulin bundle and the edge of the cell body. Bars, 10 μm.
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fig4: Live cell analysis: Flemming body lifetime and midbody length. (a–c) Cell division was visualized by live cell phase contrast microscopy at a magnification of 20×. Representative cells are shown for each stage of cell division (TTA cells closely resembled C4; unpublished data). For videos, see online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200108125/DC1. Cells were followed from interphase through initial rounding up, cell division, and reflattening until they ultimately separated. Midbodies were easily detectable in S7A and S7E cells but were smaller, and sometimes not visible, in C4. Arrows indicate cells of interest, midbodies, and newly separated cells. Times shown are hours:minutes, with the time of Flemming body appearance set to zero. (d) CENP-A mutant cell lines exhibit significantly longer midbody lifetimes than wild-type cells filmed under identical conditions of substrate attachment and cell seeding density. Images were taken once every 2 min. Midbody lifetimes were measured beginning when a visible Flemming body became visible between the two daughter cells, and ending when the midbody split, at which time the Flemming body was engulfed by one of the daughter cells and cell separation was considered complete. Standard error is shown for each cell line (bars). (e) Midbody lengths are shown in μm (± standard error) from videos of live cells (black) compared with lengths from fixed cells stained with antitubulin (gray). For live analysis, the length of the intercellular bridge was measured at each time point of 2 min, from when it first became visible to when the two cells completed separation and the Flemming body was engulfed by one of the daughter cells. These values were then averaged for each cell over time, and averaged again across the total number of cells filmed in this way (n = 7, 12, and 7 for C4, S7A, and S7E, respectively). Midbodies were observed to oscillate in a stretching motion before breaking at their maximum length. For fixed analysis, asynchronous cells were stained with antitubulin and a minimum of 150 cells were counted for each cell line. Midbody lengths for both mutants are significantly different from wild-type (P = 0) based on Chi-squared analysis. (f and g) Midbody morphology. The brightly labeled tubulin bundle (red) does not exactly match the length of the intercellular bridge (DIC). (f) In early cytokinesis, the tubulin bundle is longer than the intercellular bridge, defined as the distance between the two cell bodies. (g) In late cytokinesis, the tubulin bundle is shorter than the intercellular bridge, thus leaving an unstained gap between the end of the tubulin bundle and the edge of the cell body. Bars, 10 μm.
Mentions: We directly examined the execution of mitosis in mutant cell lines by time-lapse phase-contrast microscopy (representative images are shown in Fig. 4, a–c). One phase image was taken every 2 min overnight on a heated stage. HeLa cells characteristically round up when they enter mitosis, allowing approximate staging of cell division. The time from the onset to completion of cell rounding was similar for all cell lines. Although this analysis cannot rule out subtle differences in mitotic progression, cells in all lines appeared to progress into anaphase B (oval-shape, with visibly separated chromatin masses) and telophase with similar kinetics. However, the kinetics of the final stage of cytokinesis, scored from the first appearance of an intercellular bridge to the disappearance of the Flemming body, were quite different in CENP-A Ser7 mutant cells (Fig. 4, a–c). Wild-type or C4 cells complete this process within 30–40 min (Fig. 4 d). S7E cells exhibit a dramatic delay, requiring ∼150 min on average, whereas S7A cells took an average of ∼70 min (Fig. 4 d). The midbody structure is very dynamic during this process, oscillating in length before finally breaking (see videos available at http://www.jcb.org/cgi/content/full/jcb.200108125/DC1).

Bottom Line: The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1.Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell.These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

Show MeSH
Related in: MedlinePlus