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The transcriptional repressor Kaiso localizes at the mitotic spindle and is a constituent of the pericentriolar material.

Soubry A, Staes K, Parthoens E, Noppen S, Stove C, Bogaert P, van Hengel J, van Roy F - PLoS ONE (2010)

Bottom Line: In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody.We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex.Knockdown of Kaiso accelerated cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department for Molecular Biomedical Research, VIB, Ghent, Belgium.

ABSTRACT
Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its in vitro association with the Armadillo protein p120ctn. Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaiso's aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaiso's function as a transcriptional regulator might be linked to the control of the cell cycle and to cell proliferation in cancer.

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Kaiso localizes in the PCM.Fluorescence microscopy of methanol-fixed HeLa-GFP-centrin cells in either G2/M phase (A) or metaphase (B, C). In (A) and (B) Kaiso was immunolabeled with pAb S1337; in (C) pericentrin was detected by pAb ab4448. Alexa 594-conjugated anti-rabbit secondary antibody was used in all cases and DNA was stained with DAPI. Cells were imaged with a Leica SP5 confocal microscope. Each figure is a projection of a confocal image stack. Colocalization of centrin with Kaiso and pericentrin was confirmed in layers of 0.77 mm thickness. Scale bar, 5 µm. Arrows point at centrosomes. Insets in (A) and (B) show magnified overlay pictures of centriolar regions, each time in a second cell of the same slides. The region of Kaiso-positive staining is broader than the centrin-positive centrioles and overlaps with the mother centriole rather than the daughter centriole (arrowheads). Spindle-associated Kaiso was not obvious in the present experiments, as other fixation and staining conditions were used.
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pone-0009203-g004: Kaiso localizes in the PCM.Fluorescence microscopy of methanol-fixed HeLa-GFP-centrin cells in either G2/M phase (A) or metaphase (B, C). In (A) and (B) Kaiso was immunolabeled with pAb S1337; in (C) pericentrin was detected by pAb ab4448. Alexa 594-conjugated anti-rabbit secondary antibody was used in all cases and DNA was stained with DAPI. Cells were imaged with a Leica SP5 confocal microscope. Each figure is a projection of a confocal image stack. Colocalization of centrin with Kaiso and pericentrin was confirmed in layers of 0.77 mm thickness. Scale bar, 5 µm. Arrows point at centrosomes. Insets in (A) and (B) show magnified overlay pictures of centriolar regions, each time in a second cell of the same slides. The region of Kaiso-positive staining is broader than the centrin-positive centrioles and overlaps with the mother centriole rather than the daughter centriole (arrowheads). Spindle-associated Kaiso was not obvious in the present experiments, as other fixation and staining conditions were used.

Mentions: Evidence for specific association of Kaiso with the centrosomes was strengthened by the following observations. Confocal fluorescence images were obtained for HeLa cells expressing GFP-centrin during G2/M and metaphase. A Kaiso-specific signal was invariably observed in close proximity of the tiny GFP-centrin spots in the centrosomes (Fig. 4). Centrin is known to concentrate within the centriole distal lumen [37]. In G2/M cells, this represented only a minor fraction of Kaiso (Fig. 4A), whereas during metaphase Kaiso was largely concentrated in the centriolar regions (Fig. 4B). The Kaiso-specific signals presented as much broader spots than GFP-centrin and encompassed the mother centriole rather than the daughter centriole (Fig. 4, insets). This pattern is reminiscent of the pericentriolar material (PCM), where also pericentrin and ©-tubulin are localized (exemplified in Fig. 4C and Fig. 3C). Similar results were obtained for SK-LMS-1 cells. Further evidence for Kaiso's localization in the PCM was obtained from co-immunoprecipitation experiments. Indeed, for both HeLa-GFP-centrin and SK-LMS-1 cells such experiments reproducibly revealed that a Kaiso-containing molecular complex contained pericentrin (illustrated in Fig. 5). Also ©-tubulin was found in this complex with Kaiso, which is in line with the known interaction of pericentrin with ©-tubulin [41]. All together, this demonstrates that Kaiso is a genuine component of the PCM.


The transcriptional repressor Kaiso localizes at the mitotic spindle and is a constituent of the pericentriolar material.

Soubry A, Staes K, Parthoens E, Noppen S, Stove C, Bogaert P, van Hengel J, van Roy F - PLoS ONE (2010)

Kaiso localizes in the PCM.Fluorescence microscopy of methanol-fixed HeLa-GFP-centrin cells in either G2/M phase (A) or metaphase (B, C). In (A) and (B) Kaiso was immunolabeled with pAb S1337; in (C) pericentrin was detected by pAb ab4448. Alexa 594-conjugated anti-rabbit secondary antibody was used in all cases and DNA was stained with DAPI. Cells were imaged with a Leica SP5 confocal microscope. Each figure is a projection of a confocal image stack. Colocalization of centrin with Kaiso and pericentrin was confirmed in layers of 0.77 mm thickness. Scale bar, 5 µm. Arrows point at centrosomes. Insets in (A) and (B) show magnified overlay pictures of centriolar regions, each time in a second cell of the same slides. The region of Kaiso-positive staining is broader than the centrin-positive centrioles and overlaps with the mother centriole rather than the daughter centriole (arrowheads). Spindle-associated Kaiso was not obvious in the present experiments, as other fixation and staining conditions were used.
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Related In: Results  -  Collection

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pone-0009203-g004: Kaiso localizes in the PCM.Fluorescence microscopy of methanol-fixed HeLa-GFP-centrin cells in either G2/M phase (A) or metaphase (B, C). In (A) and (B) Kaiso was immunolabeled with pAb S1337; in (C) pericentrin was detected by pAb ab4448. Alexa 594-conjugated anti-rabbit secondary antibody was used in all cases and DNA was stained with DAPI. Cells were imaged with a Leica SP5 confocal microscope. Each figure is a projection of a confocal image stack. Colocalization of centrin with Kaiso and pericentrin was confirmed in layers of 0.77 mm thickness. Scale bar, 5 µm. Arrows point at centrosomes. Insets in (A) and (B) show magnified overlay pictures of centriolar regions, each time in a second cell of the same slides. The region of Kaiso-positive staining is broader than the centrin-positive centrioles and overlaps with the mother centriole rather than the daughter centriole (arrowheads). Spindle-associated Kaiso was not obvious in the present experiments, as other fixation and staining conditions were used.
Mentions: Evidence for specific association of Kaiso with the centrosomes was strengthened by the following observations. Confocal fluorescence images were obtained for HeLa cells expressing GFP-centrin during G2/M and metaphase. A Kaiso-specific signal was invariably observed in close proximity of the tiny GFP-centrin spots in the centrosomes (Fig. 4). Centrin is known to concentrate within the centriole distal lumen [37]. In G2/M cells, this represented only a minor fraction of Kaiso (Fig. 4A), whereas during metaphase Kaiso was largely concentrated in the centriolar regions (Fig. 4B). The Kaiso-specific signals presented as much broader spots than GFP-centrin and encompassed the mother centriole rather than the daughter centriole (Fig. 4, insets). This pattern is reminiscent of the pericentriolar material (PCM), where also pericentrin and ©-tubulin are localized (exemplified in Fig. 4C and Fig. 3C). Similar results were obtained for SK-LMS-1 cells. Further evidence for Kaiso's localization in the PCM was obtained from co-immunoprecipitation experiments. Indeed, for both HeLa-GFP-centrin and SK-LMS-1 cells such experiments reproducibly revealed that a Kaiso-containing molecular complex contained pericentrin (illustrated in Fig. 5). Also ©-tubulin was found in this complex with Kaiso, which is in line with the known interaction of pericentrin with ©-tubulin [41]. All together, this demonstrates that Kaiso is a genuine component of the PCM.

Bottom Line: In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody.We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex.Knockdown of Kaiso accelerated cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department for Molecular Biomedical Research, VIB, Ghent, Belgium.

ABSTRACT
Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its in vitro association with the Armadillo protein p120ctn. Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaiso's aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaiso's function as a transcriptional regulator might be linked to the control of the cell cycle and to cell proliferation in cancer.

Show MeSH
Related in: MedlinePlus