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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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The GFP-tagged Kaiso fragment K9, comprising SA2, localizes at the centrosomes during mitosis.HEK293 cells were transfected with construct GFP-K9 (see also Fig. 1). This fragment localizes only at the centrosomes (arrowheads), which were visualized with a polyclonal anti-γ-tubulin antibody followed by an Alexa 594-conjugated anti-rabbit secondary antibody. DNA was stained with DAPI and cells were imaged (63× objective lens) with a Leica DM IRE2 microscope.
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pone-0009203-g008: The GFP-tagged Kaiso fragment K9, comprising SA2, localizes at the centrosomes during mitosis.HEK293 cells were transfected with construct GFP-K9 (see also Fig. 1). This fragment localizes only at the centrosomes (arrowheads), which were visualized with a polyclonal anti-γ-tubulin antibody followed by an Alexa 594-conjugated anti-rabbit secondary antibody. DNA was stained with DAPI and cells were imaged (63× objective lens) with a Leica DM IRE2 microscope.

Mentions: At mitosis, the GFP-tagged protein encoded by the full-length Kaiso construct was distributed at the spindle material. Fig. 6 (lower row) shows colocalization with alpha-tubulin at one of the centrosomes of MCF-7 cells. GFP-tagged Kaiso fragments comprising AA 213–264, such as K4v (Fig. 7), K4z (Suppl. Fig. S3; metaphase and anaphase in upper and lower row, respectively), K4a (Suppl. Fig. S4, upper row) and K4b (Suppl. Fig. S4, lower row), localized at the centrosomes, mainly at the minus ends of the spindle microtubules, whereas fragments without the AA 213–264 domain (such as K1, K2, K4x and K4y) did not localize at the spindle material but disperse throughout the cell (not shown). As an exception, the GFP-tagged fragment K4, containing a conserved region CD1 as well as the 213–264 AA domain (Fig. 1), did not localize at either spindle microtubules or centrosomes. Three substages of mitosis are shown in Fig. 7, which represents HEK293 cells transfected with GFP-K4v. In cells undergoing cytokinesis, we clearly detected GFP-K4v at the midbody (Fig. 7, lower row). In contrast, fragments comprising AA 343–501 (e.g. construct K9) localized mainly at the centrosomes, where they were seen as two intense dots colocalizing with gamma-tubulin (Fig. 8). The figures shown here represent transfections of MCF-7 or HEK293 cells, but other cells studied gave the same results.


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)

The GFP-tagged Kaiso fragment K9, comprising SA2, localizes at the centrosomes during mitosis.HEK293 cells were transfected with construct GFP-K9 (see also Fig. 1). This fragment localizes only at the centrosomes (arrowheads), which were visualized with a polyclonal anti-γ-tubulin antibody followed by an Alexa 594-conjugated anti-rabbit secondary antibody. DNA was stained with DAPI and cells were imaged (63× objective lens) with a Leica DM IRE2 microscope.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821401&req=5

pone-0009203-g008: The GFP-tagged Kaiso fragment K9, comprising SA2, localizes at the centrosomes during mitosis.HEK293 cells were transfected with construct GFP-K9 (see also Fig. 1). This fragment localizes only at the centrosomes (arrowheads), which were visualized with a polyclonal anti-γ-tubulin antibody followed by an Alexa 594-conjugated anti-rabbit secondary antibody. DNA was stained with DAPI and cells were imaged (63× objective lens) with a Leica DM IRE2 microscope.
Mentions: At mitosis, the GFP-tagged protein encoded by the full-length Kaiso construct was distributed at the spindle material. Fig. 6 (lower row) shows colocalization with alpha-tubulin at one of the centrosomes of MCF-7 cells. GFP-tagged Kaiso fragments comprising AA 213–264, such as K4v (Fig. 7), K4z (Suppl. Fig. S3; metaphase and anaphase in upper and lower row, respectively), K4a (Suppl. Fig. S4, upper row) and K4b (Suppl. Fig. S4, lower row), localized at the centrosomes, mainly at the minus ends of the spindle microtubules, whereas fragments without the AA 213–264 domain (such as K1, K2, K4x and K4y) did not localize at the spindle material but disperse throughout the cell (not shown). As an exception, the GFP-tagged fragment K4, containing a conserved region CD1 as well as the 213–264 AA domain (Fig. 1), did not localize at either spindle microtubules or centrosomes. Three substages of mitosis are shown in Fig. 7, which represents HEK293 cells transfected with GFP-K4v. In cells undergoing cytokinesis, we clearly detected GFP-K4v at the midbody (Fig. 7, lower row). In contrast, fragments comprising AA 343–501 (e.g. construct K9) localized mainly at the centrosomes, where they were seen as two intense dots colocalizing with gamma-tubulin (Fig. 8). The figures shown here represent transfections of MCF-7 or HEK293 cells, but other cells studied gave the same results.

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