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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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Schematic representation of GFP-tagged Kaiso constructs.Numbers indicate amino acid residues (AA). SA1 and SA2 represent spindle-associated domains. CD1 and CD2 are well-conserved domains (see Suppl. Fig. S1). BTB/POZ, Broad Complex, Tramtrak, Bric à brac/Pox virus and Zinc finger; FL, full-length Kaiso; NLS, nuclear localization signal; pAb, epitope of the polyclonal antibody indicated; Zn, zinc finger domain.
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pone-0009203-g001: Schematic representation of GFP-tagged Kaiso constructs.Numbers indicate amino acid residues (AA). SA1 and SA2 represent spindle-associated domains. CD1 and CD2 are well-conserved domains (see Suppl. Fig. S1). BTB/POZ, Broad Complex, Tramtrak, Bric à brac/Pox virus and Zinc finger; FL, full-length Kaiso; NLS, nuclear localization signal; pAb, epitope of the polyclonal antibody indicated; Zn, zinc finger domain.

Mentions: The subcellular distribution of Kaiso was followed throughout the cell cycle by immunostaining. Human cell lines SK-LMS-1 and HEK293 were studied with the six Kaiso antibodies described in “Materials and Methods”. In agreement with the literature [1], [34], immunofluorescence microscopy of cells in interphase showed strong Kaiso staining in the nucleus as well as some positive staining in the cytoplasm. All antibodies gave similar results, with the exception of 2G, which also stains cytoplasmic particles previously described as possible artifacts [34]. Because the antibodies 6F, 12H, 2G and pAb R had been raised against the same region (Kaiso AA 1–499) [34], we tried to refine the epitope regions of these antibodies. This was done by designing different GFP-Kaiso expression constructs (Fig. 1). We found that all the available antibodies (commercial and gifts) recognize the same region (AA 213–264), present in fragment K4a, and so we made a new rabbit polyclonal antibody (pAb S1337) specifically recognizing a different Kaiso region, AA 655–671 (see “Materials and Methods”) [23]. In immunofluorescence, pAb S1337 recognized exogenously expressed Kaiso, whereas the staining pattern of endogenous Kaiso in centrosomes was similar to that obtained with the monoclonal antibodies (see below). Specificity was further confirmed by concentration-dependent peptide inhibition (data not shown). We then continued to use mainly antibodies 6F and S1337, which recognize two distinct epitopes in Kaiso. We extended our study to other cells of human origin, such as HT29, SW48, MCF-7, MCF-10A, HeLa and MDA-MB-435 cells, as well as to cells of other species, Ptk-2, L929, HL-1 and MDCK. The different cell lines showed similarly strong Kaiso staining in the nucleus besides variable positive staining in the cytoplasm. The strongest cytoplasmic staining was observed in SK-LMS-1, HeLa and MDCK cells. An image focused on the cytoplasmic content of SK-LMS-1 is shown in Fig. 2; double staining with anti-Kaiso and alpha-tubulin antibodies revealed that the fiber-like staining of cytoplasmic Kaiso colocalized with microtubules. Moreover, Kaiso's distribution was dynamic during cell cycle progression. Fig. 3 shows an overview of the mitotic stages. Kaiso was more concentrated at the centrosomes during prophase (Fig. 3A), and was seen on the entire spindle during metaphase (Fig. 3B–C). Double staining for Kaiso and gamma-tubulin showed partial colocalization at the centrosomes (Fig. 3C). During anaphase Kaiso was distributed uniformly along the elongating spindle and was also present at the centrosomal region (Fig. 3D). At telophase and cytokinesis, Kaiso localized at the midbody, but a region in the division plane was unstained (Fig. 3E). This plane is known to be inaccessible for immunodetection and represents a dense region in which microtubules interdigitate between the two daughter cells [40]. When the intercellular bridge elongated, Kaiso colocalized partly with alpha-tubulin. Kaiso was also seen at the edges (or cell protrusions) of the two forming daughter cells. Finally, Kaiso relocated to the newly formed nuclei and the cytoskeleton. In general, Kaiso staining was more intense at the spindle microtubules during mitosis than at the cytoplasmic microtubules during interphase. In Fig. 3 only Ptk-2 and SK-LMS-1 cells are presented, but all cell lines analyzed showed similar patterns, and at least one monoclonal antibody (mostly 6F) and one polyclonal antibody (S1337) were used in each cell line to confirm these 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)

Schematic representation of GFP-tagged Kaiso constructs.Numbers indicate amino acid residues (AA). SA1 and SA2 represent spindle-associated domains. CD1 and CD2 are well-conserved domains (see Suppl. Fig. S1). BTB/POZ, Broad Complex, Tramtrak, Bric à brac/Pox virus and Zinc finger; FL, full-length Kaiso; NLS, nuclear localization signal; pAb, epitope of the polyclonal antibody indicated; Zn, zinc finger domain.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009203-g001: Schematic representation of GFP-tagged Kaiso constructs.Numbers indicate amino acid residues (AA). SA1 and SA2 represent spindle-associated domains. CD1 and CD2 are well-conserved domains (see Suppl. Fig. S1). BTB/POZ, Broad Complex, Tramtrak, Bric à brac/Pox virus and Zinc finger; FL, full-length Kaiso; NLS, nuclear localization signal; pAb, epitope of the polyclonal antibody indicated; Zn, zinc finger domain.
Mentions: The subcellular distribution of Kaiso was followed throughout the cell cycle by immunostaining. Human cell lines SK-LMS-1 and HEK293 were studied with the six Kaiso antibodies described in “Materials and Methods”. In agreement with the literature [1], [34], immunofluorescence microscopy of cells in interphase showed strong Kaiso staining in the nucleus as well as some positive staining in the cytoplasm. All antibodies gave similar results, with the exception of 2G, which also stains cytoplasmic particles previously described as possible artifacts [34]. Because the antibodies 6F, 12H, 2G and pAb R had been raised against the same region (Kaiso AA 1–499) [34], we tried to refine the epitope regions of these antibodies. This was done by designing different GFP-Kaiso expression constructs (Fig. 1). We found that all the available antibodies (commercial and gifts) recognize the same region (AA 213–264), present in fragment K4a, and so we made a new rabbit polyclonal antibody (pAb S1337) specifically recognizing a different Kaiso region, AA 655–671 (see “Materials and Methods”) [23]. In immunofluorescence, pAb S1337 recognized exogenously expressed Kaiso, whereas the staining pattern of endogenous Kaiso in centrosomes was similar to that obtained with the monoclonal antibodies (see below). Specificity was further confirmed by concentration-dependent peptide inhibition (data not shown). We then continued to use mainly antibodies 6F and S1337, which recognize two distinct epitopes in Kaiso. We extended our study to other cells of human origin, such as HT29, SW48, MCF-7, MCF-10A, HeLa and MDA-MB-435 cells, as well as to cells of other species, Ptk-2, L929, HL-1 and MDCK. The different cell lines showed similarly strong Kaiso staining in the nucleus besides variable positive staining in the cytoplasm. The strongest cytoplasmic staining was observed in SK-LMS-1, HeLa and MDCK cells. An image focused on the cytoplasmic content of SK-LMS-1 is shown in Fig. 2; double staining with anti-Kaiso and alpha-tubulin antibodies revealed that the fiber-like staining of cytoplasmic Kaiso colocalized with microtubules. Moreover, Kaiso's distribution was dynamic during cell cycle progression. Fig. 3 shows an overview of the mitotic stages. Kaiso was more concentrated at the centrosomes during prophase (Fig. 3A), and was seen on the entire spindle during metaphase (Fig. 3B–C). Double staining for Kaiso and gamma-tubulin showed partial colocalization at the centrosomes (Fig. 3C). During anaphase Kaiso was distributed uniformly along the elongating spindle and was also present at the centrosomal region (Fig. 3D). At telophase and cytokinesis, Kaiso localized at the midbody, but a region in the division plane was unstained (Fig. 3E). This plane is known to be inaccessible for immunodetection and represents a dense region in which microtubules interdigitate between the two daughter cells [40]. When the intercellular bridge elongated, Kaiso colocalized partly with alpha-tubulin. Kaiso was also seen at the edges (or cell protrusions) of the two forming daughter cells. Finally, Kaiso relocated to the newly formed nuclei and the cytoskeleton. In general, Kaiso staining was more intense at the spindle microtubules during mitosis than at the cytoplasmic microtubules during interphase. In Fig. 3 only Ptk-2 and SK-LMS-1 cells are presented, but all cell lines analyzed showed similar patterns, and at least one monoclonal antibody (mostly 6F) and one polyclonal antibody (S1337) were used in each cell line to confirm these 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