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Overexpression of the human MNB/DYRK1A gene induces formation of multinucleate cells through overduplication of the centrosome.

Funakoshi E, Hori T, Haraguchi T, Hiraoka Y, Kudoh J, Shimizu N, Ito F - BMC Cell Biol. (2003)

Bottom Line: In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed; whereas in those with the nuclear dots, such aberrant nuclei were not found.Immunostaining of GFP-MNB/DYRK1A-overexpressing cells with specific antibodies against alpha- and gamma-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells.These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka 573-0101, Japan. funakos@pharm.setsunan.ac.jp

ABSTRACT

Background: Previously we cloned the human MNB/DYRK1A gene from the "Down syndrome critical region" on chromosome 21. This gene encodes a dual specificity protein kinase that catalyzes its autophosphorylation on serine/threonine and tyrosine residues. But, the functions of the MNB/DYRK1A gene in cellular processes are unknown.

Results: In this study, we examined HeLa cells transfected with cDNA encoding a green fluorescent protein (GFP)-MNB/DYRK1A fusion protein and found 2 patterns of expression: In one group of transfected cells, GFP-MNB/DYRK1A was localized as dots within the nucleus; and in the other group, it was overexpressed and had accumulated all over the nucleus. In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed; whereas in those with the nuclear dots, such aberrant nuclei were not found. Furthermore, in the latter cells, essential processes such as mitosis and cytokinesis occurred normally. Multinucleation was dependent on the kinase activity of MNB/DYRK1A, because it was not observed in cells overexpressing kinase activity-negative mutants, GFP-MNB/DYRK1A (K179R) and GFP-MNB/DYRK1A (Y310F/Y312F). Immunostaining of GFP-MNB/DYRK1A-overexpressing cells with specific antibodies against alpha- and gamma-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells.

Conclusions: These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.

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Microtubule structures in GFP-MNB/DYRK1A-overexpressing cells. HeLa cells were transfected with constructs encoding GFP or GFP-MNB/DYRK1A. After incubation for 36–48 hours, the cells were fixed, stained with anti-α-tubulin antibody and TOTO-3, and observed with a confocal laser scanning microscope as described in "Materials and Methods." (A) a and c, interphase; b and d, M phase. (B) a, prometaphase; b, metaphase; c, late-anaphase. The data are representative of those of 5 independent experiments. Scale bar, 10 μm.
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Figure 6: Microtubule structures in GFP-MNB/DYRK1A-overexpressing cells. HeLa cells were transfected with constructs encoding GFP or GFP-MNB/DYRK1A. After incubation for 36–48 hours, the cells were fixed, stained with anti-α-tubulin antibody and TOTO-3, and observed with a confocal laser scanning microscope as described in "Materials and Methods." (A) a and c, interphase; b and d, M phase. (B) a, prometaphase; b, metaphase; c, late-anaphase. The data are representative of those of 5 independent experiments. Scale bar, 10 μm.

Mentions: Next, to investigate the subcellular localization of MNB/DYRK1A protein during the cell cycle, especially during mitosis, we transiently expressed GFP-MNB/DYRK1A in HeLa cells and chased interphase cells with a speckled fluorescent pattern in a time-lapse manner (Fig. 5). At 240 minutes when the nuclear envelope disappeared, GFP-MNB/DYRK1A fluorescence gave a diffuse pattern and was located in the cytoplasm with relative exclusion from the condensed chromatin. After reformation of the nuclear envelope at telophase (see 360 minutes), GFP-MNB/DYRK1A again exhibited a speckled pattern. Thus, MNB/DYRK1A was concentrated at specific loci in the nucleus during interphase, whereas during mitosis it was located all over the cells and not condensed with either chromosome, mitotic spindle, or spindle pole. Fig. 5 also shows that the interphase cells with a speckled pattern in their nucleus progressed into M phase and produced 2 daughter cells. Further, both the condensation and subsequent segregation of chromosomes occurred in these cells as properly as in non-transfected control cells. On the other hand, chromosome missegregation was observed in HeLa cells with overexpressed MNB/DYRK1A, as will be described later (Fig. 6A, panel d).


Overexpression of the human MNB/DYRK1A gene induces formation of multinucleate cells through overduplication of the centrosome.

Funakoshi E, Hori T, Haraguchi T, Hiraoka Y, Kudoh J, Shimizu N, Ito F - BMC Cell Biol. (2003)

Microtubule structures in GFP-MNB/DYRK1A-overexpressing cells. HeLa cells were transfected with constructs encoding GFP or GFP-MNB/DYRK1A. After incubation for 36–48 hours, the cells were fixed, stained with anti-α-tubulin antibody and TOTO-3, and observed with a confocal laser scanning microscope as described in "Materials and Methods." (A) a and c, interphase; b and d, M phase. (B) a, prometaphase; b, metaphase; c, late-anaphase. The data are representative of those of 5 independent experiments. Scale bar, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC212362&req=5

Figure 6: Microtubule structures in GFP-MNB/DYRK1A-overexpressing cells. HeLa cells were transfected with constructs encoding GFP or GFP-MNB/DYRK1A. After incubation for 36–48 hours, the cells were fixed, stained with anti-α-tubulin antibody and TOTO-3, and observed with a confocal laser scanning microscope as described in "Materials and Methods." (A) a and c, interphase; b and d, M phase. (B) a, prometaphase; b, metaphase; c, late-anaphase. The data are representative of those of 5 independent experiments. Scale bar, 10 μm.
Mentions: Next, to investigate the subcellular localization of MNB/DYRK1A protein during the cell cycle, especially during mitosis, we transiently expressed GFP-MNB/DYRK1A in HeLa cells and chased interphase cells with a speckled fluorescent pattern in a time-lapse manner (Fig. 5). At 240 minutes when the nuclear envelope disappeared, GFP-MNB/DYRK1A fluorescence gave a diffuse pattern and was located in the cytoplasm with relative exclusion from the condensed chromatin. After reformation of the nuclear envelope at telophase (see 360 minutes), GFP-MNB/DYRK1A again exhibited a speckled pattern. Thus, MNB/DYRK1A was concentrated at specific loci in the nucleus during interphase, whereas during mitosis it was located all over the cells and not condensed with either chromosome, mitotic spindle, or spindle pole. Fig. 5 also shows that the interphase cells with a speckled pattern in their nucleus progressed into M phase and produced 2 daughter cells. Further, both the condensation and subsequent segregation of chromosomes occurred in these cells as properly as in non-transfected control cells. On the other hand, chromosome missegregation was observed in HeLa cells with overexpressed MNB/DYRK1A, as will be described later (Fig. 6A, panel d).

Bottom Line: In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed; whereas in those with the nuclear dots, such aberrant nuclei were not found.Immunostaining of GFP-MNB/DYRK1A-overexpressing cells with specific antibodies against alpha- and gamma-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells.These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka 573-0101, Japan. funakos@pharm.setsunan.ac.jp

ABSTRACT

Background: Previously we cloned the human MNB/DYRK1A gene from the "Down syndrome critical region" on chromosome 21. This gene encodes a dual specificity protein kinase that catalyzes its autophosphorylation on serine/threonine and tyrosine residues. But, the functions of the MNB/DYRK1A gene in cellular processes are unknown.

Results: In this study, we examined HeLa cells transfected with cDNA encoding a green fluorescent protein (GFP)-MNB/DYRK1A fusion protein and found 2 patterns of expression: In one group of transfected cells, GFP-MNB/DYRK1A was localized as dots within the nucleus; and in the other group, it was overexpressed and had accumulated all over the nucleus. In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed; whereas in those with the nuclear dots, such aberrant nuclei were not found. Furthermore, in the latter cells, essential processes such as mitosis and cytokinesis occurred normally. Multinucleation was dependent on the kinase activity of MNB/DYRK1A, because it was not observed in cells overexpressing kinase activity-negative mutants, GFP-MNB/DYRK1A (K179R) and GFP-MNB/DYRK1A (Y310F/Y312F). Immunostaining of GFP-MNB/DYRK1A-overexpressing cells with specific antibodies against alpha- and gamma-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells.

Conclusions: These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.

Show MeSH
Related in: MedlinePlus