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

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.

Figure 1

Subcellular localization of GFP-MNB/DYRK1A (A) and FLAG-tagged MNB/DYRK1A (B) in HeLa cells. HeLa cells were transfected with constructs encoding GFP, GFP-NLS, GFP-MNB/DYRK1A, or FLAG epitope-tagged MNB/DYRK1A as described in "Materials and Methods." The cells were then fixed, stained with 1 μM TOTO-3 for the counterstaining of DNA, and observed by confocal laser scanning microscopy. GFP or FITC and DNA are displayed in green and blue, respectively. Merged images are also shown in the third photo from the left. Phase-contrast images are shown at the right. The data are representative of those of 5 independent experiments. Scale bar, 10 μm.

Figure 2

Appearance of multiple nuclei in GFP-MNB/DYRK1A-overexpressing cells. HeLa cells transfected with the plasmid encoding GFP-MNB/DYRK1A were incubated for 48 hours and then fixed as described in "Materials and Methods." Fixed cells were stained with 1 μM TOTO-3 for the counterstaining of DNA and observed by confocal laser scanning microscopy. Images were collected at 0.4-μm Z axis intervals. GFP-MNB/DYRK1A (left) and DNA (middle) are displayed in green and blue, respectively. Merged images are shown in the right panel. Scale bar, 10 μm.

Figure 3

High-level expression of GFP-MNB/DYRK1A in multinucleate cells. HeLa cells were transfected with cDNA encoding GFP-MNB/DYRK1A. At 48 hours after transfection, the cells were fixed, stained with anti-MNB antibody and TOTO-3, and observed with a confocal laser scanning microscope as described in "Materials and Methods." Scale bar, 10 μm.

Figure 4

Overexpression of MNB/DYRK1A increases the number of multinucleate cells. HeLa cells were transfected with constructs encoding GFP, GFP-NLS, GFP-MNB/DYRK1A (WT), GFP-MNB/DYRK1A (K179R), or GFP-MNB/DYRK1A (Y310F/Y312F), as described in "Materials and Methods." The cells were then fixed at 24 hours (Upper panel) or 48 hours (Lower panel) and stained with 1 μM TOTO-3. The percentage of multinucleate cells was calculated by dividing the number of cells with more than 2 nuclei by the number of total cells (a total of 200 cells were examined for determining each percentage). In cells transfected with no vector, GFP cDNA, or GFP-NLS cDNA, '-' denotes cells with no fluorescent signal; '++', cells with a fluorescence signal, the level of which was comparable to that in cells with diffuse distribution of GFP-MNB/DYRK1A all over the nuclei. In cells transfected with cDNA encoding GFP-MNB/DYRK1A, GFP-MNB/DYRK1A (K179R), or GFP-MNB/DYRK1A (Y310F/Y312F), '-' denotes cells with no fluorescent signal; '+', cells with a dotted distribution of fluorescence; '++', cells with a diffuse distribution of fluorescence. The data shown are means ± S.E. of 3 independent experiments. (A) The percentages of cells expressing GFP, GFP-NLS, and GFP-MNB/DYRK1A at 24 hours after transfection were 12.24 ± 0.19%, 10.08 ± 0.37%, and 7.76 ± 0.64%, respectively (dotted distribution, 4.21 ± 0.40%; diffuse distribution, 3.55 ± 0.36%); and at 48 hours, 14.55 ± 0.21%, 11.74 ± 0.29%, and 9.34 ± 0.51%, respectively (dotted distribution, 5.42 ± 0.41%; diffuse distribution, 3.92 ± 0.15%). (B) The percentages of cells expressing GFP, GFP-MNB/DYRK1A, GFP-MNB/DYRK1A (K179R), and GFP-MNB/DYRK1A (Y310F/Y312F) at 24 hours after transfection were 13.8 ± 0.56%, 8.48 ± 0.79% (dotted distribution, 5.33 ± 0.40%; diffuse distribution, 3.15 ± 0.45%), 12.18 ± 0.55% (dotted distribution, 7.15 ± 0.80%; diffuse distribution, 5.03 ± 0.65%), and 10.43 ± 0.60% (dotted distribution, 6.10 ± 0.49%; diffuse distribution, 4.33 ± 0.38%), respectively; and at 48 hours, 15.34 ± 0.57%, 10.10 ± 0.52% (dotted distribution, 6.51 ± 0.63%; diffuse distribution, 3.59 ± 0.21%), 14.44 ± 0.30% (dotted distribution, 8.72 ± 0.76%; diffuse distribution, 5.55 ± 0.40%), and 12.07 ± 0.52% (dotted distribution, 7.45 ± 0.77%; diffuse distribution, 4.63 ± 0.54%), respectively.

Figure 5

Subcellular localization of GFP-MNB/DYRK1A in a living cell during the cell cycle. HeLa cells transfected with the plasmid encoding GFP-MNB/DYRK1A were incubated for 24 hours, stained with 100 ng/ml Hoechst33342 for the counterstaining of DNA, and then observed with a fluorescence microscope. In the merged images (right), GFP-MNB/DYRK1A and DNA are displayed in green and red, respectively. The numbers on the left side of each image represent the time after the start of microscopic observation.

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.

Figure 7

Overproduction of centrosomes in GFP-MNB/DYRK1A-overexpressing cells. HeLa cells were transfected with constructs encoding GFP or GFP-MNB/DYRK1A and then fixed as described in the legend of Fig. 6. Fixed cells were stained with anti-γ-tubulin antibody and TOTO-3 as described in "Materials and Methods." The stained cells were observed with a confocal laser scanning microscope. Arrows in panels C, D, and E point to centrosomes. A, C and D, interphase; B and E, M phase. The data are representative of those of 5 independent experiments. Scale bar, 10 μm.

Figure 8

Centrosome number in GFP-MNB/DYRK1A-overexpressing cells. HeLa cells were transfected with constructs encoding GFP, GFP-NLS, or MNB/DYRK1A as described in "Materials and Methods." The cells were then fixed, stained with anti-γ-tubulin antibody and TOTO-3, and observed by confocal laser scanning microscopy at the indicated time points. Each cell population (%) was determined by counting the centrosome number in each cell for a total of 200 cells. Paired centrosomes, which could be distinguished under microscopic observation, were scored as 2 centrosomes. The centrosome number was counted for non transfected cells (normal cells and those treated with LipofectAMINE but without a vector), for cells with fluorescence signal (GFP and GFP-NLS), and for cells with a diffuse distribution of fluorescence within their nuclei (GFP-MNB/DYRK1A). The data shown are means ± S.E. of 3 independent experiments.