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. 2003 Oct;14(10):4067-74.
doi: 10.1091/mbc.e03-04-0242. Epub 2003 Jun 13.

Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number

Ralph Gräf  1 , Ursula Euteneuer, Thi-Hieu Ho, Markus Rehberg
Affiliations

Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number

Ralph Gräf et al. Mol Biol Cell. 2003 Oct.

Abstract

The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over- and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during cytokinesis.

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Figures

Figure 1.
Figure 1.
Construction of a DdCP224 promoter replacement mutant. After insertion of the replacement vector into the endogenous DdCP224 gene by homologous recombination, the plasmid encoded DdCP224 fragment becomes completed by the missing C-terminal part and cells can only express N-terminally GFP-tagged full-length DdCP224 under control of the actin6 promoter. The endogenous promoter drives expression of an untagged N-terminal DdCP224 fragment that shows no specific cellular localization (Gräf et al., 2000). In case of ectopic plasmid insertions, only this N-terminal fragment instead of full-length DdCP224 is expressed as a GFP-fusion protein. The agarose gel on the left shows confirmation of the desired recombination event by PCR on genomic DNA. The primer combination GFP605/TOG12 leads to a 2.9-kb PCR product only in case of homologous recombination (clone A3) but not in case of ectopic integrations (clone C5), whereas the primer combination GFP605/TOG10 gives a 1.3-kb PCR product in both cases.
Figure 2.
Figure 2.
Phenotypes of altered GFP-DdCP224 expression. (A) Immunoblot of cell extracts prepared from wild-type cells (WT; strain AX2) and GFP-DdCP224 cells (clone A3) under overexpression (HL5c-medium) and underexpression (Ka-suspension) conditions. A protein equivalent of 107 cells was loaded onto each lane, and the blot was stained with the monoclonal DdCP224 antibody 2/165 (Gräf et al., 1999). To show equal loading on each lane, blots were also stained with the monoclonal Dictyostelium myosin II antibody mAb96 (Claviez et al., 1982). Endogenous DdCP224 migrates faster than GFP-DdCP224 and it is absent in A3-mutants. (B) Reduced growth of A3-mutants in Klebsiella aerogenes-suspension culture. Growth curves of GFP-DdCP224–underexpressing cells (black; A3–1, 2, and 3) and AX2 wild-type cells (red; AX-1, 2, and 3) from three independent experiments are shown. (C) Supernumerary MTOCs and nucleus-associated centrosomes nucleate microtubules. Confocal immunofluorescence microscopy of fixed A3-mutants overexpressing GFP-DdCP224. Microtubules were labeled with the α-tubulin antibody YL1/2 (yellow; Chemicon, Hofheim, Germany), and nuclei were stained with TOPRO3 (Rehberg and Gräf, 2002). (D and D′) Reversibility of the GFP-DdCP224 overexpression phenotype (D) after shift to underexpression conditions for 24 h (D′). Both specimens were processed in parallel and images were acquired with the same camera settings to allow evaluation of centrosomal GFP-fluorescence. (E and E′) GFP-DdCP224 underexpression in A3-mutants (E′) leads to less and shorter microtubules compared with wild-type cells (WT; strain AX2) (E).
Figure 3.
Figure 3.
DdCP224 is required for microtubule growth. Comparison of microtubule cytoskeletons in GFP-DdCP224–underexpressing A3-mutants and wild-type cells (WT; strain AX2) grown in Klebsiella aerogenes-suspension culture. Cells were either untreated (no noco.) or treated with nocodazole. Cells were fixed without washout of the drug or 10 and 20 min after drug removal, respectively. (A–H) Indirect immunofluorescence microscopy showing microtubules stained with the α-tubulin antibody YL1/2 and nuclei stained with DAPI. (A′–H′) Pie diagrams illustrating percentages of cells with no microtubules (light gray), an incomplete microtubule cytoskeleton with very short microtubules (dark gray), and a complete microtubule cytoskeleton with normal microtubules (i.e., as in untreated cells; A and B). The total number of cells counted for each condition is given within each diagram. One representative experiment is shown (n = 3).
Figure 4.
Figure 4.
Supernumerary MTOCs are bona fide centrosomes. Representative electron microscopic image showing one nucleus-associated centrosome on the left and one supernumerary centrosome on the right. The two insets in the upper right corner illustrate that both centrosomes contain all ultrastructural features of a bona fide Dictyostelium centrosome. N, nucleus; CS, layered core structure; Co, corona; MT, microtubules. Bar, 0.5 μm.
Figure 5.
Figure 5.
Live observation (Movie 1) of normal mitosis in a mitotic DdCP224-GFP/GFP-histone2B cell. Image acquisition started in prophase. GFP-histone2B labels chromosomes and DdCP224-GFP labels centrosomes and the midbody region. The time is indicated in seconds. Each image represents a brightest point z-projection of five confocal slices with a distance of 1 μm each. Images were acquired at the Ultraview spinning disk confocal microscope.
Figure 6.
Figure 6.
Duplication of supernumerary centrosomes. Live observation (Movie 2) of a mitotic DdCP224-GFP/GFP-histone2B cell with initially one supernumerary centrosome. Image acquisition started in metaphase after duplication of the nucleus associated centrosome. The time is indicated in seconds. Each image represents a brightest point z-projection of five confocal slices with a distance of 0.5 μm each. Images were acquired at the Ultraview spinning disk confocal microscope.
Figure 7.
Figure 7.
Supernumerary centrosome elimination by cytoplast formation. An overlay of bright field (blue) and fluorescence images (yellow) is shown. Live observation (Movie 3) of a mitotic DdCP224-GFP/GFP-histone2B cell (labeled with a red margin in the first image) with two supernumerary centrosomes. Image acquisition started in telophase where the central spindle is still present (arrowhead at midbody region). Cytokinesis yields two daughter cells and one cytoplast containing the supernumerary centrosomes. The time is indicated in seconds. Each image represents a brightest point z-projection of five confocal slices with a distance of 1 μm each. Images were acquired on a 510META confocal laser scanning microscope (Carl Zeiss). In Movie3.mov fluorescence images (left) and bright field images (right) were separated.
Figure 8.
Figure 8.
Reduction of supernumerary centrosome number by centrosome coalescence. An overlay of bright field (blue) and fluorescence images (yellow) is shown. Live observation (Movie 4) of a binuclear mitotic DdCP224-GFP/GFP-histone2B cell (labeled with a red margin in the first image) with two supernumerary centrosomes (arrowheads). Image acquisition started in metaphase. The two supernumerary centrosomes fuse into one centrosomal entity in telophase. Cytokinesis yields three daughter cells, one binuclear cell with the single supernumerary centrosome and two normal, mononucleated cells. The time is indicated in seconds. Each image represents a brightest point z-projection of five confocal slices with a distance of 1 μm each. Images were acquired on a 510META confocal laser scanning microscope (Carl Zeiss). In movie4.mov fluorescence images (left) and bright field images (right) were separated.
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