Regulated Expression of the Centrosomal Protein DdCP224 Affects Microtubule Dynamics and Reveals Mechanisms for the Control of Supernumerary Centrosome Number

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. INTRODUCTIONIn dividing cells, control of centrosome number is essential for the fidelity of mitosis and maintenance of euploidy. Supernumerary centrosomes are a hallmark of tumor cells, although it is still a matter of discussion whether their appearance is a cause or a consequence of carcinogenesis (Lingle et al., 2002;Nigg, 2002). However, it is widely accepted that supernumerary centrosomes contribute to the formation of multipolar spindles and thus to defective chromosome segregation. Although most of the daughter cells resulting from such mitotic chaos will die, some of them will acquire the potential for neoplastic growth in spite of supernumerary centrosomes and aneuploidy, as they regain the ability to form a bipolar spindle. Brinkley (2001) suggested two mechanisms how this can be achieved: first, "deamplification" of supernumerary centrosomes by elimination or inactivation, and second, fusion or "coalescence" of supernumerary centrosomes resulting in one or two compound microtubule-organizing centers (MTOCs).Herein, we have studied the fate of supernumerary centrosomes in a simple model system. In Dictyostelium amoebae, one molecular component involved in the formation of supernumerary centrosomes is DdCP224, a member of the XMAP215 family of microtubule-associated proteins (Gräf et al., 2000). In most species, these proteins are long, thin, monomeric molecules with a size of ϳ 230 kDa (Cassimeris et al., 2001). Their ubiquitous occurrence, even in plants, suggests indispensible functions (Ohkura et al., 2001). XMAP215, for instance, is a promoter of microtubule elongation due to a suppression of catastrophe events induced by the Kin I family kinesin ...

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and Cytokinesis

“…During mitosis, DdCP224 localizes at both spindle pole bodies and the spindle midzone (Figure 6, A and B;Graf et al, 2000Graf et al, , 2003. We found that DdCP224 localized normally at the interphase centrosomes in the DdINCENP null cells.…”

“…We found that some DdINCENP null cells had serious chromosome segregation defects; some of them were arrested at metaphase (Video 3), whereas some others had lagging chromosomes (Figure 4 and Video 4). The behavior of chromosomes in the DdINCENP null cells contrasted sharply with that of wild-type cells, which always displayed normal segregation of chromosomes (Graf et al, 2003; Figure 4; see Video 5). The mitotic and the cytokinetic defects displayed by the DdINCENP null cells were consistent with the observation in other organisms that INCENP plays important roles during mitosis (Kaitna et al, 2000;Adams et al, 2001b).…”

Differential Effects of Heterochromatin Protein 1 Isoforms on Mitotic Chromosome Distribution and Growth in Dictyostelium discoideum

“…These structures contain the kinetochores/centromeres of Dictyostelium chromosomes (43). Centrosomes have previously been characterized by electron microscopy and immunostaining with specific monoclonal antibodies, like DdCP224 (see below) (19).…”

“…Mitoses were identified by antibodies directed either against DdCP224, a Dictyostelium XMAP215 homologue that stains the centrosome and the mitotic spindle (18,19), or against ␣-tubulin (28).…”

DictyosteliumAurora Kinase Has Properties of both Aurora A and Aurora B Kinases

“…7,2008 ANCESTRAL AURORA KINASE FROM DICTYOSTELIUM 897 centrosomes subsequently nucleate an intranuclear spindle. DdCP224, an ortholog of XMAP215, regulates microtubule dynamics (26) and is colocalized with ␥-tubulin exactly at the mitotic spindle poles (24). Close observation of the double-stained wild-type cells revealed that, rather than displaying an overlapping colocalization with DdCP224, DdAurora localized in close proximity but either distal to DdCP224 during prometaphase and metaphase (Fig.…”