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Originally published as MBC in Press, 10.1091/mbc.E03-04-0242 on June 13, 2003

Vol. 14, Issue 10, 4067-4074, October 2003

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

Ralph Gräf *, Ursula Euteneuer, Thi-Hieu Ho, and Markus Rehberg

Adolf-Butenandt-Institut/Zellbiologie, Universität München, D-80336 Munich, Germany

Submitted April 17, 2003; Accepted May 28, 2003
Monitoring Editor: J. Richard McIntosh

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.

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03–04–0242. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03-04-0242.

Online version of this article contains video material for some figures. Online version is available at www.molbiolcell.org.

* Corresponding author. E-mail address: rgraef{at}lrz.uni-muenchen.de.




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