Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number
Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number
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DOI：
10.1091/mbc.e03-04-0242
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发表时间：
2003-10-01
影响因子：
3.3
通讯作者：
Rehberg, M
中科院分区：
文献类型：
--
作者：
Gräf, R;Euteneuer, U;Rehberg, M
Dictyostelium XMAP215家族成员DdCP224参与中心体复制和细胞质分裂，并集中在中心体和微管尖端。在此，我们创建了一个DdCP224启动子替代突变体，允许过表达和过表达。过表达导致多余的微管组织中心，并且独立地增加了多核细胞的数量。电镜显示，多余的微管组织中心代表真正的中心体。同样表达绿色荧光蛋白-组蛋白2b作为DNA标记的ddcp224 -绿色荧光蛋白突变体的活细胞成像显示，多余的中心体也能进行细胞周期依赖性复制。相比之下，低表达DdCP224抑制细胞生长，减少星状微管的数量和长度，引起诺可达唑过敏。此外，去除nocodazole后的微管再生依赖于DdCP224。低表达也导致由先前的过表达引起的多余中心体和多核细胞的显著消失。我们首次通过活细胞观察表明，在细胞质分裂过程中，通过中心体融合（合并）或含有多余中心体的细胞质的形成，可以减少多余中心体的数量。
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.