Prigozhina Natalie L, Oakley C Elizabeth, Lewis Amanda M, Nayak Tania, Osmani Stephen A, Oakley Berl R
Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, USA.
Mol Biol Cell. 2004 Mar;15(3):1374-86. doi: 10.1091/mbc.e03-06-0405. Epub 2003 Dec 10.
Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.
来自多种生物体的最新数据表明，γ-微管蛋白除了在微管组装成核过程中发挥作用外，还具有重要但尚未完全明确的功能。为了研究这些功能，我们检测了米曲霉γ-微管蛋白基因的冷敏感等位基因mipAD159的表型。对同步化材料进行免疫荧光显微镜观察发现，在限制温度下，mipAD159并不抑制有丝分裂纺锤体的形成。然而，许多细胞核中的后期A受到抑制，并且在有丝分裂稍有延迟（约占细胞周期的6%）后，大多数细胞核重新进入间期而未进行分裂。在限制温度下对染色体进行的体内观察表明，mipAD159导致有丝分裂后期事件（后期A、后期B和染色体分离）的协调失败，并且即使有丝分裂未成功完成，细胞核也会迅速重新进入间期。延时显微镜观察还发现，与对照相比，mipAD159菌株中短暂的有丝分裂纺锤体异常，特别是弯曲的纺锤体更为普遍。在用苯菌灵使微管解聚的实验中，mipAD159细胞核从有丝分裂中退出的速度明显比对照菌株中的细胞核快（根据染色体凝聚判断）。这些数据表明，γ-微管蛋白在有丝分裂后期事件的协调中起重要作用，并且在有丝分裂检查点控制中具有不依赖微管的功能。
