Evolution of cell cycle control: triangulating the last eukaryotic common ancestor

项目摘要 酵母和动物（“后鞭毛体”）中的细胞周期控制在广泛的原则以及 特定的保守机制。然而，在真核生物进化中，酵母与动物的关系更为密切 而不是其他真核生物因此，后齿兽的见解可能不适用于早期- 植物王国是地球上生命不可或缺的组成部分。具有重要 理解非后角类真核生物细胞周期控制的意义， 重要的王国，并阐明细胞周期控制的进化，从最后的真核生物共同的， 祖先，阐明了细胞周期控制的哪些特征是真正的祖先和根本。 在这里，我建议使用一种微生物“植物”，即绿色莱茵衣藻，来进行 广谱基因筛选，从分子水平鉴定细胞周期调控基因。我们将利用这些变种人 进行功能分析，以确定与后鞭毛范式的相似性和差异。那里 在这种背景下研究衣原体有两个原因：（1）它与陆地植物的关系更密切 比其他微生物模型;（2）它提供了一个模式生物，以研究古代的途径，失去了在 真菌谱系由于快速进化，如纤毛，G1/S控制网络（细胞周期蛋白D，Rb，E2 F/DP）。 为了解决这个问题，我们已经开发了一个有效的管道隔离，识别和分析 细胞周期控制基因的条件突变。该程序将经典遗传学与机器人技术相结合， 用于快速有效分子鉴定的新一代测序和新型生物信息学方法 数百个必需基因（迄今已确定约150个，还有更多的基因正在研究中）。 突变体筛选是深入分析特定生物学途径的先决条件， 基于简单的表型分析的填充的“部件”列表和初始功能分类。随着项目 进展，除了旨在全面的细胞周期收集，我们正在关注特定的基因 和途径，优先考虑那些植物王国特有的。我们将研究细胞周期蛋白-Cdk-APC 控制系统，其中我们的结果在衣原体和以前的结果在陆地植物表明， 保守性，但也与酵母/动物模型显著不同。此外，我们还将与 描述衣原体胞质分裂的机制。酵母菌/动物外的胞质分裂进行 没有肌动球蛋白收缩环。我们有证据表明已经获得的突变体 肌动蛋白和肌动蛋白相互作用成分的作用。为了实现这两个目标，我们将用荧光标记关键蛋白质。 表位，以允许在延时显微镜下在野生型和适当突变体中进行亚细胞定位 背景，并通过细胞周期分析蛋白质丰度和功能的调节。集群 突变体已经揭示了将被进一步研究的重要途径，包括控制 细胞周期定型、有丝分裂进程和胞质分裂。

Project summary Cell cycle control in yeast and animals (`Opisthokonts') is well understood, in broad principles as well as specific conserved mechanisms. However, in eukaryotic evolution, yeasts are more closely related to animals than to other eukaryotic kingdoms. Therefore, insights from Opisthokonts might apply poorly to earlier- diverging branches such as the plant kingdom, which is absolutely essential to life on earth. It is of great significance to understand cell cycle control in non-Opisthokont eukaryotes, both to understand these important kingdoms, and to elucidate the evolution of cell cycle control from the last eukaryotic common ancestor, illuminating what features of cell cycle control are truly ancestral and fundamental. Here I propose use of a microbial `plant', the green alga Chlamydomonas reinhardtii, to carry out a broad-spectrum genetic screen to molecularly identify cell cycle control genes. We will use these mutants to carry out functional analysis to determine similarities and differences from the Opisthokont paradigm. There are two reasons for studying Chlamydomonas in this context: (1) it's much more closely related to land plants than other microbial models; (2) it provides a model organism to study ancient pathways that were lost in fungal lineages due to rapid evolution, such as cilia, the G1/S control network (cyclin D, Rb, E2F/DP). To attack this problem, we have developed an efficient pipeline for isolation, identification and analysis of conditional mutations in cell cycle control genes. The procedures integrate classical genetics with robotics, next-generation sequencing and novel bioinformatics approaches for rapid and efficient molecular identification of hundreds of essential genes (~150 identified to date, with many more in the pipeline). Mutant screens are prerequisite for in-depth analysis of specific biological pathways, to provide a well- populated `parts' list and initial functional classification based on simple phenotypic assays. As the project progresses, in addition to aiming for a comprehensive cell cycle collection, we are focusing on specific genes and pathways, with priority to those that are plant-kingdom-specific. We will examine the cyclin-Cdk-APC control system, where our results in Chlamydomonas and previous results in land plants indicate substantial conservation but also significant divergence from the yeast/animal model. In addition, we will collaborate to characterize mechanisms of Chlamydomonas cytokinesis. Cytokinesis outside of yeast/animals proceeds without an actomyosin contractile ring. We have evidence from characterizing mutants already obtained for the role of actin and actin-interacting components. For both these aims, we will tag key proteins with fluorescent epitopes to allow subcellular localization in time-lapse microscopy, in wild type and appropriate mutant backgrounds, and analyze regulation of protein abundance and function through the cell cycle. Clusters of mutants have already revealed essential pathways that will be further studied, including pathways controlling cell cycle commitment, mitotic progression and cytokinesis.