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

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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.