ABSTRACT
Cell cycle control is a central aspect of the biology of proliferating eukaryotic cells. However, progression through the cell cycle relies on a highly complex network, making it difficult to unravel the core design principles underlying the mechanisms that sustain cell proliferation and the ways in which they interact with other cellular pathways. In this context, the use of a synthetic approach to simplify the cell cycle network in unicellular genetic models such as fission yeast has opened the door to studying the biology of proliferating cells from unique perspectives. Here, we provide a series of methods based on a minimal cell cycle module in the fission yeast Schizosaccharomyces pombe that allows for an unprecedented artificial control of cell cycle events, enabling the rewiring and remodeling of cell cycle progression.
Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Schizosaccharomyces/metabolism , Cell Cycle , Cell Division , Cell Cycle Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how CDK regulators coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by developing a CDK activity biosensor, Eevee-spCDK. We find that although CDK activity does not necessarily correlate with cyclin levels, it converges to the same level around mitotic onset in several mutant backgrounds, including pom1Δ cells and wee1 or cdc25 overexpressing cells. These data provide direct evidence that cells enter the M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.
Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Animals , Phosphorylation , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Mitosis , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Mammals/metabolism , Protein-Tyrosine Kinases/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
In poor nitrogen conditions, fission yeast cells mate, undergo meiosis and form spores that are resistant to deleterious environments. Natural isolates of Schizosaccharomyces pombe are homothallic. This allows them to naturally switch between the two h- and h+ mating types with a high frequency, thereby ensuring the presence of both mating partners in a population of cells. However, alteration of the mating type locus can abolish mating type switching or reduce it to a very low frequency. Such heterothallic strains have been isolated and are common in research laboratories due to the simplicity of their use for Mendelian genetics. In addition to the standard laboratory strains, a large collection of natural S. pombe isolates is now available, representing a powerful resource for investigating the genetic diversity and biology of fission yeast. However, most of these strains are homothallic, and only tedious or mutagenic strategies have been described to obtain heterothallic cells from a homothallic parent. Here, we describe a simple approach to generate heterothallic strains. It takes advantage of an alteration of the mating type locus that was previously identified in a mating type switching-deficient strain and the CRISPR-Cas9 editing tool, allowing for a one-step engineering of heterothallic cells with high efficiency.