👤 Stefano Di Talia

In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle. Show less

Molecular noise in gene expression can generate substantial variability in protein concentration. However, its effect on the precision of a natural eukaryotic circuit such as the control of cell cycle remains unclear. We use single-cell imaging of fluorescently labelled budding yeast to measure times from division to budding (G1) and from budding to the next division. The variability in G1 decreases with the square root of the ploidy through a 1N/2N/4N ploidy series, consistent with simple stochastic models for molecular noise. Also, increasing the gene dosage of G1 cyclins decreases the variability in G1. A new single-cell reporter for cell protein content allows us to determine the contribution to temporal G1 variability of deterministic size control (that is, smaller cells extending G1). Cell size control contributes significantly to G1 variability in daughter cells but not in mother cells. However, even in daughters, size-independent noise is the largest quantitative contributor to G1 variability. Exit of the transcriptional repressor Whi5 from the nucleus partitions G1 into two temporally uncorrelated and functionally distinct steps. The first step, which depends on the G1 cyclin gene CLN3, corresponds to noisy size control that extends G1 in small daughters, but is of negligible duration in mothers. The second step, whose variability decreases with increasing CLN2 gene dosage, is similar in mothers and daughters. This analysis decomposes the regulatory dynamics of the Start transition into two independent modules, a size sensing module and a timing module, each of which is predominantly controlled by a different G1 cyclin. Show less