2012;8:e1002851. shows that differential size-dependency of protein synthesis can provide an elegant mechanism to coordinate cellular functions with growth. To control size, proliferating cells tie division to growth. However, the molecular mechanisms by which growth triggers division are poorly comprehended3,9,10. In the budding yeast cells. To determine how the G1 regulatory network implements size control, we first examined how the concentration of key regulators changes through G1. We grew cells using ethanol as the carbon source to generate small daughter cells subject to strong cell size control5. We restricted our attention to these daughter cells, and used time lapse microscopy to measure the concentration of proteins tagged with the fluorescent protein mCitrine and expressed from the endogenous locus (Fig. 1b-g; Extended Data Fig. 1a). The concentration of wild-type Cln3 cannot be measured with this approach due to its rapid and constitutive degradation. We therefore examined two mutants expressing stabilized proteins (and (Fig. 2g). Thus, in diploids the biosynthetic machinery is split between the two copies of the genome. Consistently, a hemizygous diploid synthesizes mCitrine-Cln3-11A protein at a much lower rate than a similarly sized haploid or homozygous diploid (Fig. 2g). In sharp contrast, Whi5-mCitrine synthesis is similar and size-independent in hemizygous diploid and haploid cells (Fig. 2f, Extended Data Fig. 4b). Moreover, a homozygous diploid produces Whi5 at approximately twice the rate, similar to a haploid with GNE 0723 two copies of (Fig. 2f, Extended Data Fig. 4b). Thus, the rate of Whi5 synthesis is determined by the number of copies of the gene and is impartial of cell size Enpep and ploidy. While the inhibitor-dilution model takes into account cell-to-cell variability in birth size, it does not yet include the fact that cells given birth to the same size will vary in how much they grow before cells, only a fraction will pass within the short time interval between movie frames. This allows us to define a rate as this fraction divided by the time interval (Fig. 3b; see Methods). In our inhibitor-dilution model, the rate at which GNE 0723 cells pass is determined by the concentrations of Whi5 and Cln3. If Cln3 concentration is constant in pre-cells, the Whi5 concentration alone should predict the rate at which cells progress through background, where Cln3 is usually essential24. As expected, cells made up of 2 and 4 copies of produced proportionally more Whi5 protein, were larger, and exhibited a decreased size-dependent rate of progression through (Fig. 3b, Extended Data Fig. 4c-d). We note that these experiments were performed using cells expressing wild type which is usually suggested to be at constant concentration in G1 based on our measurements of Cln3-11A and Cln3-1. In complete agreement with an inhibitor-dilution model with a size-independent activator, the concentration of Whi5 alone predicts the rate at which cells progress through for all those 3 strains (Fig. 3c). Consistently, the relationship between the rate of progression through and Whi5 concentration was not changed in cells that lack a transcription factor promoting expression22 (Extended Data Fig. 7). Open in a separate window Physique 3 Whi5 concentration determines the rate at which cells progress through daughter cells (n=658). Bars denote mean and standard error. b-c, The GNE 0723 rate at which daughter cells progress through is shown as a function of cell size (b) and Whi5 concentration (c) for haploid cells with one (blue, n=658), two (green, n=310) or four (red, n=142).
