Whi5 is diluted and protein synthesis does not dramatically increase in pre-Start G1

Abstract

Whi5 is diluted and protein synthesis does not dramatically increase in pre-Start G1The Whi5 dilution model requires that the concentration of Whi5 in newborn cells decreases with their cell size at birth, and that dilution by cell growth causes a continuous decrease of Whi5 concentration during G1. While the size-dependent Whi5 concentration at birth is based on an unusual cell size-independent transcriptional mechanism and chromatin-based partitioning (Schmoller et al., 2015;Swaffer et al., 2021), the latter is explained by two key observations. First, Whi5 is a stable protein (Schmoller et al., 2015;Gomar-Alba et al., 2017). Second, transcription of WHI5 mRNA is strongly cell cycle-dependent and peaks in S/G2 (Spellman et al., 1998;Pramila et al., 2006;Granovskaia et al., 2010). Indeed, it has long been known that WHI5 is a cell cycledependent transcript as it ranks among the top 300 most periodically expressed budding yeast genes in the Cyclebase database (Santos et al., 2015). This cell cycle dependence was recently confirmed using single molecule FISH (Qu et al., 2019;Swaffer et al., 2021). Thus, unless translational control perfectly compensates for the transcriptional oscillation, cell growth during G1-the period in which Whi5 mRNA is only weakly expressed-will inevitably decrease Whi5 protein concentration. Importantly, dilution of Whi5 does not imply that there is absolutely no synthesis of Whi5 protein during G1, just that its synthesis rate in G1 is significantly lower than in S/G2/M.Whi5 dilution during pre-Start G1 was observed using live-cell wide-field fluorescence microscopy of Whi5 in asynchronously cycling single cells (Schmoller et al., 2015). This analysis required careful subtraction of background and cell autofluorescence before the total fluorescence intensity of tagged Whi5 can be extracted as a proxy for the total amount of Whi5 protein. Simultaneous estimation of cell volume based on cell segmentations obtained from phase contrast images can then be used to calculate the relative change of Whi5 concentration over time. Using this approach, we found that Whi5 protein concentrations directly reflect its transcriptional regulation. In G1, Whi5 is weakly expressed, which leads to its dilution in this phase of the cell cycle. This is followed by a strong increase in Whi5 synthesis in the budded phase of the cell cycle (Schmoller et al., 2015).

Cell-cycle regulator Whi5 shapes proliferative heterogeneity in clonal populations

“…Specifically, WHI5 was the most overexpressed gene in small microcolonies (García-Martínez et al, 2016). In budding yeast, Whi5 is a G1/S transition inhibitor (Michael Costanzo et al, 2004) that contributes to both, the determination of critical cell size at START and cell fate (Litsios et al, 2022;Palumbo et al, 2016;Schmoller et al, 2015bSchmoller et al, , 2022. We decided to confirm that this correlation between Whi5 high levels and slow-proliferating (small) microcolonies was causative, in the sense that Whi5 levels determined slow proliferation and not vice versa.…”

Whi5 hypo- and hyper-phosphorylation dynamics control cell cycle entry and progression

“…Whi5 WT was shown to be synthesized mostly during S/G2/M phases and then diluted through G1. The dilution of Whi5 in G1 enables cells to sense their size and is thus an important mechanism through which cell growth triggers the G1/S transition [21][22][23]25 . Importantly, the amount of Whi5 synthesized in S/G2/M is largely independent of cell size, and Whi5 is partitioned in nearly equal amounts to the mother and daughter cells 23 .…”

“…In contrast, a similar amount of Whi5 protein is made in rich and poor nutrient conditions 21 . Thus, Whi5 concentration directly reflects cell size prior to Start, even in different growth conditions, and is progressively diluted as cells grow in G1 [21][22][23][24][25] . In this manner, Cln3 and Whi5 transmit distinct growth rate and cell size signals that are integrated at the G1/S transition.…”

What programs the size of animal cells?

“…In contrast to Whi5 dilution, recent single cell measurements suggest that a burst in global protein synthesis in late G1 phase results in the rapid accumulation of the highly unstable G1 cyclin Cln3, which then triggers Start and G1/S transcription by the phosphorylation-dependent inactivation of Whi5 ( Litsios et al, 2019 ; Liu et al, 2015 ; Sommer et al, 2021 ). The Whi5 dilution model remains contentious as the critical underlying quantitative measurements depend heavily on appropriate controls for Whi5 signal loss ( Litsios et al, 2022 ; Schmoller et al, 2022 ). Additional regulators and mechanisms may couple cell size, growth, and Start in budding yeast.…”