Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth

  1. Robert A Sommer
  2. Jerry T DeWitt
  3. Raymond Tan
  4. Douglas R Kellogg

  • Curated by eLife

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    Evaluation Summary:

    This paper examines how cells control their size and will be interesting to scientists studying sizing mechanisms throughout biology. Using yeast cells as a model system, the authors show that an activator of the cell division cycle accumulates as cells grow until a threshold level of activator is achieved. The experiments are performed well, and the high-quality data will be useful for others in the field studying this signaling pathway.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 is thought to initiate cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that control cell growth and size. Together, the data are consistent with models in which Cln3 is a crucial link between cell growth and the cell cycle.

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  1. Version published to 10.7554/elife.64364 on eLife
  2. eLife

    Author Response:

    Reviewer #2:

    This paper investigates cell size-dependent regulation of G1/S cell cycle transition in budding yeast, with a focus on the relationship between the activator Cln3 and the inhibitor Whi5. A prominent 2015 paper proposed that cell growth dilutes the inhibitor Whi5 while Cln3 levels remain constant. This 'inhibitor dilution' model has been challenged by several recent papers. In the present paper, Sommer et al. perform a series of quantitative western blots of whole cell extracts from synchronized cell cultures. They show that Cln3 concentration increases 10-fold before bud emergence (i.e. G1/S) but Whi5 concentration is largely constant, at least in rich media. Similar results were obtained in poor carbon media with a smaller increase in Cln3. These data argue against the inhibitor-dilution model and indicate that Cln3 levels are tuned by carbon availability and cell growth rate. Interestingly, Cln3 increases are not dependent on actin-based growth or bud emergence, but rather depend on membrane trafficking and TORC-SGK signaling. A series of experiments altering ceramide synthesis identify a link with Cln3 synthesis, although it remains unclear how directly this ceramide-Cln3 connection occurs.

    The combination of results in this paper represent a significant contribution to the field. Major strengths include the careful quantitation of Whi5/Cln3 levels, and the clear effects on Cln3 from membrane trafficking events. I also appreciated the balanced tone of the text, which describes the strengths and weaknesses of each experiment and interpretation. I have a series of comments/concerns that could be addressed to strengthen the paper, as described below.

    1. I understand why cells were pre-grown in poor carbon media for these experiments, but it seems important to know how Cln3 and Whi5 levels change for cells pre-grown in rich media. Otherwise, each paper reporting different results for Cln3/Whi5 could be dismissed as using a unique set of growth conditions. Along these lines, it would be ideal for the authors to test Cln3/Whi5 levels in their western blot assay using the same strain background and media as the Schmoller paper. It would be very interesting if the inhibitor-dilution model were observed under these conditions, whereas alternative mechanisms like Cln3 accumulation were observed under other conditions.

    We attempted to grow cells in YPD, isolate small unbudded cells, and then release the cells back into YPD. However, we found that it was not possible to isolate a uniform population of small unbudded cells under these conditions. The problem is that very little growth occurs in G1 phase in YPD so that newly born cells are nearly the same size as mother cells (PMID: 28939614). This, combined with the normal variation in cell size observed in wild type yeast, means that elutriation yields a mix of unbudded and budded cells. Others have faced the same problem (PMID: 31685990, 10728640). The fact that so little growth occurs in G1 phase in YPD is an additional argument against the idea that dilution of Whi5 plays a substantial and general role in cell size control.

    As an alternative, we grew cells in complete synthetic medium (CSM) containing 2% glucose. Under these conditions, cells grow more slowly and are smaller because CSM is limiting for nutrients other than glucose. We isolated small unbudded cells and released them into the same medium so that there would not be shift in carbon source. We found that Cln3 levels increased 3-fold, while Whi5 levels did no change substantially, similar to the effects observed in YP medium containing poor carbon. These data are shown in a new figure (Figure 1 – figure supplement 2). In addition, we have included new text to highlight these issues and how they can influence interpretation of the results.

    We agree that it could be interesting to see how Cln3 and Whi5 behave in the mutant background and media conditions used by Schmoller et al. However, we were concerned that any behavior observed only in the bck2∆ background would say more about the effects of bck2∆ on accumulation of Whi5/Cln3 than it would about how cell size control works in wild type cells. Therefore, to limit the number of time-intensive elutriation experiments that we needed to complete the manuscript we would prefer to leave this experiment for others to complete if they are interested.

    1. The authors over-express WHI5 to test the inhibitor-dilution. Their results dovetail with a recent study from the Murray lab (Barber et al., PNAS) suggesting that cells are not very sensitive to Whi5 levels. However, one can envision mechanisms (e.g. PTMs) that inhibit Whi5 molecules when expressed beyond their physiological concentration. Instead, it would be interesting to know what happens in WHI5/whi5 heterozygous diploid mutants that cut Whi5 levels in half. Perhaps this experiment exists in the literature, but it would be an ideal setting for the authors to perturb the inhibitor-activator ratio, and test Cln3/Whi5 protein levels along with cell size in synchronized cultures.

    We were not able to find an analysis of the size of WHI5/whi5∆ cells in the published literature. We carried out the analysis and the data are shown in a new figure panel (Figure 3C). The effect is small – deletion of one copy of WHI5 in a diploid strain caused only a 0.9% decrease in median cell size. These data nicely complement the data showing little effect of 2xWHI5 on cell size. We were surprised that we did not think to do this simple experiment, and we were also surprised that we couldn’t find it in the literature. We thank the reviewer for suggesting the experiment. Since the heterozygous WHI5/whi5∆ cells showed minimal size defects, we have not elutriated the strain to test for changes in the Cln3/Whi5 ratio.

    1. I found the result in Figure 5E very correlative and hard to interpret. For example, Ypk1 phosphorylation is lost at 2.5 min, but Cln3 levels seem unaffected at this timepoint and the next (?). I would suggest softening the (already soft) tone of explaining these results. In general, the connection between ceramide synthesis and Cln3 levels remains quite unclear to me.

    We agree that our interpretation of the data in Figure 6E was confusing in the original version. Part of the confusion may arise from a lack of clarity in our writing and in the literature about the different phosphorylation inputs into Ypk1/2. The literature suggests that changes in the electrophoretic mobility of Ypk1 could be due largely to the Fpk1/2 kinases. TORC2 also influences Ypk1/2 phosphorylation, as detected by a phosphospecific antibody, but it remains unclear whether TORC2 also influences the electrophoretic mobility of Ypk1/2. The data suggest that the phosphorylation of Ypk1/2 that can be detected via electrophoretic mobility shifts is correlated with Cln3 levels, while TORC2-dependent phosphorylation with a phosphospecific antibody is not well correlated with Cln3 levels. We have edited the manuscript to make this more clear and to clarify what can and cannot be concluded from the data.

    1. The text would need to describe a potential role for protein localization in this pathway. All the results come from cell extracts, whereas local protein concentration in the nucleus could be changing and impact the pathway.

    The last three paragraphs of the Discussion include a discussion of potential roles for protein localization in the context of data from our work and previous studies that point to a potential role for localization of Ypk1/2 and Cln3 to the endoplasmic reticulum. In addition, we added the following sentence to the Results section to highlight potential localization issues: "Population level analysis of Cln3 and Whi5 protein levels by western blotting could miss changes in Whi5 or Cln3 concentration driven by changes in localization to specific subcellular compartments.”

  3. eLife

    Evaluation Summary:

    This paper examines how cells control their size and will be interesting to scientists studying sizing mechanisms throughout biology. Using yeast cells as a model system, the authors show that an activator of the cell division cycle accumulates as cells grow until a threshold level of activator is achieved. The experiments are performed well, and the high-quality data will be useful for others in the field studying this signaling pathway.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This study sets out to critically test two competing models of Start activation, namely an increase in the Start activator Cln3 versus a decrease in the Start repressor Whi5 as cells progress through G1 phase. Evidence has been previously published in support of both models by different groups. The authors use a dual tagged strain (CLN3-HA6, WHI5-HA3) in which both Cln3 and Whi5 can be detected on the same blot with the same antibody to enable parallel quantitative measurement of each protein in the same samples. To follow each protein as a function of cell size and cell cycle position, early G1 phase fractions of small cells are obtained by elutriation followed by inoculation into either fresh rich or poor nutrient medium. Many replicates of these elutriation experiments in different contexts show that Cln3 levels rise substantially as cells approach Start in both rich and poor nutrient conditions (about 10X and 2.5X respectively for absolute amount per cell, 7X and 2X for calculated concentrations), whereas Whi5 levels remain roughly constant throughout G1 phase in both nutrient conditions. The decrease in Cln3 occurs within minutes of a shift into poor medium, consistent with a role for Cln3 in coupling growth to division. To further test the Whi5 dilution model, the authors re-examine the genetic effects of altered WHI5 gene dosage and show that doubling the WHI5 dosage has no effect on size in wild type cells. Even very high level overexpression of WHI5 from the TEF1 or GAL1 promoters has only a modest effect on size, measured on both asynchronous and synchronized cultures. The authors then test various ways to interfere with growth in G1 phase and find that inactivation of the secretory factor SEC7 by an auxin inducible degron completely halts growth and the accumulation of Cln3, but without any apparent effect on bulk protein synthesis. The dependence of size and Cln3 accumulation on different candidate upstream kinases is then evaluated. The authors find that the redundant Yck1 and Yck2 kinases are partially required for cell growth in G1 phase and completely necessary for Cln3 accumulation in both rich and poor nutrient conditions. Cln3 accumulation thus fails to occur in inhibitor treated ypk1-as ypk2 cells. Consistently, Ypk1/2 activity as measured by Thr662 phosphorylation is rapidly lost upon shift of cells from rich to poor nutrient conditions. The loss of Ypk1/2 does not appear to affect bulk protein synthesis, suggesting that Ypk1/2 may couple growth to Cln3 accumulation. Finally, given the known role if Ypk1/2 in sphingolipid biosynthesis, the authors test for effects of sphingolipid synthesis inhibitors. The inhibitor myriocin reduces growth rate/budding onset and delays Start but only modestly attenuates Cln3 accumulation. However, genetic inhibition of ceramide production in a lac1 lag1 strain dramatically reduces Cln3 levels. Addition of exogenous phytosphingosine similarly reduces Cln3 levels, particularly in the lac1 lag1 strain. From this result, the authors conclude that feedback inhibition of Ypk1/2 by sphingosine, either due to a block in conversion to ceramides or addition of excess exogenous sphingosine, blocks the accumulation of Cln3. Overall, this study helps to resolve the on-going controversy regarding the mechanism of Start activation in budding yeast and adds new insight into Cln3 regulation by Yck1/2.

  5. eLife

    Reviewer #2 (Public Review):

    This paper investigates cell size-dependent regulation of G1/S cell cycle transition in budding yeast, with a focus on the relationship between the activator Cln3 and the inhibitor Whi5. A prominent 2015 paper proposed that cell growth dilutes the inhibitor Whi5 while Cln3 levels remain constant. This 'inhibitor dilution' model has been challenged by several recent papers. In the present paper, Sommer et al. perform a series of quantitative western blots of whole cell extracts from synchronized cell cultures. They show that Cln3 concentration increases 10-fold before bud emergence (i.e. G1/S) but Whi5 concentration is largely constant, at least in rich media. Similar results were obtained in poor carbon media with a smaller increase in Cln3. These data argue against the inhibitor-dilution model and indicate that Cln3 levels are tuned by carbon availability and cell growth rate. Interestingly, Cln3 increases are not dependent on actin-based growth or bud emergence, but rather depend on membrane trafficking and TORC-SGK signaling. A series of experiments altering ceramide synthesis identify a link with Cln3 synthesis, although it remains unclear how directly this ceramide-Cln3 connection occurs.

    The combination of results in this paper represent a significant contribution to the field. Major strengths include the careful quantitation of Whi5/Cln3 levels, and the clear effects on Cln3 from membrane trafficking events. I also appreciated the balanced tone of the text, which describes the strengths and weaknesses of each experiment and interpretation. I have a series of comments/concerns that could be addressed to strengthen the paper, as described below.

    1. I understand why cells were pre-grown in poor carbon media for these experiments, but it seems important to know how Cln3 and Whi5 levels change for cells pre-grown in rich media. Otherwise, each paper reporting different results for Cln3/Whi5 could be dismissed as using a unique set of growth conditions. Along these lines, it would be ideal for the authors to test Cln3/Whi5 levels in their western blot assay using the same strain background and media as the Schmoller paper. It would be very interesting if the inhibitor-dilution model were observed under these conditions, whereas alternative mechanisms like Cln3 accumulation were observed under other conditions.

    2. The authors over-express WHI5 to test the inhibitor-dilution. Their results dovetail with a recent study from the Murray lab (Barber et al., PNAS) suggesting that cells are not very sensitive to Whi5 levels. However, one can envision mechanisms (e.g. PTMs) that inhibit Whi5 molecules when expressed beyond their physiological concentration. Instead, it would be interesting to know what happens in WHI5/whi5 heterozygous diploid mutants that cut Whi5 levels in half. Perhaps this experiment exists in the literature, but it would be an ideal setting for the authors to perturb the inhibitor-activator ratio, and test Cln3/Whi5 protein levels along with cell size in synchronized cultures.

    3. I found the result in Figure 5E very correlative and hard to interpret. For example, Ypk1 phosphorylation is lost at 2.5 min, but Cln3 levels seem unaffected at this timepoint and the next (?). I would suggest softening the (already soft) tone of explaining these results. In general, the connection between ceramide synthesis and Cln3 levels remains quite unclear to me.

    4. The text would need to describe a potential role for protein localization in this pathway. All the results come from cell extracts, whereas local protein concentration in the nucleus could be changing and impact the pathway.

  6. eLife

    Reviewer #3 (Public Review):

    In this manuscript Sommer et al. investigate how cell growth in G1 induces cell cycle entry in budding yeast. This mechanism is fundamental to cell size homeostasis in eukaryotes and has been investigated for decades. Several models have been proposed, including a Cln3 accumulation model and a Whi5 dilution model, but conclusive evidence has been lacking mainly because Cln3 is of low abundance and difficult to detect in cells. Using a 6xHA tagged version of Cln3 and centrifugal elutriation to isolate early G1 cells, the authors are able to detect Cln3 on western blots and show that its levels increase during G1 while Whi5 levels remains nearly constant. Importantly, Cln3 levels rise more abruptly (10-fold in rich, 2.4-fold in poor carbon medium) than cell volume (1.7-fold in rich, 1.2-fold in poor carbon medium) leading to an increase in Cln3 concentration (7-fold in rich, 2.2 in poor medium) that peaks at Start. Preventing the advertised Whi5 dilution by doubling WHI5 gene dosage did not change cell size distribution (Fig. 3). These observations clearly favour the Cln3 accumulation over the Whi5 dilution model, which confirms other reports (Litsios et al., 2019; Barber et al., 2020). Interestingly, Cln3 levels drop abruptly within minutes when cells are shifted from rich to poor carbon sources (as seen previously by Perviz et al., 1998 and Hall et al., 1998), while Whi5 levels increase slightly (Fig. 2). This creates a conundrum because cells grown in poor carbon medium initiate Start at a smaller cell size despite a stark reduction of the Cln3/Whi5 ratio. The authors also looked at what might regulate cell growth and Cln3 accumulation during G1. They find that cell growth in G1 is independent of Cdk1, Pho85 and of actin or tubulin (Fig.4A and Fig.5_Sup1), but dependent on ER-Golgi trafficking (Sec7), ceramide biosynthesis (Lac1, Lag1) and the TORC2-regulated Ypk1,2 kinases. In all cases, reduced growth in G1 is accompanied by lower Cln3 levels, indicating that sustained growth might be necessary for strong Cln3 accumulation. These data bring new players to the Cln3 accumulation model.

    The experiments are in most cases well designed and performed, and the main conclusions supported by the data. Using population-based methods and western-blotting, the authors succeed at demonstrating that Cln3 levels rise during G1, in contrast to early assumptions, but which is something that has been documented previously by the same (Zapata et al. 2014) and other labs (Thornton et al., 2013). They convincingly demonstrate that Whi5 dilution during G1, as proposed by Schmoller et al. (2015), cannot be responsible for setting the critical cell size at budding. Similar conclusions were drawn by others using single-cell imaging (Litsios et al., 2019) or ectopic WHI5 expression (Barber et al., 2020). Regrettably, most studies on the coupling of cell entry to cell growth focus on Cln3 and Whi5 levels, not activity embodied by Whi5 phosphorylation's status. The key question for how cells growing on poor carbon medium reach Start at a smaller cell size despite much reduced Cln3 levels remains unanswered by the authors, and a paper (Talarek et al., 2017) proposing down-regulation of the Whi5 phosphatase PP2A-Cdc55 by the Rim15-Igo1,2 pathway to trigger Start when Cln3 levels are low in poor medium is not cited. The identification of Sec7, Ypk1,2 and Lac1,Lag1 as regulators of growth in G1 is interesting, but the link to Cln3 accumulation is not explained by a coherent model.

  7. Version published to 10.1101/2020.09.30.321182v2 on bioRxiv
  8. Version published to 10.1101/2020.09.30.321182v1 on bioRxiv