Oversized cells activate global proteasome-mediated protein degradation to maintain cell size homeostasis
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Evaluation Summary:
Kafri and colleagues assess the contribution of protein degradation to the cell size-dependent accumulation of total protein. As cells get too big, the efficiency of cell growth decreases, which the authors propose is due to increased protein degradation in larger cells. This is an interesting and novel mechanism, and its discovery is potentially useful for future research on understanding and controlling cell growth, though the data could be further strengthened and clarified to support the conclusions.
(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. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Proliferating animal cells maintain a stable size distribution over generations despite fluctuations in cell growth and division size. This tight control of cell size involves both cell size checkpoints, which delay cell cycle progression in small cells, and size-dependent regulation of mass accumulation rates. While we previously identified the p38 MAPK pathway as a key regulator of the mammalian cell size checkpoint, the mechanism of size-dependent growth rate regulation has remained elusive. Here, we quantified global rates of protein synthesis and degradation in cells of varying sizes, both under unperturbed conditions and in response to perturbations that trigger size-dependent compensatory growth slowdown. We found that protein synthesis rates scale proportionally with cell size across cell cycle stages and experimental conditions. In contrast, oversized cells that undergo compensatory growth slowdown exhibit a superlinear increase in proteasome-mediated protein degradation, with accelerated protein turnover per unit mass, suggesting activation of the proteasomal degradation pathway. Both nascent and long-lived proteins contribute to the elevated protein degradation during compensatory growth slowdown, with long-lived proteins playing a crucial role at the G1/S transition. Notably, large G1/S cells exhibit particularly high efficiency in protein degradation, surpassing that of similarly sized or larger cells in S and G2, coinciding with the timing of the most stringent size control in animal cells. These results collectively suggest that oversized cells reduce their growth efficiency by activating global proteasome-mediated protein degradation to promote cell size homeostasis.
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Author response:
Reviewer #2 (Public Review):
In this manuscript, Kafri and colleagues assess the contribution of protein degradation to the cell size-dependent accumulation of total protein. This is an interesting line of research that has not previously been explored. Most of the focus on the size-dependence of protein accumulation has been on the synthesis part of the equation. As cells get too big, the efficiency of cell growth (mass accumulation per unit mass) decreases. It is argued that this is not due to the loss of the efficiency in protein synthesis, but rather is due to the increased protein degradation in larger cells. It is an interesting hypothesis, that might well be true, but there are some issues with key aspects of the data and other supporting data are quite indirect. More work needs to be done to support the …
Author response:
Reviewer #2 (Public Review):
In this manuscript, Kafri and colleagues assess the contribution of protein degradation to the cell size-dependent accumulation of total protein. This is an interesting line of research that has not previously been explored. Most of the focus on the size-dependence of protein accumulation has been on the synthesis part of the equation. As cells get too big, the efficiency of cell growth (mass accumulation per unit mass) decreases. It is argued that this is not due to the loss of the efficiency in protein synthesis, but rather is due to the increased protein degradation in larger cells. It is an interesting hypothesis, that might well be true, but there are some issues with key aspects of the data and other supporting data are quite indirect. More work needs to be done to support the central claims.
We thank the reviewer for appreciating the work is interesting and previously unexplored.
The major issue is that the data supporting the proportional increase in protein synthesis with cell size need to be strengthened. Protein synthesis is measured by the amount of a methionine analog that is incorporated in a fixed amount of time. Fig. 2 then plots this amount as a function of cell size, which is presumably measured using a total protein dye (this information is not included; incidentally the axis labels should note what the measurement is 'total protein' or 'forward scatter' rather than the more ambiguous 'cell size'). In any case, something is wrong with the cell size measurements in Figure 2 because many cells basically have almost negligible size (near 0) while others have sizes up to 5 or 6 arbitrary units. It makes no sense that there should be a 10-fold or even 100-fold range in cell sizes. For this reason, I can't interpret the data in Figure 2, which is unfortunate since that is a crucial figure for the authors' argument.
The data supporting higher rates of protein degradation per unit mass in large cells suffers from a similar problem as Figure 3E has the same issue as Figure 2 with too many tiny 'cells'.
Yes, the reviewer is correct that we are using a total protein dye (Alexa fluorophore-conjugated succinimidyl ester, abbreviated as SE) to measure cell size. We have included details regarding the methods of cell size (total protein content) measurement in both the Methods (line 463-466) and Results (line 100-102) sections.
Regarding the reviewer’s concern on the cell size range, we apologize for the confusion the figures may have caused. These cell size measurements are within reasonable range and not 10-fold or 100-fold. Please refer to our detailed response above to essential point #1.
Moreover, the reliance on cycloheximide to treat cells and measure reduction in mass isn't ideal since shutting off all protein synthesis is a pretty drastic perturbation. It would have been better to shut off synthesis of a specific protein and measure its degradation in large and small cells while keeping the cells otherwise intact.
We acknowledge that relying on cycloheximide to measure changes in mass has limitations, as acute inhibition in protein synthesis is a significant perturbation. Ideally, we would measure the degradation of specific proteins in large and small cells while keeping the rest of the cellular processes intact. However, this presents considerable technological challenges. While our evidence clearly shows increased protein degradation and compensatory growth slowdown in large cells, we have not yet identified the specific proteins/genes being targeted. Implementing the reviewer's suggestion would require first screening for a suitable protein/gene to serve as a reporter for compensatory degradation. A significant proteomics screen may allow identification of potential targets, but further validation would necessitate substantial effort, including the generation and validation of a reporter system. We agree that this is a valuable experiment to pursue, but it will likely be part of a follow-up study focused on characterizing the specific protein targets and E3 ligases involved in these processes. In the revised manuscript, we discuss these open questions and future directions in line 380-410.
Reviewer #3 (Public Review):
The authors report a previously undocumented role for UPS-mediated protein turnover in size control in human cells. The study builds on previous observations made by the Kafri group that large cells undergo size compensation by reducing their rate of growth. In particular, recent published work by Ginzberg et al showed that CDK2 inhibition is accompanied by long term size compensation in the form of reduced cell growth whereas CDK6 inhibition is not. The authors investigate the basis for this effect and demonstrate in both unperturbed and perturbed growth/division contexts, using both fixed cells and time lapse microscopy, that the rate of protein synthesis increases proportionately in large cells that undergo size compensation even though mass accumulation is attenuated. The authors show that this effect appears to be mediated by increased proteasomal activity, as demonstrated by proteasome-dependent K48-ubiquitin chain turnover. Intriguingly, this degradation-mediated size compensation mechanism appears to be most active at the G1/S transition, the primary point at which size control operates. The experiments are well controlled, and the conclusions of the study are in general well supported by the data. The authors present an interesting set of discussion points that relate their observations to size control mechanisms in dividing and non-dividing cells. While specific mechanisms are not pursued, this study nevertheless adds an important new insight into the still unsolved problem of size control.
We thank the reviewer for appreciating the novelty of the work.
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Evaluation Summary:
Kafri and colleagues assess the contribution of protein degradation to the cell size-dependent accumulation of total protein. As cells get too big, the efficiency of cell growth decreases, which the authors propose is due to increased protein degradation in larger cells. This is an interesting and novel mechanism, and its discovery is potentially useful for future research on understanding and controlling cell growth, though the data could be further strengthened and clarified to support the conclusions.
(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. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this paper, Liu, Kafri and colleagues seek to understand how human RPE1 cells maintain size homeostasis during cell cycle progression. This is a commonly discussed issue in cell biology that has not been resolved. Their primary experimental approach is to partially inhibit CDK2 to slow the cell cycle. Using advanced single cell measurement techniques, they show that this treatment slows the cell cycle during G1 progression, and that the cells first enlarge, but then later continue to divide whilst maintaining a constant size. By tracking cellular protein amounts in single cells, they show that cell growth rates and protein synthesis scale linearly with cell size, as expected, and that normalized growth rate is not different between control and CDK2-inhibited cells (Figs 1, 2). This part of the manuscript, …
Reviewer #1 (Public Review):
In this paper, Liu, Kafri and colleagues seek to understand how human RPE1 cells maintain size homeostasis during cell cycle progression. This is a commonly discussed issue in cell biology that has not been resolved. Their primary experimental approach is to partially inhibit CDK2 to slow the cell cycle. Using advanced single cell measurement techniques, they show that this treatment slows the cell cycle during G1 progression, and that the cells first enlarge, but then later continue to divide whilst maintaining a constant size. By tracking cellular protein amounts in single cells, they show that cell growth rates and protein synthesis scale linearly with cell size, as expected, and that normalized growth rate is not different between control and CDK2-inhibited cells (Figs 1, 2). This part of the manuscript, which sets the stage, is robust and convincing. They go on to present data indicating that rates of protein degradation increase in cells that have become enlarged due to long term CDK2 inhibition (Fig 3, 4). This data is interesting, novel, and consistent with their conclusions that cell enlargement enhances protein degradation. However the presentation was unclear in certain aspects, and some obvious experiments are missing. Despite the use of several innovative tests and a number of interesting provocative results, due to the lack of control experiments the data on protein degradation are insufficient to support the author's conclusions to the degree I'd like to see. Another general issue is that, although the authors attribute the increased protein degradation to cell enlargement, they present very little data from experiments in which cells were enlarged using treatments other than CDK2 inhibition. Including examples of enhanced protein degradation after enlarging cells by alternative means is important to support the authors' conclusions (e.g. in Fig 5), which are very general. Providing examples using alternative modes of cell enlargement is also important to rule out the possibility that CDK2 inhibition directly affects protein turnover rates, for instance by altering degradation substrate phosphorylation. This issue is discussed, but not sufficiently resolved.
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Reviewer #2 (Public Review):
In this manuscript, Kafri and colleagues assess the contribution of protein degradation to the cell size-dependent accumulation of total protein. This is an interesting line of research that has not previously been explored. Most of the focus on the size-dependence of protein accumulation has been on the synthesis part of the equation. As cells get too big, the efficiency of cell growth (mass accumulation per unit mass) decreases. It is argued that this is not due to the loss of the efficiency in protein synthesis, but rather is due to the increased protein degradation in larger cells. It is an interesting hypothesis, that might well be true, but there are some issues with key aspects of the data and other supporting data are quite indirect. More work needs to be done to support the central claims.
The major …
Reviewer #2 (Public Review):
In this manuscript, Kafri and colleagues assess the contribution of protein degradation to the cell size-dependent accumulation of total protein. This is an interesting line of research that has not previously been explored. Most of the focus on the size-dependence of protein accumulation has been on the synthesis part of the equation. As cells get too big, the efficiency of cell growth (mass accumulation per unit mass) decreases. It is argued that this is not due to the loss of the efficiency in protein synthesis, but rather is due to the increased protein degradation in larger cells. It is an interesting hypothesis, that might well be true, but there are some issues with key aspects of the data and other supporting data are quite indirect. More work needs to be done to support the central claims.
The major issue is that the data supporting the proportional increase in protein synthesis with cell size need to be strengthened. Protein synthesis is measured by the amount of a methionine analog that is incorporated in a fixed amount of time. Fig. 2 then plots this amount as a function of cell size, which is presumably measured using a total protein dye (this information is not included; incidentally the axis labels should note what the measurement is 'total protein' or 'forward scatter' rather than the more ambiguous 'cell size'). In any case, something is wrong with the cell size measurements in Figure 2 because many cells basically have almost negligible size (near 0) while others have sizes up to 5 or 6 arbitrary units. It makes no sense that there should be a 10-fold or even 100-fold range in cell sizes. For this reason, I can't interpret the data in Figure 2, which is unfortunate since that is a crucial figure for the authors' argument.
The data supporting higher rates of protein degradation per unit mass in large cells suffers from a similar problem as Figure 3E has the same issue as Figure 2 with too many tiny 'cells'. Moreover, the reliance on cycloheximide to treat cells and measure reduction in mass isn't ideal since shutting off all protein synthesis is a pretty drastic perturbation. It would have been better to shut off synthesis of a specific protein and measure its degradation in large and small cells while keeping the cells otherwise intact.
-
Reviewer #3 (Public Review):
The authors report a previously undocumented role for UPS-mediated protein turnover in size control in human cells. The study builds on previous observations made by the Kafri group that large cells undergo size compensation by reducing their rate of growth. In particular, recent published work by Ginzberg et al showed that CDK2 inhibition is accompanied by long term size compensation in the form of reduced cell growth whereas CDK6 inhibition is not. The authors investigate the basis for this effect and demonstrate in both unperturbed and perturbed growth/division contexts, using both fixed cells and time lapse microscopy, that the rate of protein synthesis increases proportionately in large cells that undergo size compensation even though mass accumulation is attenuated. The authors show that this effect …
Reviewer #3 (Public Review):
The authors report a previously undocumented role for UPS-mediated protein turnover in size control in human cells. The study builds on previous observations made by the Kafri group that large cells undergo size compensation by reducing their rate of growth. In particular, recent published work by Ginzberg et al showed that CDK2 inhibition is accompanied by long term size compensation in the form of reduced cell growth whereas CDK6 inhibition is not. The authors investigate the basis for this effect and demonstrate in both unperturbed and perturbed growth/division contexts, using both fixed cells and time lapse microscopy, that the rate of protein synthesis increases proportionately in large cells that undergo size compensation even though mass accumulation is attenuated. The authors show that this effect appears to be mediated by increased proteasomal activity, as demonstrated by proteasome-dependent K48-ubiquitin chain turnover. Intriguingly, this degradation-mediated size compensation mechanism appears to be most active at the G1/S transition, the primary point at which size control operates. The experiments are well controlled, and the conclusions of the study are in general well supported by the data. The authors present an interesting set of discussion points that relate their observations to size control mechanisms in dividing and non-dividing cells. While specific mechanisms are not pursued, this study nevertheless adds an important new insight into the still unsolved problem of size control.
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