Control and maintenance of mammalian cell size: Rejoinder
Stephen Cooper, University of Michigan Medical School
30 November 2004
Control and maintenance of mammalian cell size: Rejoinder
Stephen Cooper, Department of Microbiology and Immunonlogy, University of Michigan Medical School, Ann Arbor, MI 48109-0620 USA (cooper@umich.edu)
I appreciate the reply by Drs. Conlon and Raff to my article on Control and Maintenance of Cell Size [1]. All of the comments and points raised in their Reply are answered in the original article. That article [1] did deal with the conclusions they drew and the arguments they did make. I trust that the readers interested in this subject will read the original Conlon/Raff article [2] and my critique [1] to understand the issues. However, for the sake of clarity and efficiency I will comment on new issues raised by the reply so that the differences are clearly joined.
Regarding the comment that I use the term “cell growth” ambiguously, that quote is taken out of context. Thus, the question noted by Drs. Conlon and Raff occurs in the following context [1] where I write:
“The key question studied by Conlon and Raff asks, "How do cells maintain a constant cell size and cell size distribution during extended cell growth?" In a cell culture growing over many generations, the cell size distribution neither varies nor broadens. Cells do not get progressively larger nor do they get progressively smaller. One formulation of this result is that cell mass increase is regulated during the cell cycle so that there is no disparity between the rate of cell mass increase and the rate of cell number increase. Total cell number and total cell mass increase in parallel during unlimited exponential growth. If there were any disparity or disproportion in the rate of mass and cell number increase, cells would get either larger or smaller during extended growth.”
It is quite clear that from this context that the question described by Drs. Conlon and Raff, is unambiguously discussing cell growth and cell number increase over many generations.
Regarding their statement that I attribute the notion of linear growth incorrectly to their experiments, I can only point out that in Figure 4 of my paper [1] the attribution of linear growth to successive cell cycles is not possible if cell size constancy is to be maintained. Thus, in the caption to Figure 4 it is written: “It is not proposed that mass increases linearly, but merely that even linear synthesis should exhibit, in an uninhibited situation, exponential mass growth.” Linear growth is only logically possible as a topic of discussion within the cell cycle. Over many generations linear growth is logically impossible if cell size is maintained relatively constant.
Regarding “synchronization” at the start of S phase with aphidicolin, I can only point out that this method cannot synchronize cells. The details of this argument are presented in recent papers and I recommend the reader to these papers [3-7].
Regarding the “shift-up” and other experimental manipulations, I refer the reader to the original paper. The slow part of the shift-up is explained clearly in Figure 10 and I hope that the reader will read this caption and the associated text. In particular I note the point made with regard to Figure 10 that: “…one can postulate that for reasons unrelated to the cell cycle but merely related to cellular metabolism occurring continuously throughout the cell cycle, the change in external conditions does not immediately lead to the new rate of mass increase.” This is a simple explanation for the delayed size change during a shift-up.
The IGF-1/GGF experiments [8] are very complex and would require a lengthy discussion to point out all of the problems with this work However, in this space let me briefly discuss the experiments and analyses related to the proposal that one can dissociate cell growth and “progression through the cell cycle”.
1. There is a confusion of the notion of “cell growth” with “cell size”. This is best seen in the analysis of their conclusion that “…GGF stimulated entry into S phase, even though it did not promote cell growth.” Cell growth in this case was determined (Fig. 2 in [8]) by looking at cell size distributions before and after a treatment. But merely because cell size did not change from one point to a second point does not lead to a conclusion that cell growth did not occur. Cell size is determined by a combination of cell mass increase and cell division. A constant cell size may therefore mask true mass increase (leading to initiation of S phase) with an associated division (not observed because only beginning and end point were determined).
2. It is suggested [8] that “as the total cell-cycle time is longer in low GGF than in high GGF [i.e., Fig. 3 shows a greater cell number increase in high GGF than in low GGF] a higher proportion of the cells in low GGF would therefore be expected to be in S and G2.” One experimental result is therefore viewed as a paradox as it is observed, by FACS analysis, that “…the proportion of cells in G1 was lower rather than higher in low concentrations of GGF [compared to high GGF].” My analysis of the relationship of growth rate and proportion of cells with a G1-phase amount of DNA predicts that slower cells should have more cells with a G1-phase amount of DNA. This is simply because S and G2-phase times are relatively invariant, and increasing the interdivision time increases the time for G1-phase disproportionately [9]
3. The observation that only half of cells at maximal induction of initiation into S phase are found to replicate DNA suggests that the cells are quite heterogeneous and not suitable for analysis of the problem.
4. The use of primary Schwann cells to study these questions when there are so many unknowns can only lead to confusion. A discussion of why such cells are inappropriate for analysis has been published ([10] (this paper can be read at my web site at www.umich.edu/~cooper).
In summary, I hope that the readers of the reply by Drs. Conlon and Raff [11] compare their views with the views in the original article [1].
1. Cooper S: Control and Maintenance of Mammalian Cell Size. BMC Cell Biol 2004, 5:35.
2. Conlon I, Raff M: Differences in the mammalian cell and yeast cells coordinate cell growth and cell-cycle progression. J Biol 2003, 2:7.
3. Cooper S: Is Whole-culture synchronization Biology's "Perpetual Motion Machine"? Trends in Biotechnology 2004, 26:266-269.
4. Cooper S: Whole-culture Synchronization Can Not, and Does Not, Synchronize Cells. Trends in Biotechnology 2004, 22:274-276.
5. Cooper S: Rethinking synchronization of mammalian cells for cell-cycle analysis. Cell Mol Life Sci 2003, 6:1099-1106.
6. Cooper S: Reappraisal of serum starvation, the restriction point, G0, and G1-phase arrest points. FASEB J 2003, 17:333-340.
7. Cooper S: Mammalian cells are not synchronized in G1-phase by starvation or inhibition: considerations of the fundamental concept of G1-phase synchronization. Cell Prolif 1998, 31:9-16.
8. Conlon IJ, Dunn GA, Mudge AW, Raff MC: Extracellular control of cell size. Nat Cell Biol 2001, 3:918-921.
9. Cooper S: On the interpretation of the shortening of the G1-phase by overexpression of cyclins in mammalian cells. Exp Cell Res 1998, 238:110-115.
10. Cooper S: Toward a standard system for the mammalian cell cycle. ASM News 2000, 66:71-75.
11. Conlon I, Raff M: Control and maintenance of mammalian cell size: Response. BMC Cell Biol 2004, 5.
Competing interests
No competing interests not indicated in this comment
Brooks' hypothesis on size control
Akos Sveiczer, Budapest University of Technology and Economics
3 March 2005
Nearly two years ago, Conlon and Raff (J Biol 2003, 2:7) proposed that mammalian cells do not need any checkpoint to maintain size homeostasis. An "evidence" was that cell volume increased linearly, irrespectively of cell size, under an S-phase arrest. Conlon and Raff referred to an old and clever (albeit never proved) hypothesis of Brooks (in The Cell Cycle. Edited by John PCL. Cambridge: Cambridge University Press; 1981), arguing that linearly growing cells not necessarily require any size control mechanism. Last year, Cooper (BMC Cell Biol 2004, 5:35) and we (Sveiczer A, Novak B, Mitchison JM. Theor Biol Med Model 2004, 1:12) criticised the conclusions of Conlon and Raff. We both pointed out that even if S-phase blocked cells grow linearly, it does not mean that these cells normally grow linearly during their cell cycle. In their response to Cooper here (BMC Cell Biol 2004, 5:36), Conlon and Raff state that they had never spoken about linear growth during the normal cycle, because this question had simply never been addressed by them. At the first sight, this is a smart answer to disclaim the criticism, however, it generates a serious problem. Namely, in his old paper Brooks proposed that the cells grew linearly, irrespectively of their size, therefore they increased their volume by the same amount during the cycle, if the cycle time was the same. In that special case, Brooks could suppose even a large scattering in the cycle time, and division size was finally convergent during consecutive cycles, even after a large perturbation. He was definitely thinking of normal cycles, during which the linear rate of growth extension is independent of both size and of cell cycle stage! As a consequence, I think that the answer given here by Conlon and Raff generates an even larger discrepancy and, afterwards, the authors have two possibilities. One of them is to explain in detail how their data and assumptions fulfil the general requirements, where the Brooks' hypothesis might be valid. The other one is to withdraw their conclusions about the lack of size control in mammalian cells.
Control and maintenance of mammalian cell size: Rejoinder
30 November 2004
Control and maintenance of mammalian cell size: Rejoinder
Stephen Cooper, Department of Microbiology and Immunonlogy, University of Michigan Medical School, Ann Arbor, MI 48109-0620 USA (cooper@umich.edu)
I appreciate the reply by Drs. Conlon and Raff to my article on Control and Maintenance of Cell Size [1]. All of the comments and points raised in their Reply are answered in the original article. That article [1] did deal with the conclusions they drew and the arguments they did make. I trust that the readers interested in this subject will read the original Conlon/Raff article [2] and my critique [1] to understand the issues. However, for the sake of clarity and efficiency I will comment on new issues raised by the reply so that the differences are clearly joined.
Regarding the comment that I use the term “cell growth” ambiguously, that quote is taken out of context. Thus, the question noted by Drs. Conlon and Raff occurs in the following context [1] where I write:
“The key question studied by Conlon and Raff asks, "How do cells maintain a constant cell size and cell size distribution during extended cell growth?" In a cell culture growing over many generations, the cell size distribution neither varies nor broadens. Cells do not get progressively larger nor do they get progressively smaller. One formulation of this result is that cell mass increase is regulated during the cell cycle so that there is no disparity between the rate of cell mass increase and the rate of cell number increase. Total cell number and total cell mass increase in parallel during unlimited exponential growth. If there were any disparity or disproportion in the rate of mass and cell number increase, cells would get either larger or smaller during extended growth.”
It is quite clear that from this context that the question described by Drs. Conlon and Raff, is unambiguously discussing cell growth and cell number increase over many generations.
Regarding their statement that I attribute the notion of linear growth incorrectly to their experiments, I can only point out that in Figure 4 of my paper [1] the attribution of linear growth to successive cell cycles is not possible if cell size constancy is to be maintained. Thus, in the caption to Figure 4 it is written: “It is not proposed that mass increases linearly, but merely that even linear synthesis should exhibit, in an uninhibited situation, exponential mass growth.” Linear growth is only logically possible as a topic of discussion within the cell cycle. Over many generations linear growth is logically impossible if cell size is maintained relatively constant.
Regarding “synchronization” at the start of S phase with aphidicolin, I can only point out that this method cannot synchronize cells. The details of this argument are presented in recent papers and I recommend the reader to these papers [3-7].
Regarding the “shift-up” and other experimental manipulations, I refer the reader to the original paper. The slow part of the shift-up is explained clearly in Figure 10 and I hope that the reader will read this caption and the associated text. In particular I note the point made with regard to Figure 10 that: “…one can postulate that for reasons unrelated to the cell cycle but merely related to cellular metabolism occurring continuously throughout the cell cycle, the change in external conditions does not immediately lead to the new rate of mass increase.” This is a simple explanation for the delayed size change during a shift-up.
The IGF-1/GGF experiments [8] are very complex and would require a lengthy discussion to point out all of the problems with this work However, in this space let me briefly discuss the experiments and analyses related to the proposal that one can dissociate cell growth and “progression through the cell cycle”.
1. There is a confusion of the notion of “cell growth” with “cell size”. This is best seen in the analysis of their conclusion that “…GGF stimulated entry into S phase, even though it did not promote cell growth.” Cell growth in this case was determined (Fig. 2 in [8]) by looking at cell size distributions before and after a treatment. But merely because cell size did not change from one point to a second point does not lead to a conclusion that cell growth did not occur. Cell size is determined by a combination of cell mass increase and cell division. A constant cell size may therefore mask true mass increase (leading to initiation of S phase) with an associated division (not observed because only beginning and end point were determined).
2. It is suggested [8] that “as the total cell-cycle time is longer in low GGF than in high GGF [i.e., Fig. 3 shows a greater cell number increase in high GGF than in low GGF] a higher proportion of the cells in low GGF would therefore be expected to be in S and G2.” One experimental result is therefore viewed as a paradox as it is observed, by FACS analysis, that “…the proportion of cells in G1 was lower rather than higher in low concentrations of GGF [compared to high GGF].” My analysis of the relationship of growth rate and proportion of cells with a G1-phase amount of DNA predicts that slower cells should have more cells with a G1-phase amount of DNA. This is simply because S and G2-phase times are relatively invariant, and increasing the interdivision time increases the time for G1-phase disproportionately [9]
3. The observation that only half of cells at maximal induction of initiation into S phase are found to replicate DNA suggests that the cells are quite heterogeneous and not suitable for analysis of the problem.
4. The use of primary Schwann cells to study these questions when there are so many unknowns can only lead to confusion. A discussion of why such cells are inappropriate for analysis has been published ([10] (this paper can be read at my web site at www.umich.edu/~cooper).
In summary, I hope that the readers of the reply by Drs. Conlon and Raff [11] compare their views with the views in the original article [1].
1. Cooper S: Control and Maintenance of Mammalian Cell Size. BMC Cell Biol 2004, 5:35.
2. Conlon I, Raff M: Differences in the mammalian cell and yeast cells coordinate cell growth and cell-cycle progression. J Biol 2003, 2:7.
3. Cooper S: Is Whole-culture synchronization Biology's "Perpetual Motion Machine"? Trends in Biotechnology 2004, 26:266-269.
4. Cooper S: Whole-culture Synchronization Can Not, and Does Not, Synchronize Cells. Trends in Biotechnology 2004, 22:274-276.
5. Cooper S: Rethinking synchronization of mammalian cells for cell-cycle analysis. Cell Mol Life Sci 2003, 6:1099-1106.
6. Cooper S: Reappraisal of serum starvation, the restriction point, G0, and G1-phase arrest points. FASEB J 2003, 17:333-340.
7. Cooper S: Mammalian cells are not synchronized in G1-phase by starvation or inhibition: considerations of the fundamental concept of G1-phase synchronization. Cell Prolif 1998, 31:9-16.
8. Conlon IJ, Dunn GA, Mudge AW, Raff MC: Extracellular control of cell size. Nat Cell Biol 2001, 3:918-921.
9. Cooper S: On the interpretation of the shortening of the G1-phase by overexpression of cyclins in mammalian cells. Exp Cell Res 1998, 238:110-115.
10. Cooper S: Toward a standard system for the mammalian cell cycle. ASM News 2000, 66:71-75.
11. Conlon I, Raff M: Control and maintenance of mammalian cell size: Response. BMC Cell Biol 2004, 5.
Competing interests
No competing interests not indicated in this comment
Brooks' hypothesis on size control
3 March 2005
Nearly two years ago, Conlon and Raff (J Biol 2003, 2:7) proposed that mammalian cells do not need any checkpoint to maintain size homeostasis. An "evidence" was that cell volume increased linearly, irrespectively of cell size, under an S-phase arrest. Conlon and Raff referred to an old and clever (albeit never proved) hypothesis of Brooks (in The Cell Cycle. Edited by John PCL. Cambridge: Cambridge University Press; 1981), arguing that linearly growing cells not necessarily require any size control mechanism. Last year, Cooper (BMC Cell Biol 2004, 5:35) and we (Sveiczer A, Novak B, Mitchison JM. Theor Biol Med Model 2004, 1:12) criticised the conclusions of Conlon and Raff. We both pointed out that even if S-phase blocked cells grow linearly, it does not mean that these cells normally grow linearly during their cell cycle. In their response to Cooper here (BMC Cell Biol 2004, 5:36), Conlon and Raff state that they had never spoken about linear growth during the normal cycle, because this question had simply never been addressed by them. At the first sight, this is a smart answer to disclaim the criticism, however, it generates a serious problem. Namely, in his old paper Brooks proposed that the cells grew linearly, irrespectively of their size, therefore they increased their volume by the same amount during the cycle, if the cycle time was the same. In that special case, Brooks could suppose even a large scattering in the cycle time, and division size was finally convergent during consecutive cycles, even after a large perturbation. He was definitely thinking of normal cycles, during which the linear rate of growth extension is independent of both size and of cell cycle stage! As a consequence, I think that the answer given here by Conlon and Raff generates an even larger discrepancy and, afterwards, the authors have two possibilities. One of them is to explain in detail how their data and assumptions fulfil the general requirements, where the Brooks' hypothesis might be valid. The other one is to withdraw their conclusions about the lack of size control in mammalian cells.
Competing interests
None declared