Is whole-culture synchronization biology's ‘perpetual-motion machine’?

doi:10.1016/j.tibtech.2004.04.009

Trends in Biotechnology

Volume 22, Issue 6, June 2004, Pages 266-269

https://doi.org/10.1016/j.tibtech.2004.04.009 Get rights and content

Abstract

Whole-culture or batch synchronization cannot, in theory, produce a synchronized culture because it violates a fundamental law that proposes that no batch treatment can alter the cell-age order of a culture. In analogy with the history of perpetual-motion machines, it is suggested that the study of these whole-culture ‘synchronization’ methods might lead to an understanding of general biological principles even though these methods cannot be used to study the normal cell cycle.

Section snippets

The lure and persistence of perpetual-motion machines

For centuries researchers have attempted to develop or invent the Holy Grail of energy production – a perpetual-motion machine. Recorded attempts to achieve perpetual motion date back at least 15 centuries. Many simply think that it must be possible, and that if they only try hard enough, they will find a solution. Up until 200 years ago, this belief could be forgiven. Even Leonardo da Vinci hoped to develop such an instrument. But with our understanding of the nature of energy, and the first

Whole-culture synchronization and the law of conservation of cell-age order

Whole-culture synchronizing methods are those in which all of the cells in a culture are treated identically to produce a set of cells that are therefore presumed to be of the same cell-cycle age; that is, the cells are forced to be of the same age and are thus ‘synchronized’. Numerous methods have been proposed for the ‘synchronization’ of a population of cells by treating a cell culture to arrest cells at a particular point in the cell cycle. Releasing the arrested cells is then proposed to

Why whole-culture synchronization does not work

The simplest example of this law in application is in the case of cell-growth arrest such that after treatment all cells have G1-phase amount of DNA. 5, 6, 7, 8, 9, 10, 11. Consider that in the exponentially growing culture the cells range between sizes 1.0 (newborn size) and 2.0 (dividing cell size). Now inhibit cell mass increase so that all cells remain fixed in their cell mass. That is, the mass of a cell remains fixed for the entire treatment period. Cells that have not reached the size

Selective methods can synchronize cells

Of course, these strictures regarding synchronization do not apply to selective procedures in which cells are not all treated identically. A subset of cells of a certain age selected from the original asynchronous population could constitute, in theory, a synchronized culture. Two well-known selective methods for producing synchronized cultures are elutriation (hydrodynamic separation in a centrifuge) or mitotic selection (selection of cells released from a substrate during mitosis). Although

The development of the eukaryotic ‘baby machine’

There have been many arguments raised against the proposal that forced synchronization methods are unable to produce a synchronized culture. Some have said that perhaps methods are not very ‘efficient’ at synchronizing cells, but that some other whole-culture method does it better. Or some have said that one should not expect good synchronization of cell division because stochastic variation in interdivision times would lead to a rapid decay of cell division synchrony. All of these arguments

Whole-culture synchronization and perpetual motion

We can now see the analogy of whole-culture synchronization and perpetual-motion machines. First, both have been with us a long time and it appears that they will be with us in the future – although both projects are doomed to failure. Second, both projects are doomed because they violate fundamental laws.

The final part of the analogy with perpetual-motion machines is to study these synchrony methods to glean from them some basic laws of biology. The main idea to be taken from the failure of

Acknowledgements

Dr Charles Helmstetter has been extremely helpful with comments on the problem of synchronization of eukaryotic cells. Additional papers on this subject can be studied and read at www.umich.edu/~cooper. I wish to thank a very astute anonymous reviewer for suggesting that the LCCAO is not a law accepted as universal as are the laws of conservation of mass and energy or the laws of thermodynamics. I agree with this idea, but the use of the perpetual-motion metaphor was for pedagogical and

References (17)

S. Cooper
The Schaechter-Bentzon-Maaløe experiment and the analysis of cell cycle events in eukaryotic cells
Trends Microbiol.
(2002)
S. Cooper
Reappraisal of G1-phase arrest and synchronization by lovastatin
Cell Biol. Int.
(2002)
S. Cooper
Bacterial Growth and Division
(1991)
C.E. Helmstetter
Synchrony in human, mouse, and bacterial cell cultures
Cell Cycle
(2003)
M. Thornton
Production of minimally disturbed synchronous cultures of hematopoietic cells
Biotechniques
(2002)
S. Cooper
Minimally disturbed, multi-cycle, and reproducible synchrony using a eukaryotic “baby machine
Bioessays
(2002)
S. Cooper
Rethinking synchronization of mammalian cells for cell-cycle analysis
Cell. Mol. Life Sci.
(2003)
S. Cooper
Reappraisal of serum starvation, the restriction point, G0, and G1-phase arrest points
FASEB J.
(2003)

There are more references available in the full text version of this article.

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Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods.
Experimental reconsideration of the utility of serum starvation as a method for synchronizing mammalian cells
2009, Cell Biology International
Citation Excerpt :
A well-synchronized culture is one in which the cells move as a uniform cohort through the cell cycle; truly synchronized cells should accurately reflect the events occurring in unperturbed cells during normal passage through the cell cycle. The sine qua non of synchronization is that the cells move uniformly through the cell cycle and divide synchronously over a relatively narrow span of time (Cooper, 2004a; Cooper and Shedden, 2003). Synchronization methods can be separated into two classes, whole-culture methods and selective methods (Cooper, 1991).
Accurate cell-size determinations support the prediction that serum starvation and related whole-culture methods cannot synchronize cells. Theoretical considerations predict that whole-culture methods of synchronization cannot synchronize cells. Upon serum starvation, the fraction of cells with a G1-phase amount of DNA increased, but the cell-size distribution is not narrowed. In true synchronization, the cell-size distribution should be narrower than the cell-size distribution of the original culture. In contrast, cells produced by a selective (i.e. non-whole-culture) method have a specific DNA content, a narrow size distribution, and divide synchronously. The general theory leading to the conclusion that whole-culture methods for synchronization do not work implies that one can generalize these serum-starvation results to other cell lines and other whole-culture methods, to conclude that these methods do not synchronize cells.
Invariant mRNA and mitotic protein breakdown solves the Russian Doll problem of the cell cycle
2009, Cell Biology International
Citation Excerpt :
First, the clear presence of cell-cycle variation in protein content does not mean that cyclical mRNA variation is expected. Second, the data on mRNA variation during the cell cycle has to be reconsidered, with attention to problems of synchronization of cells and perturbations when whole-culture methods are used (Cooper, 1998c, 2002, 2003a,b, 2004c,d, 2005, 2006), as well as problems with microarrays (Cooper and Shedden, 2003; Shedden and Cooper, 2002a,b). And finally, one must consider the logical and theoretical problems with postulating mRNA variation during the cell cycle as exemplified by both the infinite regression problem and the minor affect of mRNA variation on protein variation.
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Continuous and constant gene expression during the cell cycle, continuous protein accumulation, and protein breakdown only within the mitotic window solves the Russian Doll problem or infinite regression problem. These results, and theoretical ideas support an alternative view of the cell cycle where many of the proposed control systems do not exist.
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Microarray analyses have led to the postulated existence and identification of numerous genes that are believed to be expressed and presumably to act in a cell-cycle-specific manner because their expression varies during the cell cycle. It is important to see how protein variation can be produced from mRNA variation. We have calculated the protein content throughout the cell cycle resulting from cell-cycle-specific mRNA expression, and compared the result to protein content resulting from constant, cell-cycle independent, mRNA expression. For stable proteins, cell-cycle-specific mRNA expression leads to a maximum 2-fold change in protein content compared to proteins synthesized from constantly expressed mRNA. More realistic sinusoidal patterns of mRNA expression exhibit much smaller ratios of 1.25 or lower, even for extremely large amplitudes in mRNA expression. For unstable proteins that have a cycle-independent half-life, only at extremely short protein half-lives does mRNA variation have a significant impact on variation of protein content during the division cycle. We also apply these findings to proteins with a cycle-specific decay pattern. mRNA variations during the eukaryotic division cycle variation of mRNA during the cell cycle can have only a minimal affect on the variation of protein content during the cell cycle. We conclude that mRNA variations during the division cycle, as measured by microarrays, cannot by themselves, identify cycle-specific functions related to protein variations.
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View full text

Trends in Biotechnology

Is whole-culture synchronization biology's ‘perpetual-motion machine’?

Abstract

Section snippets

The lure and persistence of perpetual-motion machines

Whole-culture synchronization and the law of conservation of cell-age order

Why whole-culture synchronization does not work

Selective methods can synchronize cells

The development of the eukaryotic ‘baby machine’

Whole-culture synchronization and perpetual motion

Acknowledgements

Trends Microbiol.

Cell Biol. Int.

Bacterial Growth and Division

Synchrony in human, mouse, and bacterial cell cultures

Cell Cycle

Production of minimally disturbed synchronous cultures of hematopoietic cells

Biotechniques

Minimally disturbed, multi-cycle, and reproducible synchrony using a eukaryotic “baby machine

Bioessays

Rethinking synchronization of mammalian cells for cell-cycle analysis

Cell. Mol. Life Sci.

Reappraisal of serum starvation, the restriction point, G0, and G1-phase arrest points

FASEB J.