School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK1
Author for correspondence: Simon V. Avery. Tel: +44 115 9513315. Fax: +44 115 9513251. e-mail: Simon.Avery{at}nottingham.ac.uk
Keywords: cell-to-cell variability, flow cytometry, cell cycle, ageing, non-genetic variation
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Overview |
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Background |
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That there have been few direct studies of phenotypic heterogeneity to date in part reflects the inability to purify phenotypically divergent subpopulations for study by subculture; since the determinants of phenotypic heterogeneity are not usually inherited, such subpopulations again generally show variable phenotype after subculture (Davis et al., 1990 ; Steels et al., 2000
). Thus characterization of phenotypic variants requires growth-independent means of differentiation at the single-cell level. Techniques such as flow cytometry provide the necessary tools for these analyses (Howlett & Avery, 1999
; Attfield et al., 2001
).
S. cerevisiae is an excellent candidate for studies of phenotypic heterogeneity since it commonly exists in isogenic populations, particularly in settings relevant to human use (see above), and its experimental tractability and widespread adoption as a eukaryotic model are well documented. Thus several recent studies of phenotypic heterogeneity have focused on S. cerevisiae, and this organism provides the main focus for this mini-review.
The potential mechanisms underlying phenotypic heterogeneity have been speculated upon in several papers. The purpose of this mini-review is to address each of the possible cell variables that may contribute to non-genetic heterogeneity and to discuss the roles of these variables in determining differential stress resistance.
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Genetic versus non-genetic variation in isogenic cultures |
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The simplest way to discriminate experimentally whether a phenotype is genetically determined is to test for inheritance of the phenotype in progeny. Of all the possible factors (see sections below) that might determine non-genetic resistance of a fraction of cells to a short-term stress, none, with the possible exception of epigenetic regulation, are sustainable through the multiple rounds of cell division and physiological changes associated with growth. However, if maintained with stressor, one way in which such cells could appear to sustain resistance during growth would be if initial survival allowed them time to mount an adaptive response, i.e. induction of genes whose products conferred resistance. Such inducible resistance would still be lost on removal of the stressor however. Therefore, only genetically determined phenotypes should be inherited indefinitely in the absence of selection. In this way, rare reversion to oxytetracycline resistance in sod1 S. cerevisiae was shown to be genetic (Avery et al., 2000
), whereas sorbic-acid resistance of some cells in Zygosaccharomyces bailii cultures was non-genetic (Steels et al., 2000
).
Thus phenotypic heterogeneity is not related to differential gene possession. However, it could arise from differential basal gene expression. Heterogeneity in basal gene expression could be linked to most of the cell variables discussed in the following sections. It is stressed that genes revealed to influence a phenotype through comparison of non-isogenic organisms (e.g. mutant versus wild-type) are only potential contributors to heterogeneity in a phenotype. True determinants of phenotypic heterogeneity are decisive, in a non-engineered system.
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Cell division cycle |
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One problem with using cell cycle arrest to establish the cell cycle dependency of a phenotype is that synchronization methods commonly exert secondary perturbing effects on cell physiology. Flow cytometry can be used to circumvent this problem, and provides an excellent tool for examining cell-to-cell heterogeneity generally. In our laboratory, flow cytometry has been used to demonstrate that heterogeneous sensitivity to Cu in exponential-phase S. cerevisiae is at least partly cell-cycle-dependent (Howlett & Avery, 1999 ). Cells in the G2/M phases of the cell cycle were predominant in Cu-sensitive subpopulations, whereas G1/S-phase cells were predominant in Cu-resistant subpopulations (Fig. 1
). Furthermore, with the use of an oxidant-sensitive fluorescent probe in conjunction with flow cytometry, it was established that initial (pre-Cu exposure) cellular oxidant status was predictive of Cu sensitivity (Howlett & Avery, 1999
).
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Rhythms |
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Growth rate of individual cells and cell size |
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Cell age |
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A range of physiological changes is associated with ageing in S. cerevisiae. Gross parameters such as cell size and generation time are influenced by cell age (discussed above), as well as more specific phenotypes many of which may arise from differential gene transcription that occurs during ageing of S. cerevisiae (Jazwinski, 1996 ). Stress sensitivity is affected by cell age. Kale & Jazwinski (1996
) prepared purified cells of different ages and compared their resistances to the DNA-alkylating agent ethyl methanosulphate (EMS) and to UV irradiation. Susceptibility to EMS increased steadily with age, whereas resistance to UV peaked in middle-aged cells (approx. eight generations old) and declined markedly in older cells. This biphasic pattern of UV resistance was closely correlated with the level of RAS2 mRNA; Ras2p is involved in the repair of UV-induced DNA damage (but not EMS-induced DNA damage). The authors concluded that age-dependent changes in stress sensitivity were related directly to the ageing process rather than incidental changes in cell physiology (e.g. via cell volume changes). It was suggested that age-dependent variation in stress sensitivity might have arisen to ensure persistence of clonal populations against naturally occurring stresses such as UV irradiation. Such a scenario would be in keeping with the proposed general role of phenotypic heterogeneity in enabling the persistence of isogenic populations (see Background section).
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Mitochondrial function |
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The influence of mitochondrial activity on yeast phenotype can be readily assessed by comparison of petite and grande cultures. Altered stress responses in petite cells have been widely reported, although such phenotypes can be strain-specific. Altered susceptibility may be related to certain of the parameters discussed elsewhere in this review. For example, owing to their fermentative metabolism, petite mutants generally display slow growth rates. Moreover, mitochondrial activity is directly related to the generation of highly damaging reactive oxygen species (ROS, which are mostly free radicals) that are formed as by-products of respiration. Many types of stress promote ROS action or formation in cells. For example, freezethaw stress is considered to increase cytosolic ROS activity in yeast via an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain (Park et al., 1998 ). Metals such as Cu, Cd and Cr also may promote the production of ROS in a mitochondria-dependent manner (Avery, 2001
). Petite mutants actually exhibit enhanced susceptibility to certain types of free-radical-generating stressors, and this has been attributed to lack of some energy-generating capability that may be required for active free radical detoxification (Grant et al., 1997
).
In addition to the above effects, a pathway termed the retrograde response signals mitochondrial dysfunction to the nucleus in S. cerevisiae and results in modulation of the nuclear gene expression profile. A recent microarray analysis of a petite strain entirely lacking mitochondrial DNA revealed increased expression of heat-shock proteins and members of the ABC family of drug transporters. Accordingly, the mutant was resistant to severe heat shock and to several drugs (Traven et al., 2001 ).
Thus the presence of petite cells in normal yeast cultures and the heterogeneous mitochondrial activity of grande cells together have the potential to generate considerable heterogeneity in cell phenotypes that are influenced by mitochondrial function. Such heterogeneity should be absent in petite cultures.
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Epigenetic regulation and prions |
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Although the focus of this review is on S. cerevisiae, in the context of epigenetic regulation of phenotypic variation in yeasts it is worth addressing the important subject of phenotypic switching that occurs in certain Candida spp., including pathogenic Candida albicans (for a review, see Soll, 1997 ). There is evidence that phenotypic switching may serve as a virulence factor in C. albicans. Such phenotypic switching is readily observed as changes in colony appearance that may typically occur at frequencies in the order of 1 in 104. Moreover, switching may be accompanied by a broad range of phenotypic changes at the single cell level also (e.g. antigen expression, tissue affinity). It is thought that heritable but reversible changes in heterochromatic state and/or possible chromosomal rearrangements underpin these phenotypic switches. Thus deletion of the SIR2 gene that is involved in maintaining chromatin silencing yielded a switching frequency in C. albicans as high as 1 in 10 (Perez-Martin et al., 1999
).
A protein-based form of epigenetic inheritance involves the so-called yeast prions. Prion-like proteins exist in two heritable conformational states normal and prion. Much recent work has focused on a prion element that results from aggregation of the Sup35 translation termination factor in S. cerevisiae. Conversion from the normal form, [psi-], to prion, [PSI+], is self-perpetuating and may result in reduced fidelity of protein synthesis and enhanced variation at the proteome level (Pal, 2001 ). Recent studies have revealed that [PSI+] strains exhibit altered susceptibility to a wide array of stresses compared to co-isogenic [psi-] strains (Eaglestone et al., 1999
; True & Lindquist, 2000
). For example, in roughly half of over 150 different environmental conditions tested, significant differences in growth rate were noted between co-isogenic [PSI+] and [psi-] strains, and [PSI+] strains showed enhanced growth relative to [psi-] strains in over 25% of these tests (True & Lindquist, 2000
). It has been suggested that [PSI+]-induced variability may be selectively maintained in yeast as a means of enhancing the survival of clonal populations in the event of adverse environmental conditions (True & Lindquist, 2000
). However, the presence of prions can explain only limited phenotypic variation. Prions do not appear to be widespread and in S. cerevisiae may be restricted to laboratory strains; prion-forming ability has not been identified in natural or industrial strains (Chernoff et al., 2000
). Furthermore, as with spontaneous mutation (see above), the frequencies of changes in phenotype of [PSI+] cells and of spontaneous conversion between normal and prion states (True & Lindquist, 2000
) are too low to account for much of the phenotypic heterogeneity evident within yeast cultures.
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Stochastic variation |
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Concluding remarks |
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ACKNOWLEDGEMENTS |
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