MINIREVIEW
Aging and differentiation in yeast populations: elders with
different properties and functions
1, Derek Wilkinson1 & Libuse Va
chova
2,1
Zdena Palkova
1
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague 2, Czech Republic; and 2Institute of
Microbiology of the ASCR, v.v.i., Prague 4, Czech Republic
Correspondence: Zdena Palkova,
Department of Genetics and Microbiology,
Charles University, Vinicna 5, 128 44 Prague
2, Czech Republic.
Tel.: +420 221951721;
fax: +420 221951724;
e-mail: [email protected]
Received 9 June 2013; revised 23 September
2013; accepted 26 September 2013. Final
version published online 25 October 2013.
DOI: 10.1111/1567-1364.12103
Editor: Dina Petranovic
Keywords
yeast colonies; stationary-phase liquid
cultures; comparison of differentiated cell
subpopulations; Saccharomyces cerevisiae;
chronological aging and quiescence;
starvation.
Abstract
Over the past decade, it has become evident that similarly to cells forming
metazoan tissues, yeast cells have the ability to differentiate and form specialized cell types. Examples of yeast cellular differentiation have been identified
both in yeast liquid cultures and within multicellular structures occupying solid
surfaces. Most current knowledge on different cell types comes from studies of
the spatiotemporal internal architecture of colonies developing on various
media. With a few exceptions, yeast cell differentiation often concerns nongrowing, stationary-phase cells and leads to the formation of cell subpopulations differing in stress resistance, cell metabolism, respiration, ROS
production, and others. These differences can affect longevity of particular
subpopulations. In contrast to liquid cultures, where various cell types are dispersed within stationary-phase populations, cellular differentiation depends on
the specific position of particular cells within multicellular colonies. Differentiated colonies, thus, resemble primitive multicellular organisms, in which the
gradients of certain compounds and the position of cells within the structure
affect cellular differentiation. In this review, we summarize and compare the
properties of diverse types of differentiated chronologically aging yeast cells
that have been identified in colonies growing on different media, as well as of
those found in liquid cultures.
YEAST RESEARCH
Introduction
Yeast, although considered for decades to be unicellular
organisms, can organize themselves into multicellular
communities that, to some extent, behave as primitive
multicellular organisms. Yeast cells within these populations can undergo differentiation that results in the formation of specialized cell types (Vachova & Palkova,
2005; Vachova et al., 2009a, b, 2011; Piccirillo et al.,
2010; Cap et al., 2012; Traven et al., 2012). While yeast
cells occur mostly in a relatively homogeneous state in
exponentially growing populations, differentiation processes typically occur during chronological aging as a
result of a changed environment, such as starvation and
stress. In contrast to replicative aging, which counts the
number of divisions of an individual cell (Jazwinski et al.,
1989), chronological aging is a process whereby yeast cells
change their properties (metabolism, physiology) and survive for long periods in a nondividing or very slowly
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
dividing state (MacLean et al., 2001). In this review, we
show diverse types of nondividing cells that can be
formed during yeast chronological aging under various
sets of conditions, such as during liquid cultivation under
limited nutrients and in the form of colonies developing
on solid media.
What are the possible fates of cells that suffer nutrient
limitation and undergo chronological aging? There is a
great deal of information (often obtained by highthroughput screenings) on the genes that are expressed,
proteins that are produced, and some metabolites that
accumulate during chronological aging. However, these
data often differ between individual screenings (Matecic
et al., 2010), and only a few of the identified features
appear repeatedly. In addition, inconsistencies exist
regarding the terminology used to define chronologically
aged yeast cells. In mammals, the term ‘quiescence’ is
usually used for diverse kinds of cells, the main characteristic of which is a nondividing/G0 state (Coller et al.,
FEMS Yeast Res 14 (2014) 96–108
Differentiation of aging yeast populations
2006; Table 1); thus, the term ‘quiescence’ does not imply
a specific metabolism or cell physiology in mammals. In
yeast, the term ‘quiescence’ is often reserved for a subset
of nondividing (presumably G0) cells in stationary-phase
cultures (Gray et al., 2004), and increasingly, it is used
for such cells that have gained some stress resistance and
longevity features, such as the accumulation of storage
compounds (Allen et al., 2006; Table 1). In other words,
it is usually only some nondividing yeast cells with specific physiology that are now termed ‘quiescent cells’.
However, other yeast cell types are found within stationary-phase populations (nongrowing or even G0), which
are chronologically aged and gain-specific properties,
97
some of which are specified in this review (Table 2). In
addition, some of the cells of these stationary-phase populations could even divide, but with very low frequencies
(e.g. lasting several days), which of course is too infrequent to be monitored by standard techniques.
Another term ‘persisters’ is often used for a cell subpopulation (bacteria, yeast, fungi) that survives unpleasant conditions such as antibiotic treatment in a ‘dormant’
state and is able to regrow under more propitious conditions (LaFleur et al., 2006; Table 1). It, thus, differs from
another dormant form of microorganisms named the
VBNC (viable but not culturable) state (Whitman et al.,
1998; Table 1). Microorganisms are able to survive for a
Table 1. Definitions of some terms used in this article
Term
Definition
Elders
Any type of nondividing yeast cells that enter chronological aging; their growth arrest is caused by
external factor(s) (not by a mutation); different types of elders can mutually differ by their features
(longevity, physiology, respiration, etc.)
A reversible growth/proliferation arrest (Coller et al., 2006)
Defined, for example, as the state of cells in a saturated culture (Gray et al., 2004) or as the state of a
subpopulation of dense, unbudded, G0-arrested daughter cells formed after glucose exhaustion
(Allen et al., 2006)
A type of elder; localized to upper regions of central parts of S. cerevisiae colonies entering the alkali
phase of colony development (linked to ammonia production); exhibit specific physiology as described
by Cap et al. (2012) and Vachova et al. (2013)
A type of elder; localized to lower regions of central parts of S. cerevisiae colonies entering the alkali
phase of colony development (linked to ammonia production); exhibit specific physiology as described
by Cap et al. (2012) and Vachova et al. (2013)
Cells forming the outermost layers of yeast microcolonies, which have a specific physiology as described
by Traven et al. (2012); a significant proportion of these cells are probably nondividing cells
representing another type of elder
Cells forming the innermost layers of yeast microcolonies, which have a specific physiology as described
by Traven et al. (2012); very probably nondividing cells representing another type of elder
A type of elder; separated from stationary-phase liquid culture by gradient centrifugation; exhibit
specific physiology as described in Allen et al. (2006), Aragon et al. (2008) and Davidson et al. (2011)
A type of elder; separated from stationary-phase liquid culture by gradient centrifugation; exhibit
specific physiology described by Allen et al. (2006), Aragon et al. (2008) and Davidson et al. (2011)
Cells that do not regrow when replated on the fresh medium, but are still living cells and may carry out
metabolic activity and may be resuscitated by an external stimulus provided for example by other cells.
These cells have been characterized mostly in bacteria, but indications of their occurrence in yeast exist.
Belong to the category of elders
A subpopulation of dormant cells with increased resistance to stress (often to drug treatment); active
growth of persisters may be restored by improved environment such as replating on fresh medium.
Typical of bacteria, but described, for example, also in yeast biofilms (LaFleur et al., 2006). Belong to
the category of elders
A colony derived from a drop of cell suspension spotted on the agar (Richards, 1966;
Kockova-Kratochvilova, 1990; Palkova et al., 1997)
A colony derived from a single cell
Colonies exhibiting structured morphology (when compared with smooth colonies formed by
laboratory strains). These colonies have some features typical of biofilms as described by
Vachova et al. (2011)
A stage of yeast colony development during which the pH of the surrounding media is decreased
(Palkova et al., 1997; Vachova et al., 2009b)
A stage of yeast colony development during which ammonia is produced by yeast cells and the pH of
the surrounding media increases (Palkova et al., 1997; Vachova et al., 2009b)
Quiescence (mammalian)
Quiescence (yeast)
U cells
L cells
Outer cells
Inner cells
Q (quiescent) cells
NQ (nonquiescent) cells
Viable but nonculturable (VBNC) cells
Persister cells
Giant colony
Microcolony
Structured biofilm colonies
Acidic phase
Alkali phase
FEMS Yeast Res 14 (2014) 96–108
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N/A
WD
N/A
WD
Higher
Inactive
Higher
N/A
N/A
N/A
N/A
WD
N/A
WD
Lower
Active
Lower
N/A
N/A
N/A
Vital/long living
N/A
WD
Less
WD
WD
N/A
Lower
N/A
N/A
N/A
Higher
Higher
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Lower
WD
Higher
Higher
(E), Analyzed on gene expression level; N/A, Not analyzed; WD, Without difference (difference between both cell types was not reported).
*According to Shi et al. (2010), **(according to Peters et al. (2012).
Budding
Degradation/protein sequestration
Noncoding RNA
Recombination and repair
Protein synthesis
Ribosomal proteins(E)
Translation proteins(E)
mRNA in insoluble
protein–RNA complexes
rRNA(E)
General transcription(E)
Noncoding transcripts
Recombination and repair genes(E)
Transposones(E)
Autophagy
Proteasome(E)
Insoluble proteins
Unbudded cells
Budded cells
Higher level:
Ala, Lys, GABA
Lower
Lower
N/A
Higher level:
Gln, Glu, Arg
Higher
Higher
N/A
Amino acids/nucleotides
Glucose metabolism
Mitochondria/respiration
Storage compounds
Less vital
Lower
Lower
Lower
Higher
Lower
N/A
Lower
Higher
Higher
Lower
Higher
Lower
Vital/long living
Higher
Higher
Higher
Lower
Higher
N/A
Higher
Lower
Lower
Higher
Lower
Higher
Zymolyase resistance
Heat shock resistance
Ethanol resistance
ROS production
Glycogen
Trehalose
Lipid droplets
Oxygen consumption
Oxidative phosphorylation(E)
Glycolysis(E)
Gluconeogenesis(E)
Amino acid and nucleotide
metabolism(E)
Amino acid concentration
Vitality
Stress/resistance
Outside cells
Microcolonies
Giant colonies
L cells
Traven et al. (2012)
Cap et al. (2012)
U cells
Cell types
Biological process
Reference(s)
Table 2. Selected properties of different types of elders
N/A
WD
More
WD
WD
N/A
Higher
N/A
N/A
N/A
Lower
Lower
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Higher
WD
Lower
Lower
Less vital
Inside cells
Lower
Lower
N/A
Lower
Lower
No
WD
No**
Yes
No
WD
Lower
Higher
N/A
N/A
Higher
N/A
Lower
Higher*
Higher*
N/A
Higher
WD
WD
WD
WD
Vital/long living
Q cells
Higher
Higher
N/A
Higher
Higher
Yes
WD
Yes**
Yes
Yes
WD
Higher
Lower
N/A
N/A
Lower
N/A
Higher
Lower*
Lower*
N/A
Lower
WD
WD
WD
WD
Less vital
NQ cells
Stationary-phase liquid
cultures
Allen et al. (2006), Aragon
et al. (2008) and
Davidson et al. (2011)
98
Z. Palkov
a et al.
FEMS Yeast Res 14 (2014) 96–108
99
Differentiation of aging yeast populations
very long time as VBNC, and a specific factor (e.g. a peptide (Kell & Young, 2000)) is required for ‘resuscitation’
(Rice et al., 2000) that could be provided either by growing cells of the same microorganism or even by other
microorganisms in the immediate vicinity. The VBNC
state has mainly been reported in bacteria, but there are
also a few examples in yeast (Divol & Lonvaud-Funel,
2005; Palkova et al., 2009; Serpaggi et al., 2012).
The strains, ages, growing conditions, and other
parameters used in studies of yeast cell diversification
display some similarities, but many differences. The
nomenclature used to describe cell types and the criteria
for dividing a population into subpopulations is widely
divergent. In this review, we therefore discuss these similarities and differences to work toward a consensus
regarding the categorization of differentiating yeast cells.
We introduce the new term ‘elders’ (Table 1) for all types
of nondividing chronologically aging yeast cells (irrespective of their other features), and we show examples of
currently known types of yeast elders that gain varying
properties and can have different fates, such as survival
capability and capabilities to undergo metabolic reprogramming and differentiation to other types of elders
(Figs 1 and 2, Tables 1 and 2). We focus mostly on those
examples of elder populations that have already been at
least partially characterized with respect to their gene
expression, metabolism, survival rate, and/or other properties. In addition to colonies and stationary phase liquid
cultures in which various types of relatively homogeneous
elder subpopulations have already been identified, the
Fig. 1. Time line of differentiation of liquid culture and colony
populations. After inoculation to/on fresh medium, yeast cells grow
and divide exponentially (G) in both types of populations. After
expending the nutrients, the majority of cells in the liquid cultures
(upper part) and colonies (lower part) enter the stationary phase and
become elders (NG, non-growing cells), which afterward differentiate
to different types of elder such as vital Q or U cells and less vital NQ
or L cells, respectively. Differentiated cell subpopulations specifically
localize within the colony structure, which enables the formation of
gradients of regulatory compounds; specific localization is impossible
in liquid shaken cultures (neither specific cell localization nor the
formation of gradients is possible).
FEMS Yeast Res 14 (2014) 96–108
effect of some forms of nutrient starvation on properties
of mixtures of diverse elders is also briefly discussed.
Nutrient starvation is a commonly used means of achieving stationary phase. Opinion is divided as to whether
starvation for different nutrients (amino acids, glucose,
sulfates, and phosphates) triggers a single aging program,
different programs, or a variety of programs that converge toward a common end point. More recently, shortterm nutrient limitation has revealed that some of the
characteristics attributed to stationary-phase cells are
actually indicators of slow growth. We discuss here therefore the similarities and differences to highlight one of
the factors that could affect formation of different types
of elders.
Formation of different cell types in
chronologically aged liquid cell
population
Some of the current knowledge regarding the properties
of yeast elders in liquid cultures is based on studies in
which yeast entry to a stationary phase is induced by the
Fig. 2. Examples of cell differentiation within Saccharomyces
cerevisiae populations: different types of elders. Differentiation of
nongrowing stationary-phase cells (blue cells in the center) to
quiescent (Q) and nonquiescent (NQ) cells in liquid cultivations (left
upper part), to subpopulations of U (U) and L (L) cells in giant
colonies of laboratory haploid strains (left lower part), to
subpopulations of sporulating (S) and nonsporulating (NS) cells in
colonies of diploid strains (right lower part) and differentiation of part
of growing (G) cells of young microcolonies of wild strains to
stationary-phase (Sf) cell subpopulation (right upper part). Different
types of elders gain different properties (see the text and Table 2). M,
margin colony areas.
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100
limitation of a particular nutrient source (see the final
section). In these studies, a whole population of cells is
usually studied without any separation of subpopulations
that could potentially occur in the population. Knowledge
of the presence of subpopulations of elders in liquid cultures came mostly from studies of Werner-Washburne’s
group, which showed two major cell types that are
formed in stationary-phase liquid cultures of laboratory
strains of S. cerevisiae (Allen et al., 2006; Fig. 1). From
about 20 h after cell inoculation in rich glucose medium
when glucose has already been exhausted, two types of
elders differing in a range of properties can be separated
from the liquid culture (Fig. 2, Table 2). These cells have
been termed ‘quiescent/G0 (Q) cells’ and ‘nonquiescent
(NQ) cells’ according to the predicted occurrence of Q
cells in a G0 phase that are able to synchronously re-enter
mitosis and the asynchronous state of NQ cells (Allen
et al., 2006).
The vacuoles of 7-day-old Q cells are more electrondense, and the cellular glycogen and trehalose contents are
higher than those of NQ cells (Allen et al., 2006; Shi et al.,
2010). Q cells are up to four times more viable than NQ
cells during long-term persistence in the culture, and they
have better prospects for regrowth after nutrient replenishment (monitored up to day 28, by which CFU ability
had dropped significantly to 12% in Q and 3% in NQ
cells; Allen et al., 2006). Q cells are also more resistant to
heat shock than NQ cells. NQ cells are a mixture of budded and unbudded, less-dense cells with large vacuoles
and numerous mitochondria. These cells probably have
active autophagy, accumulate insoluble proteins, and produce more ROS than Q cells (Allen et al., 2006; Peters
et al., 2012). Counting the number of bud scars suggested
that Q cells are mostly virgin daughter cells, while the NQ
cell population contains equal numbers of mother cells
(i.e. of cells that underwent at least one additional division
after they were born) and virgin daughter cells (Allen
et al., 2006). Transcriptome analyses showed that Q cells
upregulate genes involved in stress response, in vesiclemediated transport and exocytosis, and in membrane
organization and biogenesis as well as genes for fatty acid
oxidation and lipid metabolism (Allen et al., 2006; Aragon
et al., 2008). In addition, Q cells contain a fraction of
their mRNAs in insoluble protein–RNA complexes (in P
bodies or other RNA granules) from which RNA can be
released by proteinase K treatment (Aragon et al., 2008).
In contrast, NQ cells increase the transcription of genes
involved in DNA recombination, Ty-element transposition, DNA metabolism, and some other genes related to
cell damage and cell cycle arrest (Allen et al., 2006; Aragon
et al., 2008). Analysis of the protein abundance in Q and
NQ cells using a GFP-fusion library led to the identification of some typical Q and NQ proteins. Proteins most
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Published by John Wiley & Sons Ltd. All rights reserved
Z. Palkov
a et al.
abundant in Q cells include proteins involved in mitochondrial functions, which is consistent with the finding
that Q cells consume oxygen six times more effectively
than NQ cells (Davidson et al., 2011).
These findings indicate that elders in liquid cultures are
able to differentiate to subpopulations with different
properties and subsequent fates (including survival rate)
similarly to colonies growing on solid media.
Various types of elders specifically
localize within developing colonies
Studies of yeast liquid cultures often cover a relatively
short time period from several hours to several days, and
little is known about the changes that occur within
already formed elders over a longer period. It is known,
however, that yeast populations developing within colonies are able to change their behavior several times over a
period of 20–30 days (or even longer; Palkova et al.,
2002). Only a short interval of about 40–45 h after the
onset of colony development is linked to exponential
growth of the whole colony population (Meunier &
Choder, 1999). After 2 days, the number of budding cells
within colonies drops to about 15% (Meunier & Choder,
1999), and the majority of cells within the colony enter
the stationary (or slow-growing) phase and thus begin to
undergo chronological aging and become elders. Colonies
continue their slow linear growth over the next
16–18 days (Vachova & Palkova, 2005; Palkova et al.,
2009), although they are largely formed by nondividing
cells. Such slow colony growth significantly decreases
later, but does not completely cease even in 25-day-old
colonies. During the reported periods of colony development, different types of elders are formed within the colonies, some of which have already been characterized at
least partially (Figs 1 and 2, Table 2).
Differentiation of aging Saccharomyces
cerevisiae colonies and formation of
specialized cell subpopulations
Aging yeast giant colonies [colonies originating from a
cell suspension spotted onto the agar (Richards, 1966;
Kockova-Kratochvilova, 1990; Palkova et al., 1997)]
growing on complex glycerol medium pass through developmental phases that are characterized by changes in the
surrounding pH from acidic to alkali and vice versa
(Palkova et al., 1997, 2002). The acid-to-alkali transition
of colonies formed by laboratory strains of S. cerevisiae
starts between days 7 and 10 of colony development, after
elders have already become the largest cell subpopulation
of the colony. Alkalization is accompanied by the
production of volatile ammonia, which functions as a
FEMS Yeast Res 14 (2014) 96–108
Differentiation of aging yeast populations
quorum-sensing signal able to induce all of the surrounding colonies to initiate their own ammonia production
and transit to the alkali phase. Ammonia, thus, contributes to the synchronizing of subsequent colony development (Palkova & Forstova, 2000). The acid-to-alkali
transition is accompanied by extensive metabolic reprogramming of the colony population (Palkova et al., 2002)
and by the differentiation of a relatively homogeneous
population of elders forming the central parts of the colonies during the acidic phase, into two major elder subpopulations of U and L cells that are in distinct locations
within the colony (Figs 1 and 2, Table 2; Vachova et al.,
2009a; Cap et al., 2012).
Elders localized to the upper regions of alkali-phase
colonies (U cells) of the strain BY4742 gain a stress resistance and longevity phenotype, do not produce ROS, and
accumulate storage compounds such as glycogen and
lipid droplets (Cap et al., 2012). Together with the thick,
zymolyase-resistant cell wall of U cells, these features have
been also specifically attributed to Q cells of liquid populations (Allen et al., 2006; Table 2). However, U cells also
behave as metabolically active cells and gain features that
have not been described in Q cells. U cells, thus, express
a large group of genes involved in translation, glycolysis,
amino acid metabolism, the pentose shunt, and other
processes (Cap et al., 2012). These cells also have active
autophagy, which could be important for metabolic
reprogramming and the removal of cell components that
are no longer needed. Some of the metabolic pathways
appear to be controlled by the TOR pathway that is active
in U cells and others, such as amino acid biosynthetic
genes, by the transcription regulator Gcn4p (Cap et al.,
2012). As TORC1 is usually linked to active cell growth
under nutrient abundance and Gcn4p is active under
amino acid starvation (Smets et al., 2010), the characteristics of U cells indicate that these elders are regulated by
an unusual combination of nutrient-sensing pathways
(Cap et al., 2012). In addition, although U cells are
located close to the air, they surprisingly decrease their
respiratory capacity (Cap et al., 2012). Such a downregulation of mitochondrial respiration may contribute to the
decrease in ROS in U cells compared with the ROS level
observed in elders that form younger acidic-phase colonies (Vachova & Palkova, 2005). The active TORC1 and
decreased respiration in vital, long-living U cells (Cap
et al., 2012) contrast studies that show increased yeast cell
longevity in liquid populations to be related to permanently reduced TORC1 signaling (by TOR1 gene deletion), leading to a transient increase in mitochondrial
oxidative phosphorylation (Pan & Shadel, 2009; Pan
et al., 2011). The increased respiration is also a characteristic of long-living Q cells in liquid populations (Davidson
et al., 2011).
FEMS Yeast Res 14 (2014) 96–108
101
L cells, a second major subpopulation of elders, localize
to the interior and lower parts of alkali-phase colonies. In
contrast to U cells, L cells are more sensitive to stress,
produce more ROS, and decrease their viability over time.
These cells contain thin cell walls and do not store glycogen and lipid droplets (Cap et al., 2012). According to
these characteristics, L cells resemble the NQ cells
described in liquid cultures (Allen et al., 2006). L cells,
however, gain additional features that are found neither
in NQ nor in Q cells, such as the expression of genes
involved in hydrolytic mechanisms, such as proteolysis.
Autophagy is not active in L cells (Cap et al., 2012),
although it was described in NQ cells in stationary-phase
liquid cultures (Allen et al., 2006). Some characteristics of
L cells indicate that these cells decrease their cell content
over time and provide compounds important for the
feeding of U cells (Cap et al., 2012).
The above data thus show that two groups of elders
(each harboring-specific features) have different functions
and different fates (related to their metabolic program
and longevity) when coexisting in the central parts of
aging giant colonies. In addition, analyses of colonies
formed by specific knockout strains (Cap et al., 2012)
suggested that these two subpopulations somehow mutually interact and affect each other over the course of
long-term colony development. When, for example, typical L cells are not developed, U cells decrease their longevity (in some cases). Thus, the long-term perspective of
a colony population is dependent on the formation of
different types of elders.
It was shown recently that microcolonies of S. cerevisiae
laboratory strains (growing on complex glycerol medium
as giant colonies) also differentiate and form two major
layers of upper and lower cells. Similarly to giant colonies, upper/lower cell formation in microcolonies is
linked to ammonia signaling, and accordingly, these cells
can be observed as early as day 4 in densely plated microcolonies that have already started to produce ammonia
(Vachova et al., 2013). Upper cells of microcolonies gain
most of the features of U cells of giant colonies, while
only some features of L cells are acquired by microcolony
lower cells. These findings indicate that some features of
L cells of giant colonies (such as stress sensitivity) are
related to long-term chronological aging of these cells.
Other features, particularly those typical of U cells, are
independent of the length of chronological aging (being
the same in 15-day-old giant colonies and 4-day-old
microcolonies) and probably dependent on ammonia
signaling and related metabolic reprogramming (Vachova
et al., 2013).
Some of the features of U and L cells have been
described in ‘outside’ and ‘inside’ cells, respectively, the
cells separated by FACS from 4-day-old microcolonies of
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Published by John Wiley & Sons Ltd. All rights reserved
102
the laboratory strain BY4741 growing on complex glucose
medium (Traven et al., 2012; Table 2). In contrast to
studies of upper cells from microcolonies and U cells of
giant colonies, the ‘outside cell’ population also includes
cells from margin colony areas (M in Fig. 2). Comparison
of the transcriptomes of ‘outside’ and ‘inside’ cells
showed that similarly to U cells, ‘outside’ cells express
genes coding for proteins of the translational machinery
and ribosomal proteins, amino acid metabolic genes,
genes for glycolytic enzymes, and the genes for enzymes
involved in cell wall biosynthesis. ‘Outside’ cells also seem
to produce all three Ato proteins that are typical markers
of U cells and are related to ammonia signaling. ‘Inside’
cells upregulate the expression of some genes involved in
the mitochondrial respiratory chain and genes for cellwall-degrading enzymes, as we also observed in L cells.
Interestingly, a high proportion of genes upregulated in
either ‘outside’ (17% of genes) or ‘inside’ (27% of genes)
cells could be controlled by the transcription factor Sok2p
(Traven et al., 2012), which is important for ammonia
signaling and colony entry to the alkali phase (Vachova
et al., 2004). ‘Outside’ and ‘inside’ cell analysis also
showed a large number of noncoding transcripts
expressed differently in these two cell types (Traven et al.,
2012). This observation suggests a role for post-transcriptional regulation in yeast colony development that could
be involved in the diversification of elders in aging
colonies.
Early differentiation of structured biofilm
colonies of wild Saccharomyces cerevisiae
strains – first elders are formed
The spatiotemporal development of smooth colonies
formed by S. cerevisiae laboratory haploid strains (Vachova
et al., 2009a, b; Cap et al., 2012) significantly differs from
the development of structured biofilm colonies of
S. cerevisiae natural diploid strains (Vachova et al., 2011).
Although structured colony development starts with an
exponential phase similar to smooth colonies, after about
40 h of colony growth on complex respiratory medium,
layers of nongrowing cells start to be formed at the colony surface (Vachova et al., 2011). These cells are thus
formed when the colony has sufficient nutrients that, in
addition, can diffuse effectively into surface cell layers as
shown by confocal microscopy (Vachova et al., 2011). In
contrast, in acidic-phase smooth colonies, elders typically
appear in internal areas at first (Cap et al., 2012). Over
the next several hours, the elders cover the whole of the
structured colony from the side facing the air, while the
cells inside, as well as those cells invading the agar and
differentiating to pseudohyphae, still continue their
growth and division (Vachova et al., 2011; Fig. 2). As
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Published by John Wiley & Sons Ltd. All rights reserved
Z. Palkov
a et al.
stationary-phase cells are more resistant to certain stresses, the transition of the surface cells to the elder state
could contribute to the mechanisms of biofilm colony
protection. Interestingly, as the surface of the structured
colony significantly increases over the following hours
(and days), new elders should differentiate from growing
cells over time. In addition, as only the uppermost surface layer of elders produces multidrug resistance transporters able to remove toxic compounds (Vachova et al.,
2011), elders seem to be diverse and form at least two
subpopulations differing in location within the colony. At
present, no more information on the further development
and chronological aging of elders in biofilm colonies is
available.
Within a Candida albicans biofilm, the cells producing
the stationary-phase-specific proteins Sno1p and Snz1p
(Uppuluri et al., 2006) localize mostly to the basal,
acrylic, surface-adhered cell layers. This suggests that an
acrylic-adhered biofilm contains a few stationary-phase
cells located in the basal adhered cell layer, while the rest
of the biofilm consists of a range of slow-growing proliferating cells. It is interesting to note that a population of
multidrug-tolerant C. albicans persister cells has only
been detected in biofilms (LaFleur et al., 2006). Development of these persisters seems to be dependent upon
adherence. There are insufficient data at present to determine whether any of the cell types observed in complex
yeast colonies (such as elders producing multidrug resistance transporters) constitutes a typical persister cell
phenotype.
Sporulation, another type of elder cell
differentiation
In contrast to haploid yeast cells that can only enter a stationary phase under nutrient starvation, S. cerevisiae diploid cells have an additional possibility. These cells can
activate an alternative developmental program, which
includes meiotic division and cell conversion to resistant
spores. Interestingly, it was shown that in contrast to
liquid cultures where cells sporulate randomly, in colonies
of diploid S. cerevisiae strains grown on sporulation
inducing acetate agar, cells located at specific positions
within the colony initiate the sporulation program
(Lindegren & Hamilton, 1944; Piccirillo et al., 2010).
Thus, after cells have ceased growth, sporulating cells (as
documented by the production of sporulation-specific
proteins and subsequent formation of spores) appeared at
two positions within the colony, one near the agar at the
colony bottom and a second one in cell layers inside the
colony. Later, the sporulation extends from the interior
to all cells located in upper colony areas. The boundary
between this upper part of the colony composed of
FEMS Yeast Res 14 (2014) 96–108
Differentiation of aging yeast populations
sporulating cells and internal parts containing cells that
do not sporulate becomes sharp (Piccirillo et al., 2010).
Thus, interestingly, the area of sporulating upper cells
roughly corresponds to the U cell area, and the area of
internal nonsporulating cells, to the area of L cells
(Fig. 2). In addition, a c. 1–3 cell thick layer of sporulating cells on the bottom of the colony (Piccirillo et al.,
2010) corresponds to a thin layer of bottom cells found
in differentiated alkali-phase colonies (Vachova et al.,
2009a). These very bottom cells have not been thoroughly
characterized yet, but some of their properties (such as
the production of some proteins including Ato proteins)
resemble the properties of U cells.
In addition to pattern similarities between sporulating
colonies of diploids and alkali-phase colonies of haploids,
indications exist that some alkali signals are also involved
in the formation of the sporulation pattern. These indications come from an analysis of chimeric colonies formed
by two mixed strains, which revealed a role for alkali
signals (and maybe also for CO2) that are sensed by the
Rim101p pathway (Piccirillo et al., 2010). This pathway is
involved in extending sporulation to cells in upper colony
areas. In addition, the mutual transmission of some signals between the two different cell types within a chimeric
colony has been observed (Piccirillo et al., 2010).
A similar sporulation pattern was observed in differently
structured colonies including typical smooth colonies of
laboratory strains, more structured colonies formed by a
Σ1267-derived strain, and structured colonies of natural
S. cerevisiae isolates. In structured colonies, pseudohyphae
invading the agar also efficiently sporulate (Piccirillo &
Honigberg, 2010).
Starvation of various nutrient sources
induces S. cerevisiae entry into
stationary phase and partially affects
the metabolic state of early-stage
stationary-phase cells
The different types of elders discussed above are formed
under conditions when a yeast population (within a colony or in liquid medium) gradually consumes nutrients,
and the cells accordingly change their physiology and
enter the slow-growing and stationary phases. In other
words, as nutrients, such as carbon and nitrogen sources,
are gradually diminished, cells adapt to such situations
and then persist for a long time in a ‘nongrowing’ stationary phase. Additional changes linked to colony differentiation could occur much later in chronologically aging
cells of 7- to 10-day-old giant colonies (Cap et al., 2009,
2012; Vachova et al., 2009b). At present, little is known
of whether early-stage starvation and aging could affect
differentiation occurring much later. Such questions are
FEMS Yeast Res 14 (2014) 96–108
103
related to speculation concerning whether one or more
different programs of aging could occur in yeast, as well
as if and how they are potentially affected by shortages of
particular nutrients (Klosinska et al., 2011). For example,
some effects seen in batch culture are not observed in
chemostat culture (Saldanha et al., 2004), and the absence
of some amino acids may activate or inhibit important
nutrient signaling pathways (Powers et al., 2006). Furthermore, studies of starvation for ammonia or other
nitrogen sources may have failed to consider the effects of
concomitant starvation for particular amino acids, especially in auxotrophic strains (Cakar et al., 2000). In this
section, we therefore briefly show only some examples of
the consequences of yeast entry to a stationary phase,
which is not balanced, but is evoked by a shortage of one
particular nutrient.
Cells of early stationary phase resemble slowgrowing cells with some characteristics
independent of the type of limiting nutrient
Short-term starvation often induces a stress response,
which may enable a cell to survive by reducing growth,
recycling nutrients, and reprogramming metabolism, and
this response depends upon the specific nutrient for
which the cell is starved (a lack of nitrogen, carbon,
phosphate, sulfate, or some other nutrient; Boer et al.,
2010; Klosinska et al., 2011; Table 3). There is some evidence that the early stage of a starvation-induced stationary phase may actually constitute an extended period of
slow growth. This evidence comes from observations that
a number of cellular properties including increased stress
resistance and thickened cell wall are typical of slowgrowing cells and persist in cells in an early stationary
phase (Elliott & Futcher, 1993; Lu et al., 2009; Klosinska
et al., 2011). In addition, it was shown that the stress
resistance of slow-growing cells was not related to a particular phase of their cell cycle (Elliott & Futcher, 1993).
These findings also indicate that the differences in stress
sensitivity observed in some types of elders develop later
during cell development and differentiation and are probably not directly related to cell entry to a G0 stage of the
cell cycle (or cell cycle arrest).
Many transcriptional changes during starvation in an
early stationary phase also resemble those occurring during
slow growth under nutrient limitation, where the expression of many genes, including most environmental stressresponsive genes, is proportional to the growth rate (Brauer
et al., 2008; Klosinska et al., 2011). As the growth rate
decreases, the expression of many general stress-response
genes, genes involved in autophagy, and peroxisomal genes
increases, while the expression of many ribosomal,
mitochondrial protein transport and cell-cycle-related
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Z. Palkov
a et al.
104
Table 3. Some properties of stationary-phase cells under different starvation
Reference(s)
Boer et al. (2010), Gresham et al. (2011) and Klosinska et al. (2011)
Biological process
Limiting nutrient
Glucose
Ammonia
Phosphate
Leu
Ura
Stress/resistance*
Zymolyase resistance
Heat shock resistance
Ethanol resistance
Trehalose
Amino acids
Nucleotides
Autophagy
Higher
Higher
N/A
High
Low
High
Higher
Higher
Higher
Very high
Low
High
Higher
Higher
N/A
High
High
Low
Yes†
Higher
High
High
Mixed
Yes†
Higher
High
Storage compounds
Intracellular metabolite concentrations
Degradation
Low
*Compared with exponential-phase cells.
†
Autophagy mutants were less viable than wild type when starved for phosphate or leucine.
genes decreases, irrespective of the type of starvation
(Brauer et al., 2008; Klosinska et al., 2011).
Metabolic profile of cells in early stage of
stationary phase depends on type of limiting
nutrient
In contrast to cell properties and gene expression (characteristics that are similar under different types of starvation), metabolic profiling showed that other changes in
starving yeast cells initially depend upon the nutrient
being withheld, being probably caused by changes in
metabolic pathways due to a particular type of starvation
(Klosinska et al., 2011). Intracellular metabolite concentration is, thus, often dependent upon the nature of the
starvation (nucleoside and base biosynthesis is dependent
upon nitrogen level, and the accumulation of TCA cycle
intermediates upon carbon level). Glycolysis is, however,
affected by most types of starvation, and so the downregulation of glycolysis may be a general starvation
response (Klosinska et al., 2011).
By comparing the viability of mutants exposed to specific nutrient limitations, subsets of genes were identified,
which facilitated survival during starvation of a particular
nutrient. Thus, genes involved in mitochondrial function
and respiration facilitate survival during glucose starvation, while those mediating vacuolar function and autophagy enable cells to survive nitrogen starvation
(Klosinska et al., 2011). In addition to metabolic genes,
the importance of regulatory genes also differs in relation
to the particular type of starvation. For example, Rim15p,
the TORC1 complex, and downstream signaling components (Kog1p and Sch9p) facilitate the survival of nitrogen-starved cells (Boer et al., 2008; Klosinska et al.,
2011), while the AMP kinase and RAS/PKA pathways are
involved in the survival of cells under glucose starvation
(Klosinska et al., 2011).
These data show that many characteristics of yeast cells
in an early stationary phase are similar to those of
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
slow-growing cells. In addition, although changes related
to general stress response occur in cells under conditions
of any type of starvation, other early changes, in particular those related to metabolism, are starvation-typespecific. In a similar manner, early-stage quiescence in
mammalian fibroblasts is accompanied by transcriptional
changes, depending upon the means by which quiescence
was induced (Coller et al., 2006). The absence of mitogens induces the downregulation of genes involved in the
synthesis of DNA, RNA, and proteins and the upregulation of antiproliferation genes. Loss of cell adhesion
induces the downregulation of genes involved in lipid,
carbohydrate, and nucleotide metabolism and the upregulation of MAP kinase inhibitors. Contact inhibition (due
to high cell density) induces the upregulation of E2F4, a
quiescence-specific inhibitor of transcription (Coller
et al., 2006).
Viability, morphology, and carbohydrate
accumulation in starved cells is dependent
upon limiting nutrient
The viability of starved cells depends upon the type of
starvation. It seems that the deprivation of phosphorus or
sulfur is less detrimental for yeast cell survival than that
of nitrogen, carbon, or auxotrophic starvation (Boer
et al., 2008; Brauer et al., 2008). Auxotrophic starvation
of auxotrophic strains results in a highly enhanced loss of
viability, compared with nonauxotrophic phosphorus and
sulfur starvation, and the degree of lethality was greater
on fermentable than on nonfermentable carbon sources
(Boer et al., 2008). Cells deprived of an auxotrophic
nutrient also fail to execute proper cell cycle arrest
(Saldanha et al., 2004) and thus seem to be unable to
reprogram for the subsequent stationary phase. This finding may explain the low viability of cells under auxotrophic starvation. Deleting TOR1 or PPM1 at least partially
abrogates auxotrophic starvation-induced death (Boer
et al., 2008).
FEMS Yeast Res 14 (2014) 96–108
Differentiation of aging yeast populations
A number of morphological changes occur in cells during a starvation-induced stationary phase. Limiting sulfate
or phosphate levels lead to a reduced or increased cell
volume, respectively, which may result from changes in
the relative rates of growth and division of cells prior to
their entry into the stationary phase. Thus, for example,
when phosphate is limited, the cells can continue to
grow, but not divide (Saldanha et al., 2004). It has been
shown in laboratory strains that when a leucine auxotroph is deprived of leucine a single, large vacuole forms
during the exponential phase, remaining until the stationary phase, and many cells are arrested in G1. However,
where leucine is plentiful, the vacuole fragments early in
the exponential phase and fewer cells are arrested in G1
(Cakar et al., 2000). Restricting calories in an anaerobic
retentostat culture (a culture in which cells are retained,
and nutrients replaced) increases the incidence of intracellular lipid droplets, but it is likely the inability of cells
in an anaerobic culture to carry out beta oxidation that
leads to lipid accumulation (Boender et al., 2011). Limiting the carbon or nitrogen source in a culture induces
the accumulation of trehalose and glycogen; the accumulation of both is influenced by the length of the G1 phase.
Trehalose level is directly correlated with the length of
the G1 phase, but the accumulation of glycogen is also
dependent upon Cln3p, which mediates cell cycle progression (Paalman et al., 2003). When carbon-limited
yeast in chemostat-grown cultures are starved of either
carbon or nitrogen, there is a linear relationship between
the poststarvation fermentation capacity and the prestarvation accumulation of storage carbohydrate, but this is
not the case for nitrogen-limited yeast (Thomsson et al.,
2005). It appears therefore that stationary-phase cellular
development is highly dependent upon prestationaryphase nutrition and that the limiting nutrient could have
profound effects upon the experimental outcome.
Conclusions and perspectives
Although we are still very much at the beginning, knowledge obtained over the last few years has clearly shown
that yeast cells can occur in many more different developmental stages than was previously thought. In addition to
cells in different stages of the mitotic cycle, nondividing
yeast cells in stationary phase that we have designated
‘elders’ are also not uniform and can differentiate to various cell types. Different types of elders, differing in their
properties and subsequent fates, are formed in both liquid
cultures and within colonies developing on solid media.
Some examples of elders derived from either haploid or
diploid strains are depicted in Figs 1 and 2. However, the
properties of elders created under each of these conditions (liquid cultures vs. colonies) are rather different.
FEMS Yeast Res 14 (2014) 96–108
105
Under both conditions, elders could be affected by their
‘history’. Such history may include which medium was
available in the past, which nutrients have been limiting,
how the cell culture has been provided with oxygen.
Another relevant factor in an elder’s history could be
whether the particular cell divided during the exponential
growth phase and became a mother cell or was blocked
somehow as a daughter cell. In both conditions (liquid
cultures and colonies), the cells could also be affected by
some signaling molecules functioning as quorum-sensing
molecules, that is, the molecules that can accumulate during the liquid culture/colony growth and activate changes
based on the population density. In colonies, however, in
contrast to liquid cultures, the cells could sense their
position within the population, they could be affected by
precisely localized gradients of signaling compounds, and
they can even mutually communicate via oriented cell–
cell interactions. From this point of the view, elders
within colonies have more possibilities available to them
that could be involved in their differentiation. These possibilities include mechanisms similar to mechanisms that
are involved in the development of metazoa.
How does position within the colony affect the differentiation of particular cells? Interestingly, both vital haploid U cells (Cap et al., 2012; Vachova et al., 2013) and
sporulating diploid cells (Piccirillo et al., 2010) mostly
localize to upper parts of differentiating colonies. In contrast to margin cells that, according to their position
within colonies, have relatively good access to nutrients,
upper central cells are located further from the nutritive
agar than less viable inner and lower central cells. However, when taking into account those colonies occupying
solid surfaces in a natural environment, their upper cells
have a greater probability of being spread to the surroundings (e.g. by water) than cells within the colony
interior. Thus, the higher fitness of upper elders is profitable for the population, because it provides these cells
with a better chance of reaching and colonizing new territories, which, when rich in nutrients, enable the elders to
renew growth and form new colonies. In addition, the
stress resistance of upper elders provides them with a better chance of surviving various harmful environmental
attacks such as changes in temperature or exposure to
extracellular toxic compounds.
How do signaling molecules on one side and nutrients
on the other side affect the survival, resistance, and longevity of elders as well as the formation of different types
of elders? This is an important question that is also
related to discussions of how starvation of a particular
nutrient affects yeast cell features and longevity (Klosinska
et al., 2011). In contrast to potential signaling molecules
that are currently mostly unknown, there are data indicating the effects of nutrients. As shown by a number of
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Z. Palkov
a et al.
106
studies, the early cell response to impending starvation of
glucose, ammonia, and other nutrient sources is similar
in terms of cell stress resistance and the expression of
some prominent gene groups. Thus, stress resistance and
increased cell wall thickness belong to the small group of
features that are similar in long-living elders both in
liquid media and in colonies. On the other hand, the
content of metabolites, such as amino acids, nucleosides,
and bases, differs during nutrient exhaustion, depending
on the type of the starvation. Similarly, different genes
have been shown to support cell survival under different
starvation conditions. This means that at least some of
the features of elders could be induced by a particular
starvation and thus by the nutrient sources available.
Thus, for example, a large, single vacuole in L cells of
colonies could be reminiscent of leucine starvation (Cakar
et al., 2000), and the upregulation of genes for mitochondrial respiration (Cap et al., 2012; Traven et al., 2012)
could facilitate L cell survival during glucose starvation
(Klosinska et al., 2011).
As shown here mature colonies consist of differentiated
cell subpopulations with specific properties, ranging from
different types of elders to still proliferating cells. Cell
groups with longevity features could enhance the survival
prospects of the population as a whole, while some other
cells could function as ‘supporting’ cells that potentially
could die after accomplishing their task. Different cell
types have characteristics, which suggest differences in the
availability of (or ability to utilize) nutrients: how nutrients are sensed and imported, how intercellular signaling
affects gene expression and metabolism, and how the
signaling pathways interact is still being deduced. Future
experiments will need to be carried out under strictly
defined culture conditions (liquid or solid media, microcolonies or giant colonies, batch or chemostat, aerobic or
anaerobic, standard media and precise age of culture) to
compare the results of different studies and to elucidate
the mechanisms by which yeast are induced to form different cell types with differing viabilities and transcriptional profiles, as well as the signaling pathways that
orchestrate such differentiation.
Acknowledgements
This work was supported by the Grant Agency of the Czech
Republic (13-08605S), Charles University in Prague (UNCE
204013), the Ministry of Education (MSM0021620858),
RVO 61388971, and the European Social Fund and the state
budget of the Czech Republic (CZ.1.07/2.3.00/30.0061).
This publication is also supported by the project ‘BIOCEV –
Biotechnology and Biomedicine Centre of the Academy of
Sciences and Charles University’ (CZ.1.05/1.1.00/02.0109),
from the European Regional Development Fund.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Authors’ contribution
Z.P. and L.V. contributed equally to this work.
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