Biosci. Biotechnol. Biochem., 74 (1), 195–198, 2010
Note
Cell Size Regulation during Telomere-Directed Senescence
in Saccharomyces cerevisiae
Aiko M ATSUI and Akira M ATSUURAy
Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University,
1-33 Yayoi, Inage-ku, Chiba 263-8522, Japan
Received August 25, 2009; Accepted September 25, 2009; Online Publication, January 7, 2010
[doi:10.1271/bbb.90627]
DNA replication without telomerase leads to telomere
shortening and induces replicative senescence. We
found that in a telomerase-deficient budding yeast
mutant, the volume of each telomere-shortened cell
increased as its growth capacity decreased, and that this
process was associated with changes in vacuolar morphology. Senescence-induced cell expansion required
Mec1, a DNA damage-responsive kinase, but not
vacuolar SNARE Vam3.
Key words:
telomere; senescence; budding yeast; cell
size; vacuole
Normal human cells generally have a low level of
telomerase activity, which means that their telomeres are
gradually shortened as the cells continue to proliferate.
The process of declining proliferation potential due to a
loss of telomere sequences, called replicative senescence, is accompanied by a series of physiological
changes. The senescent cells are enlarged and flattened,
and this is associated with increases in the size of the
nucleus and the number and/or size of the organelles,
including the lysosomes and mitochondria.1) In addition,
senescent cells express a marker protein, senescenceassociated -galactosidase.2) These functional changes
are thought to contribute at least in part to the
irreversible alterations in cell physiology that occur
during senescence, although the precise mechanisms and
the contributions of individual factors remain unknown.
The budding yeast Saccharomyces cerevisiae is
widely used as a model organism in analyzing the
mechanisms of telomere-directed cellular senescence.
TLC1 encodes an RNA component of the telomerase that
is essential for its catalytic activity. We constructed a
strain (LSS305) in which the chromosomal allele of
TLC1 is deleted, and in which the defect is complemented by plasmid-borne TLC1. After the plasmid was
eliminated by growing the cells in a counter-selection
medium, a mutant that lacked the telomerase activity was
isolated. Then the cells were successively cultured in
liquid media following the liquid growth potential (LGP)
assay protocol.3) As the mutant cells were cultured
successively, there was a gradual decrease in telomere
repeat lengths as detected with Southern blot (Fig. 1A),
and this was accompanied by a gradual reduction in the
growth rate (Fig. 1B). We measured the volume of each
cell quantitatively using the CalMorph image-processing
program,4) and found that the cell volume increased
y
dramatically as growth capacity was reduced during this
process (Fig. 1C). The growth rate of the mutant was
rescued with the appearance of survivor cells, i.e., cells
that had acquired the ability to maintain their chromosomal ends through a telomerase-independent, homologous recombination-mediated process5) (Fig. 1A and B).
These rescued cells had almost the normal cell volume
(Fig. 1C). This indicates that telomere shorteninginduced senescence in yeasts is associated with the
expansion of cellular volume, and this phenotype is
coupled with impaired growth potential.
Previous studies have found that telomere shortening
activates the DNA damage checkpoint system, resulting
in cell-cycle arrest.3) This process is regulated by
evolutionarily conserved ATM family protein kinases,
including Mec1 and Tel1, in budding yeast. We
examined the role of these kinases in senescenceinduced cell expansion.
MEC1 is essential for viability, and the lethality of the
mec1 mutation is suppressed by the sml1 mutation. As
reported previously,3) the combination of mec1 sml1 with
tlc1 affected the senescence process, showing higher
growth potential than the tlc1 mutant (Fig. 2A). In
addition, the tlc1 mec1 sml1 mutant scarcely increased
the volume, even when growth capacity reached its
lowest level (day 9) (Fig. 2A and B). In contrast, the tlc1
tel1 mutant decreased growth potential and increased the
volume, although the tel1 mutation appeared to affect
cell expansion to some degree (Fig. 2C and D). These
results suggest that the cell enlargement induced by
telomere erosion is mediated by a DNA damage
checkpoint pathway that involves Mec1.
During the senescence process, we observed a
dramatic change in vacuole morphology. In the normal
cells the vacuoles were relatively small, and were
usually comprised of multiple lobes6) (Fig. 3A, a and b).
In contrast, the senescent cells tended to have a large,
prominent vacuole (Fig. 3A, c and d). This morphological abnormality disappeared with the emergence of
survivor cells. We conceived the possibility that vacuole
size is critical to the determination of cell volume upon
senescence, and we examined this possibility using the
vam3 mutant. VAM3 encodes vacuolar SNARE, which
facilitates vacuolar homotypic fusion, and its loss of
function leads to the accumulation of small vesicles with
vacuolar marker proteins.7) The vam3 mutant contained
a number of FM4-64-positive vesicles regardless of
telomere status (Fig. 3A, e–h). Moreover, we found that
To whom correspondence should be addressed. Fax: +81-43-290-3706; E-mail: [email protected]
A. M ATSUI and A. MATSUURA
A
TLC1
(kbp) (day) 0 1 2 3 4 5 6 7 8 9 10 11 12 1 12
23.1
9.4
6.6
4.4
A
Number of cells
(cells/ml)
196
2.3
2.0
10
8
10
7
10
6
10
5
10
4
5
7
9
11
13
Day
1.5
1.0
B
10 8
10 7
10
8000
Cell volume (pixel)
Number of cells
(cells/ml)
B
6
10 5
0
2
4
6
8
10
6000
4000
2000
0
(day) 5
7
9
5
7
9
5
7
9
5
7
9
12
Day
C
C
1500
Number of cells
(cells/ml)
Cell volume (pixel)
2000
1000
500
0
1
2
3
4
5
6
7
8
9 10 11 12
Day
the vam3 mutation did not affect the expansion of cell
size by telomere shortening; the volume of the tlc1 vam3
double-mutant was increased at day 7, as compared with
the volume of the tlc1 single-mutant (Fig. 3B and C).
We conclude that vacuolar fusion and/or morphology is
dispensable for the size regulation of yeast cells with
shortened telomeres. The vam3 mutation exacerbated
growth potential during senescence, and reduced the
appearance of survivor cells (Fig. 3B and D). This
suggests a possible contribution of the VAM3 function to
physiology of telomere-shortened cells.
The finding that expansion of senescent yeast cells is
dependent upon the Mec1-dependent signaling cascade
is consistent with a previous study indicating that Mec1,
but not Tel1, is involved in cell-cycle arrest during
8
10
7
10
6
10
5
10
4
TLC1
5
7
9
11
Day
D
Cell volume (pixel)
Fig. 1. Reduced Growth Rate of Telomerase-Defective tlc1 Cells
Was Associated with Increased Cell Volume.
A, Telomere shortening in tlc1 cells. LSS305 (tlc1) and
LSS296 (TLC1) cells were cultivated successively using the LGP
assay protocol, and telomere length was determined by Southern
blotting using the telomere repeat probe. Each cell was cultured in
SC-Ura medium for 1 d and in SC plus 5-FOA medium for 12 d. B,
Growth potential measurements. Cultures of LSS305 (square) and
LSS296 (diamond) cells were analyzed by LGP assay. The samples
are the same as those described in A. C, Cell volume measurements.
The volumes of the cells stained with FITC-Con A were measured
using the CalMorph program for LSS305 cells from the corresponding days in A. The box-plot shows the medians (central cross figure)
with the 25th and 75th percentiles (box). The lower line protruding
from the box ends at the 10th percentile, whereas the upper line ends
at the 90th percentile.
10
10000
8000
6000
4000
2000
0
(day)
5
7
5
7
5
7
5
7
TLC1
Fig. 2. Deletion of MEC1 Affected Cell Size and Rescued the
Growth Potential of Telomerase-Negative Cells.
A, Effect of the mec1 mutation on the growth potential of
telomere-shortened cells. Segregants of AMY101 (TLC1/tlc1
MEC1/mec1 sml1/sml1) were cultivated successively using
the LGP assay protocol. Growth potential was measured for
AMY101-1B (tlc1 mec1 sml1), AMY101-1D (tlc1 sml1),
AMY101-9C (mec1 sml1), and AMY101-9D (sml1) cells. B,
Effects of the mec1 mutation on cell enlargement. The volumes of
the cells from the corresponding days in A were determined as in
Fig. 1C. C, Effects of the tel1 mutation on growth potential of
telomere-shortened cells. Segregants of AMY82 (TLC1/tlc1
TEL1/tel1) were cultivated successively using the LGP assay
protocol. Growth potential was measured for AMY82-7A (tlc1
tel1), AMY82-1A (tlc1), AMY82-1D (tel1), and AMY82-1C
(TLC1 TEL1) cells. D, Effects of the tel1 mutation on cell
enlargement. The volumes of the cells from the corresponding days
in C were analyzed.
Regulation of Cell Size in Senescent Yeast Cells
A
a
b
e
f
c
d
g
h
B
Number of cells
(cells/ml)
10 8
∆tlc1
∆tlc1 ∆vam3
∆tlc1 ∆vam3
10 7
10 6
TLC1
∆vam3
10 5
10 4
10 3
5
7
9
11
Day
C
Cell volume (pixel)
8000
6000
4000
2000
0
(day)
5
7
∆tlc1
5
7
5
7
∆tlc1 ∆vam3 ∆tlc1 ∆vam3
5
7
TLC1
5
7
∆vam3
D
102
Viability (%)
197
8)
∆tlc1
∆tlc1 ∆vam3
∆tlc1 ∆vam3
TLC1
∆vam3
10
1
10-1
senescence. It appears likely that the increase in cell
size is the result of continued metabolic growth of the
arrested cells. In addition to cellular senescence triggered by telomere erosion, wild-type yeast mother cells
undergo a limited number of divisions due to the process
known as replicative senescence.9) Genome instability
has been found to be involved in this type of senescence,
and replicatively senesced yeast cells also showed
increases in cell volume.10) It would be interesting to
examine the possible contribution of Mec1 to cell
morphology during replicative senescence.
Previous studies have suggested a functional relationship between telomeres and vacuoles in yeast; vps
mutations, which cause defects in vacuolar protein
sorting, reduce the steady-state lengths of telomeric
repeats,11,12) although the underlying mechanism remains unknown. Adding to previous results showing that
vacuolar function is somewhat important to the maintenance of telomeres in telomerase-positive cells, our
results suggest that the vacuole is of specific importance
when the telomeres are critically shortened. The morphology of the yeast vacuole is dynamically changed in
response to extracellular and intracellular stimuli.6)
Since signaling pathways that are activated upon
exposure to environmental stresses have been linked to
senescence-induced gene expression,13) the activation of
a stress-responsive pathway might contribute to the
observed vacuolar enlargement in senescent cells.
Further studies are required to elucidate the mechanism
and the functional significance of changes in vacuolar
morphology during senescence.
In plant cells, vacuoles contribute to increasing cell/
body size during growth.14) For example, VAM3 has
been identified as an allele that confers a dwarf
phenotype upon Arabidopsis.15) In contrast, cell-size
control in the yeast vam3 mutant operates in an orderly
fashion under normal physiological conditions. Moreover, we found that the senescent cells expanded in
volume without vacuolar homotypic fusion, which
suggests that yeasts exert cell-volume regulation without
vacuolar fusion. Thus the mechanisms and/or strategies
for cell size regulation might differ between plants and
unicellular fungi.
Acknowledgments
10-2
5
7
9
11
Day
Fig. 3. VAM3 Was Dispensable for Senescence-Induced Cell Enlargement.
A, Vacuolar morphology. The vacuolar membranes of the AMY1
(tlc1) and AMY2 (tlc1 vam3) cells were stained with FM4-64
and observed by fluorescence microscopy. Each cell was cultured in
SC-Ura medium for 1 d, in SC plus 5-FOA medium for 2 d, and in
YPD medium for 10 d. The panels show AMY1 cells on day 1 (a, b)
and day 5 (c, d), and AMY2 cells on day 1 (e, f) and day 5 (g, h). a,
c, e, and g, differential interference contrast microscopy; b, d, f, and
h, fluorescence microscopy. Bar, 10 mm. B, Growth potential
measurements. Cultures of AMY32-9A (tlc1), AMY32-10C and
9D (tlc1 vam3), AMY82-1C (TLC1 VAM3), and AMY32-9C
(vam3) cells were analyzed by LGP assay. The cells were cultured
in YPD medium for 8 d. C, Cell volume measurements. The volumes
of the cells stained with FITC-ConA were measured for the cells
from the corresponding days in B. D, Viability. Colony-forming
units (CFUs) were measured for the cells from the corresponding
days in B. Bar, standard deviation.
We thank Dr. K. Fujimura-Kamada for gifts of
strains. This work was supported in part by a Grant-inAid from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan, and a grant from the
Toray Science Foundation.
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