Department of Biology, Queen's University, Kingston, ON, Canada, K7L
3N6
*
Author for correspondence (e-mail:
youngpg{at}biology.queensu.ca
)
Accepted May 18, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Fission yeast, Intracellular pH, Cell cycle, Ratiometric pH-sensitive GFP
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intriguingly, artificially raising pHi in Dictyostelium
results in an increased rate of both protein and DNA synthesis (Aerts et al.,
1985). This has led to the
suggestion that cytoplasmic alkalinization serves as an on/off trigger for
passage through `start'. In mammalian cells, however, artificially raising the
pHi of mitogen-deprived cells does not by itself trigger division,
although blocking the rise can stop cellular proliferation upon exposure to
mitogens (Moolenaar, 1986
;
L'Allemain et al., 1984
;
Pouyssegur et al., 1985
; Lucas
et al., 1988
).
Typical methods for intracellular pH measurement have included the use of
H+-selective microelectrodes (Gerson and Burton,
1976), 31P-NMR
(Gillies et al., 1981
; Civan
et al., 1986
; Barton et al.,
1980
; Navon et al.,
1979
), radiolabeled
membrane-permeable weak acids (Gillies and Deamer,
1979
; Schuldiner and
Rozengurt, 1982
; L'Allemain et
al., 1985
; Anand and Prasad,
1989
) and pH-sensitive
fluorescent dyes (Grinstein et al.,
1984
; Gillies et al.,
1990
; Haworth and Fliegel,
1993
; Pena et al.,
1995
). In all of these cases
the methods used have required the extensive manipulation of cells. This has
ranged from centrifugation and re-suspension at high density in
non-physiological buffers, to the electroporation (or de-energizing) of cells
to allow entrance of fluorescent dyes. The discrepancy in the timing and
duration of pHi fluctuations reported for different organisms thus
raises the question of the possible consequences of such pre-treatments on the
accurate estimation of pHi.
Furthermore, experiments addressing the role of pHi in cell-cycle progression have called for the synchronization of cultures through starvation, heat shock, or the use of temperature-sensitive mutants. These experiments have thus failed to address the simple question of the status of intracellular pH in an unperturbed exponentially growing culture. In this report we have used a non-invasive method of intracellular pH measurement to definitively determine the limits of pH homeostasis and whether or not pH fluctuations are associated with progression through the cell cycle under physiologically relevant conditions.
The method chosen involves the use of a ratiometric pH-sensitive GFP (as
opposed to non-ratiometric alternatives; Kneen et al.,
1998; Llopis et al.,
1998
; Robey et al.,
1998
; Elsliger et al.,
1999
). This engineered
derivative of the wildtype GFP (referred to as `phluorin') has a bimodal
excitation spectrum, but unlike wildtype GFP, the relative emission
intensities at the excitation wavelengths used show striking pH dependence
(Miesenbock et al., 1998
). By
expressing phluorin in fission yeast cells, and examining them microscopically
under native conditions in a flow cell, we have an exquisitely sensitive and
stable measure of pHi. We are thus able to examine wild-type and
mutant cells either during growth or following various experimental
treatments.
The use of S. pombe offers a distinct advantage for these studies.
Since S. pombe grows only by extension in length at its growing tips,
it is possible to quickly and easily determine cell-cycle position by the
measurement of cell length (Mitchison,
1957; Mitchison,
1990
). Thus pHi and
cell length can be measured in a random population of unsynchronized,
logarithmically growing cells, and the individual cell data easily correlated
with cell-cycle position.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids
The ratiometric pH-sensitive GFP gene (Miesenbock et al.,
1998), a gift from J. E.
Rothman, was PCR amplified (forward primer: 5'-ggg gga tta ata tga gta
aag gag aag aac ttt tca ctg g-3'; reverse primer: 5'-ggg ggg tcg
act tat ttg tat agt tca tcc atg cca tgt g-3') and cloned into the unique
NdeI and SalI sites of pREP1, or pREP41 (Maundrell,
1993
; Basi et al.,
1993
), using standard
techniques (Maniatis et al.,
1989
). Nuclear-targeted
phluorin was constructed in the same way, but with PCR primers (forward:
5'-gga gga tta ata tga gta aag gag aag aac ttt tca ctg g-3';
reverse: 5'-ggg ggg tcg act tag acc tta cgc ttc ttc tta ggt ttg tat agt
tca tcc atg cca tgt g-3') incorporating the SV40 large T antigen nuclear
localization signal, PKKKRKV (Gorlich and Mattaj,
1996
), to the C-terminus. All
plasmids were transformed into fission yeast strains through electroporation
(Toone et al., 1998
).
Standard curve
A standard curve of the fluorescence of ratiometric phluorin at different
pH values was generated by growing a leu1-32 h+ strain,
expressing phluorin behind the thiamine-repressible nmt1 promoter, in
thiamine-free media to a concentration of 1x107
cells/ml. These phluorin-expressing cells were then permeabilized to
H+ ions in highly buffered media at various pHs as follows. 1 ml
aliquots of culture were collected and washed three times (3000 rpm, 5
minutes) with 1 ml of either succinate pH 5.6, MES pH 6.0, MES pH 6.4, PIPES
pH 6.6, PIPES pH 6.8, MOPS pH 7.0, MOPS pH 7.15, MOPS pH 7.3, HEPES pH 7.45,
HEPES pH 7.6, or Tris pH 8.0. All buffers were at a concentration of 200 mM
and pH-adjusted with NaOH or HCl. Protease inhibitors were added (final
concentrations 15 µg/ml pepstatin A, 1 mM PMSF, 1 µg/ml
o-phenanthroline, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml
antipain), followed by addition of the protonophore CCCP (final concentration
50 µM), sarcosine (final concentration 0.05%) and toluene (final
concentration 0.1%) to aid in permeabilization. Samples were then incubated
for 20 minutes on a rotary shaker, collected, and again washed three times
with 1 ml of the appropriate pH-adjusted buffer. 40 µl aliquots were then
placed onto poly-L-lysine coated coverslips, and the permeabilized cells
allowed to settle for 40-60 minutes. Coverslips were inverted onto a
depression slide filled with the appropriate buffer and sealed with vaseline
before image acquisition (see below). Differences of temperature in the range
of 20-36°C did not alter phluorin calibration curves.
Image acquisition and analysis
All cells were prepared for image acquisition by aliquoting 40 µl of an
early log phase culture to coverslips that had been treated with 0.1%
poly-L-lysine (Pringle et al.,
1991). A 40-minute incubation
was sufficient time for the cells to settle and adhere to the poly-L-lysine
coated surface. Coverslips were subsequently inverted onto depression slides,
sealed with vaseline, and the cells examined. Under these conditions randomly
sampled individual cells monitored under the microscope grew at a rate of
1.61±0.28 µm/hour. This correlates favourably with the calculated
rate of 1.73 µm/hour based on a generation time of 260 minutes
(Table 1) and 7.5 µm of
extension growth per generation. All solutions and glassware were kept at
25°C (with the exception of experiments dealing with the effects of
temperature, where solutions and glassware were maintained at the temperature
being assayed). Images were acquired using a Leitz DMRB fluorescence
microscope (Leica Microsystems), a Lambda 10-2 filter wheel controller (Sutter
Instruments), and a high performance cooled CCD camera (Cooke SensiCam)
operated by Slidebook image analysis software (Intelligent Image Innovations).
Some variation in excitation intensity and/or spectra was noted with the Hg
light source depending on the age of the bulb. This necessitated repeating the
standard curve every time an absolute measure of internal pH was being made.
Excitation was through D420/30X (`low') or D460/20X (`high') excitation
filters (Chroma Technology Corporation) and emission monitored using a JP3 BS
dichroic and D535/50M emission filter (Chroma Technology Corporation).
Emission intensities were determined from the acquired images (using
Slidebook's masking and statistical functions) by randomly sampling a block of
24 to 112 pixels per cellular compartment (i.e. cytoplasm, nucleus, spore or
vacuole) and subtracting background. Cell lengths could be calculated directly
from images using the Slidebook ruler function. Images were taken using either
a 100x or 40x objective (Leica).
|
Flow chamber experiments
80 µl aliquots of an early log phase culture were transferred to
poly-L-lysine-coated 25 mm circular coverslips. After 40-60 minutes, to allow
adherence of cells, coverslips were fitted to the imaging chamber/chamber
heater platform (RC-21BR/PH-2, Warner Instrument Co.), which contained
400 µl of the same media. The chamber was subsequently sealed with
vacuum grease and fitted to the microscope stage. All experiments (unless
otherwise noted in the text) were carried out at 25°C and preceded by a
30-60 minute equilibration time during which the initial media was perfused.
Temperature was controlled using a heater controller (TC-344B, Warner
Instrument Co.) and in-line heater (IA SF-28, Warner Instrument Co.). Changes
in media were controlled using a perfusion valve controller (VC-6, Warner
Instrument Co.). Flow rates were set with a peristaltic pump (P-3, Pharmacia).
The pump was halted during image acquisition. Each image was acquired from a
different field of view to eliminate photobleaching as a variable.
FACS analysis
FACS analysis was performed as described (Alfa et al.,
1993).
Stress tests
Cells were grown in EMM pH 5.5 to early logarithmic phase
(<1x106 cells/ml) and subjected to the following stresses:
(1) centrifugation (3000 rpm, 5 minutes) followed by resuspension in the same
media at the same cell density; (2) centrifugation (3000 rpm, 5 minutes)
followed by resuspension in the same media at a density of
1x108 cells/ml for 3 hours; (3) incubation for 1 hour at
4°C; (4) centrifugation (3000 rpm, 5 minutes) followed by resuspension at
the same density in sterile water buffered to pH 5.5 with 20 mM MES for 1
hour; or (5) electroporation as described (Pena et al.,
1995). Cells were then
transferred to poly-L-lysine-coated coverslips, incubated for a minimum time
(5-10 minutes), and inverted onto depression slides before images were
acquired. The effects of heat shock were assayed in the flow chamber by
shifting the temperature from 25°C to 36°C. The effects of
hyper-osmotic shock were also assayed in the flow chamber by shifting the
growth medium from EMM pH 5.5 to EMM pH 5.5 supplemented with 1 M sorbitol.
Cells were also examined by transferring 2 µl aliquots to a conventional
slide and overlaying with a coverslip. Unperturbed controls were prepared as
described in `Image acquisition and analysis'.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Intracellular pH of wild-type cells was then determined as a function of the extracellular pH of liquid minimal growth media (EMM). Cultures were first grown to early logarithmic phase in the appropriate pH-adjusted media and the images acquired as described (Fig. 1D). Adherence to the coverslip through poly-L-lysine did not appear to have any adverse effects on the cells as judged by their ability to grow normally on the coverslip surface for extended periods of time.
Since fluorescent intensity varied greatly from cell to cell (Fig. 1D) it was critical to ensure that any observed measurements were in fact independent of phluorin levels. We thus expressed the data normalized to the most weakly fluorescing cell in each individual data set (Fig. 1E). Emission intensity varied up to eightfold, but the majority of the data points (89%) were within a fourfold intensity range of the weakest expressing cell. As expected (based on the ratiometric nature of our reporter) pHi values were independent of intensity of signal.
When expressed as a function of external pH, pHi showed a
statistically significant decrease of only 0.14 pH units (from 7.31 to 7.17)
over a change in external H+ ion concentration of three orders of
magnitude (degrees of freedom (df)=4, T=6.93, P<0.05;
Fig. 1F). These estimates are
very similar to the internal pH estimated for higher eukaryotes and are
generally greater than previous estimates for the budding yeast
Saccharomyces cerevisiae (which have ranged anywhere from between 6
and 7.5; see Discussion). This estimate is also in a general agreement with
the only other available estimate in S. pombe, an internal pH value
of 7.0 estimated using carboxy-seminaphthorhodafluor-1 (SNARF) (Haworth and
Fliegel, 1993).
Since our pHi measurements showed remarkable homeostasis, and to test that the system could in fact detect pHi fluctuations in vivo, wild-type cells were prepared as above and fitted into a flow chamber (see Materials and Methods). EMM was used as the initial perfusion medium and thereafter a simple valve switch allowed the medium to be changed to EMM (adjusted to a final pH of 5.5) containing either 15 mM acetic acid, or 200 mM bicarbonate (to lower and raise internal pH, respectively). As shown in Fig. 1G, positive or negative changes of 0.2-0.3 pH units were easily resolved by the system. In both cases the response was transient and the cells regulated back towards their homeostatic set point of 7.2-7.3 while remaining in the acetic acid or bicarbonate media.
Intracellular pH and the cell cycle
To determine whether pH fluctuations were associated with cell-cycle
progression we first attempted to follow the internal pH of single cells over
successive cell cycles. Not surprisingly, we found that repeated exposure to
the high and low excitation wavelengths resulted in a cessation of growth
within 4-5 hours, making the methodology unsuitable. We also observed that
fission yeast cells were exquisitely sensitive to even mild stresses.
Treatments such as centrifugation, temperature shifts, prolonged suspension at
high density, or changes in medium osmolarity, all resulted in statistically
significant changes in pHi (Fig.
2; t-tests assuming unequal variance all gave
P-values <0.05). Since typical methods of syncronization (e.g.
elutriation, lactose gradients, temperature-sensitive mutations) would require
such treatments, we were concerned about their possible artefactual effects on
pHi.
|
As an alternative we made use of the fact that cell-cycle position in
S. pombe can be easily monitored by the measurement of cell length
(Mitchison, 1957; Mitchison,
1990
). This allows us to
photograph populations of cells with only one exposure at each wavelength, and
then to determine pHi in cells of different size. Examples of
S. pombe cells in different phases of the cell cycle can be easily
distinguished in Fig. 1D (see
legend).
As seen in Fig. 3 (upper
left panel), a typical scatter plot of an individual replicate
(n=1000, mean pHi=7.32, s.d.=0.05) clearly shows that
pHi is constant irrespective of cell length. Using the
non-parametric Spearman's rank correlation test (since cell length is not
itself normally distributed), no association between cell length and
pHi was detected (df=998, R=0.008, P>0.05). The slope
of the line of best fit was -0.0002 and attempts to fit 2nd to 6th order
polynomials to the data did not produce curves that deviated significantly
from a straight line. Furthermore, upon classifying the cells into arbitrary 1
µm size classes (Fig. 3,
lower left panel), analysis of variance demonstrated that the means of the
populations were not significantly different (1=9,
2=990, F=1.61, P>0.05).
|
Since S. pombe has an unusually long G2 phase
comprising 70% of the cell cycle, and a G1 comprising only
10% it was conceivable that a fluctuation of pHi in
G1 might be difficult to resolve, and thus formally possible that a
pHi change at the G1/S boundary might be missed. To
examine these possibilities the same methods were applied to a strain carrying
the cdc2-1w mutation (Carr et al.,
1989
). Strains carrying this
mutation maintain a smaller cell size and have an extended G1 phase
(Fig. 1B). The scatter plot of
an individual replicate of this experiment (n=998, mean
pHi=7.29, s.d.=0.05) is shown in
Fig. 3 (upper right panel).
Just as in the wildtype, a linear relationship between cell length and
internal pH is observed. Spearman's rank correlation test showed no
association between pHi and length (df=996, R=-0.002,
P>0.05), and upon classification into 1 µm size classes
(Fig. 3, lower right panel),
analysis of variance showed no significant difference between the means
(
1=6,
2=991, F=1.58, P>0.05).
Having shown that pHi changes were not associated with normal
progression through the cell cycle, we next asked if an association existed
between internal pH fluctuations and discrete delays, or advancements, in
cell-cycle progress. To do this we made use of the fact that entry into
mitosis in S. pombe is governed by a nutritionally sensitive cell
size control. For example, upon shift from a growth media containing a
relatively rich nitrogen source to a media containing a relatively poor
nitrogen source, cells will reduce the size threshold they must attain to
initiate division. In the converse shift the opposite is true (Fantes and
Nurse, 1977). Glutamine
(generation time (tgen)=309 minutes) and proline
(tgen=475 minutes) were chosen as the rich and poor
nitrogen sources, respectively, as both are non-ionizable polar amino acids
and would not be expected to acidify or alkalinize the cytoplasm per se.
The results of an EMM(glutamine) to EMM(proline) nutritional shift are shown in Fig. 4 (left panel). In EMM(glutamine) cell length ranges from approximately 7-15.5 µm. Forty to eighty minutes after the shift one can see a stimulation of division as shown by the increase in the proportion of binucleate cells (open diamonds). This is a result of the cells in the upper portion of the size distribution already being over the new, reduced threshold for mitotic initiation. This is followed by a corresponding shift to the left of the cell size distribution at later time points.
|
In the converse shift (Fig.
4, right panel), cell size in EMM(proline) is initially
distributed from approximately 6-13.5 µm. Forty to eighty minutes after the
shift a delay in division is visible as the proportion of binucleate cells
(open diamonds) falls to zero. This is accompanied by a rightward shift of the
cell size distribution at later time points as the cells grow until they
attain the new, increased size threshold for mitotic initiation. Notably,
internal pH showed no significant changes in the short- or long-term during
shift from glutamine to proline (1=13,
2=378,
F=1.14, P>0.05) or from proline to glutamine (
1=13,
2=378, F=1.69, P>0.05).
Although concerned about the artefactual effects caused by shifts in
temperature, as well as the possible confounding effects caused by loss of
gene function, we ultimately measured pHi in various cell-cycle
mutants arrested at their restrictive temperatures. These mutants included
cdc10-129 (G1 arrest prior to `start'; Aves et al.,
1985), cdc22-M45
(S-phase arrest; Fernandez-Sarabia et al.,
1993
), cdc25-22
(G2 arrest prior to mitosis; Russell and Nurse,
1986
) and nda3-KM311
(mitotic arrest; Hiraoka et al.,
1984
). As seen in
Fig. 5A, temperature-sensitive
mutants as well as wild-type cells shifted to 36°C demonstrated
statistically significant decreases in internal pH (analysis of variance for
each strain gave P-values <0.05). However, wild-type cells
maintained at 25°C showed no significant changes in pHi
(
1=6,
2=143, F=0.138, P>0.05). Thus,
the observed pHi changes are consistent with the effects of heat
shock, as opposed to arrest at different points in the cell cycle. Wild-type
and cold-sensitive nda3-KM311 cells maintained at 36°C and then
shifted to 20°C for 8 hours showed no statistically significant
differences in pHi (Fig.
5B; df=38, T=0.449, P>0.05).
|
Intracellular pH in stationary phase and during sporulation
We next examined the status of pHi in cells in stationary phase,
or G0 of the cell cycle. Cells were grown to early log phase in EMM
and then transferred to media lacking either nitrogen or glucose to allow
entrance to G0. Intriguingly, when starved of nitrogen, it was
found that the phluorin signal moved from the cytoplasm and nucleus, to small
spherical structures located in the cytoplasm. These structures were shown to
be vacuoles (Fig. 6A) by
starving phluorin-expressing ade6-210 cells for nitrogen, and
subsequently localizing a fluorescent intermediate of adenine metabolism
(rhodamine channel) that localizes to the vacuolar compartment and accumulates
in the ade6-210 mutant (Szankasi et al.,
1988).
|
In an attempt to prevent or delay this vacuolar localization the SV40 nuclear localization signal was fused to the phluorin C-terminus. The fusion protein (phluorin-NLS) was correctly targeted to the nucleus, but strong expression from the nmt1 promoter resulted in a slower growth phenotype. This necessitated placing the construct under control of the weaker nmt41 promoter (Fig. 6B). Expression from this promoter did not produce any observable phenotypes and the signal was restricted to the nucleus.
Although one would not expect nuclear pH to differ from cytoplasmic pH
based on the size of nuclear pores, there have been reports asserting pH
differences between the two compartments (Dubbin et al.,
1993; Seksek and Bolard,
1996
). To formally test this,
phluorin and phluorin-NLS-expressing wild-type cells were grown concurrently
to early log phase (i.e. as a mixed population in the same culture) in order
to minimize any potential variation. This was possible since phluorin and
phluorin-NLS-expressing cells are easily distinguished upon image acquisition
based on the localization of the GFP signal. Under these conditions no
statistically significant difference between nuclear and cytoplasmic pH was
observed (Fig. 6C; df=78,
T=0.529, P>0.05). We thus continued with the use of the
phluorin-NLS construct to provide us with an estimate of internal pH.
Upon 36 hours of nitrogen starvation, the phluorin-NLS signal was distributed in the vacuole, as well as in the nucleus (Fig. 6D), allowing both nuclear and vacuolar pH measurements to be made. Interestingly, we found that nitrogen-starved cells maintained a reduced nuclear pHi relative to logarithmically growing cells (pH 6.6 versus 7.3; Fig. 6E). We also found that these cells were able to tightly regulate their internal pH in response to wide changes in external pH. Over a change in H+ ion concentration of three orders of magnitude from pH 6.5 to 3.5, nuclear pH dropped by only 0.13 pH units. Nuclear pH in logarithmically growing cells, although maintained approximately 0.60 to 0.65 pH units higher, decreased to a similar extent (0.15 pH units) over the same external pH range. Vacuolar pH was maintained approximately 0.4 units lower than nuclear pH and was also tightly controlled over wide changes in external pH (Fig. 6E).
We next nitrogen-starved a homothallic phluorin-NLS-expressing strain to induce mating, and the formation of asci (Fig. 6F). We found that quiescent spores, just as nitrogen-starved cells, maintained a lower pHi than logarithmic phase cells, and that, remarkably, pHi decreased to an extent similar (0.05 pH units) to that seen in logarithmically growing cells over the external pH range of 6.5 to 3.5 (Fig. 6E). To ensure the validity of these results, the viability of spores and of nitrogen-starved cells was determined by plating samples to rich media. Viability as measured by colony formation was >85% at all external pHs tested.
Intriguingly, glucose-starved cells demonstrated no translocation of the phluorin signal to the vacuoles, and an even greater reduction in pHi. These cells also demonstrated a compromised ability to regulate their internal pH in response to even moderate changes in external pH (Fig. 6G). Over an external pH range of 6.6 to 5.7 the internal pH of glucose-starved cells decreased by 0.46 pH units (from 6.21 to 5.75), whereas, over the same external pH range, exponentially growing cells did not show a statistically significant difference.
Since cells starved of glucose lack both a carbon and an energy source, we
repeated the experiment by growing cells in EMM, and shifting them to
EMM(minus glucose) in the presence of 1% ethanol. Although fission yeast cells
are unable to utilize ethanol as a sole carbon source for growth (since they
lack a functional glyoxylate cycle; Fiechter et al.,
1981; Tsai et al.,
1987
) ethanol can be used as
an energy source through its conversion to acetyl-CoA and entrance into the
TCA cycle (Tsai et al., 1987
).
Remarkably, under these conditions, glucose-starved cells behaved similarly to
cells starved of nitrogen. The phluorin signal moved to the vacuoles, and the
cells demonstrated a reduced, but homeostatically maintained, internal pH (as
measured using the phluorin-NLS construct). As in nitrogen-starved cells
vacuolar pH was maintained
0.4 units lower, and was also homeostatically
maintained (Fig. 6G,H). These
results show that the loss of homeostasis upon glucose starvation is a result
of the lack of an energy source.
Since wild-type cells show a cell-cycle response as well as a
downregulation of pHi when starved of a nitrogen source, we
performed a short-term nutritional shift in which logarithmically growing
cells were transferred from EMM to EMM(minus nitrogen)
(Fig. 7). This is a nutritional
down-shift analogous to the EMM(glutamine) to EMM(proline) shift performed
previously and causes cells to initiate mitosis at a smaller cell size. After
40-80 minutes, entry into mitosis is stimulated as demonstrated by the
increase in the proportion of binucleate cells. This is followed by a
subsequent shift in the cell size distribution to the left (i.e. a decrease in
cell size) at later time points. Notably, no significant change in
pHi was observed in this time frame (1=13,
2=378, F=1.65, P>0.05).
|
Longer term nitrogen starvation studies showed that pHi begins to decrease only between 6 and 12 hours of incubation in EMM(minus nitrogen) (data not shown). Thus the decrease in pHi seen after long-term nitrogen starvation is independent of the initial changes in the regulation of cell-cycle progression. Cells starved of glucose (in the presence of 1% ethanol) do not show any changes in the regulation of cell-cycle progression, but do show a similar pHi decrease starting between 6 and 12 hours (data not shown). These data thus strongly suggest that the drop in pHi is associated with metabolic quiescence as opposed to changes in the progression of the cell cycle.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
pHi is homeostatically maintained at a value around
7.3
These studies have revealed that under optimal growth conditions
intracellular pH is tightly regulated at a value of approximately 7.25-7.35.
This estimate is similar to estimates from higher eukaryotes (Madshus,
1988; Ober and Pardee,
1987
; Schuldiner and
Rozengurt, 1982
; Moolenaar et
al., 1983
; Musgrove et al.,
1987
; Kneen et al.,
1998
; Llopis et al.,
1998
) and suggests that, like
other fundamental biological processes, the regulation of intracellular pH at
or around this value is conserved. This is not surprising considering the
extensive similarity between S. pombe and higher eukaryotes, and the
importance of pHi for protein structure, stability and function
(Madshus, 1988
). This is also
supported by examples of cross-species complementation, which suggest
similarity between intracellular environments (Lee and Nurse,
1987
; Campbell et al.,
1995
; Rodel et al.,
1997
; Jimenez et al.,
1990
).
Comparisons of our pHi estimates to those in S.
cerevisiae are difficult to interpret in this respect owing to the great
variation in internal pH (from 6.0 to 7.5) estimated by different researchers
using different methodologies. These methodologies have included incubation
(2x108 cells/ml) in nitrogen, and glucose-free buffers at
2°C for periods of up to a week (Gillies et al.,
1981), washes with ice-cold
water (Anand and Prasad, 1989
),
centrifugation and suspension in nitrogen- and glucose-free buffers at
temperatures ranging from 0 to 22°C (Navon et al.,
1979
; Carmelo et al.,
1997
; Haworth and Fliegel,
1993
; Sychrova et al.,
1999
; Pena et al.,
1995
), incubation at densities
up to 3x109 cells/ml (Greenfield et al.,
1987
; Gonzalez et al.,
2000
), and electric shocks of
1.5-2.0 V (Pena et al., 1995
;
Sychrova et al., 1999
).
These methodologies, together with our own data, which demonstrate the artefactual effects caused by some of these treatments, strengthen the notion that differences in methodology can have serious consequences for the accurate estimation of pHi. We believe our method to be superior. No S. cerevisiae study employing a ratiometric GFP (or other probe) to monitor internal pH in unsynchronized cells as a function of bud size or cell volume has been reported.
Data also demonstrate that robust mechanisms exist to maintain
pHi at a homeostatic value of 7.3
Fig. 1F shows that artificially
raising or lowering pHi by 0.2-0.3 units elicits an immediate
response that returns internal pH towards initial values within 10-20 minutes.
Furthermore, intracellular H+ ion concentration varies only from
4.90x10-8 M (pH 7.31) to 6.76x10-8 M (pH
7.17) over a 1000-fold increase in external H+ ion concentration, a
small but statistically significant (38%) change in concentration.
Not surprisingly, our data also reveals that the mechanism of pH homeostasis is dependent on the presence of an energy source, since cells starved of glucose (in the absence of ethanol) demonstrate a greatly compromised ability to regulate pHi in response to even minor changes in external pH. In the absence of an energy source, H+ ion concentration shows approximately a threefold increase over a tenfold rise in external H+ concentration.
pHi regulation during the cell cycle
To determine whether pH fluctuations were associated with progression
through the cell cycle, we decided to use methods in which cultures did not
need to be synchronized by any of the commonly used techniques (i.e. lactose
gradients, elutriation, starvation), or even the use of temperature-sensitive
mutants. This was done because relatively minor stresses and perturbations
could have drastic effects on pHi homeostasis
(Fig. 2). Instead we made use
of the simple fact that since S. pombe cells grow only by extension
at their tips, it is possible to monitor cell-cycle position by monitoring
cell length (Mitchison, 1957;
Mitchison, 1990
).
Using large sample sizes in which the pHi and length of individual cells were measured, we found no relationship between intracellular pH and cell-cycle position in wild-type cells. Similar experiments in cdc2-1w mutants, which exhibit a relatively longer G1 and shorter G2 than wildtype, also revealed no relationship between pHi and cell-cycle position. This result further supports the notion that intracellular pH is tightly regulated during logarithmic growth independently of cell-cycle-related parameters. It is thus unlikely to play any signaling or regulatory role.
We next asked if pH fluctuations were associated with induced alterations in cell-cycle progression by employing the S. pombe nutritionally sensitive cell size control. These experiments (Fig. 4) also do not support the involvement of intracellular pH fluctuations in promoting (or being associated with) delays or advancements in cell-cycle progression. Furthermore (despite the artefactual changes in pHi that arose due to heat shock), experiments with temperature-sensitive cell-cycle mutants also showed no relationship between pHi and cell-cycle position. Taking all data together, we conclude that changes in intracellular pH are not part of, and play no role in, progression through the cell cycle during logarithmic growth. By contrast, all data is consistent with a very tight regulation of internal pH around a homeostatic value of 7.3 at all points in the cell cycle.
These results are in contrast to data from several other organisms that
purport to show the existence of cell-cycle-related oscillations of internal
pH (Anand and Prasad, 1989;
Gillies and Deamer, 1979
;
Aerts et al., 1985
; Gerson and
Burton, 1976
). However, it is
important to note that these studies have employed synchronized, and therefore
perturbed, cell populations. Considering the sensitivity of fission yeast to
different stresses, the methods of pHi measurement also raise
concerns as to the possible detrimental effects on normal cellular function
(these methods included treatments such as centrifugation, suspension in
nitrogen- and/or glucose-free buffers, repeated insertions of
micro-electrodes, heat shock, and washes with ice-cold water). In this
respect, it is interesting to note that relatively mild disturbances, such as
gentle centrifugation (800 g, 5 minutes), initiate the stress
response in S. pombe as assayed by the activation of the
spc1/sty1 stress-induced MAPK pathway (Shiozaki et al.,
1998
).
Furthermore, an examination of these studies reveals several fundamental
differences including discrepancies in the magnitude, timing, and duration of
pHi changes. Considering the conservation of the fundamental
mechanisms of cell-cycle control from yeast to humans (Nurse,
1990), we believe that our
results probably represent the true behavior of pHi under these
conditions.
pHi regulation during changes in growth state
We next attempted to investigate pHi in cells that had entered
G0 of the cell cycle through starvation for nitrogen. However,
under these conditions we found that phluorin became targeted to the vacuole.
This result is not surprising, and probably represents an attempt by the cell
to recycle proteins under starvation conditions. Although not studied in
detail in fission yeast, the vacuolar targeting and proteolysis of both
cytoplasmic and plasma-membrane-bound proteins has been well documented in
budding yeast in response to starvation (Van den Hazel et al.,
1996; Volland et al.,
1994
).
The construction of a phluorin-NLS fusion allowed sufficient GFP to remain in the nucleus after 36 hours of starvation to obtain an estimate of internal pH. A signal was also present in the vacuole. These two compartments were easily differentiated and demonstrated two clear and distinct signals with pH estimates of approximately 6.2 for the vacuole, and 6.6 for the nucleus (at an external pH of 5.5). Furthermore, pHi in these compartments was tightly regulated and not very responsive to large changes in the pH of the external media. Quiescent spores demonstrated a reduction in internal pH similar to that seen in cells starved for nitrogen. Moreover, cells starved of carbon, but not of an energy source, demonstrated a similar and homeostatically maintained decrease in pHi.
A reduction in pHi in starved relative to proliferating cells
has been documented in both mammalian cells (Musgrove et al.,
1987), and budding yeast
(Navon et al., 1979
; Gillies
et al., 1981
). In addition,
mitogen deprivation (although physiologically distinct from nutrient
starvation) has been well documented to result in quiescence and a lowering of
pHi (Schuldiner and Rozengurt,
1982
; Moolenaar et al.,
1983
; Busa,
1986
; Moolenaar,
1986
). In this respect, it is
interesting to note research that suggests a correlation of pHi to
simple metabolic changes in budding yeast. For instance, Portillo and Serrano
demonstrated a correlation between reduced growth rate, lowered
H+-ATPase activity, and intracellular pH (Portillo and Serrano,
1989
). Furthermore, Gonzalez
et al. have shown internal pH to decrease from 7.5 to 6.8 upon shift from
aerobic to anaerobic growth (Gonzalez et al.,
2000
).
Intriguingly, our short-term experiments in which the phluorin-NLS construct was used to monitor intracellular pH upon shift from EMM to EMM(minus nitrogen) showed no statistically significant intracellular pH changes. This indicates that the drop in pHi seen upon nitrogen starvation is associated with the longer term transition in metabolic state to quiescence, as opposed to the shorter term changes in cell-cycle control that advance progression into mitosis, and thereby shunt cells into G1 to prepare for mating. This hypothesis is further supported by pHi measurements in cells starved of glucose (in the presence of ethanol), which do not advance progression into mitosis, but show a similar reduction in pHi.
Cytoplasmic, nuclear pH and vacuolar pH
The phluorin-NLS construct allowed a formal testing of the question of
whether nuclear pH differs from cytoplasmic pH. Although one would not expect
a difference based on the sheer size of nuclear pores (which allow entry of a
27 kDa protein such as phluorin itself), there have been reports asserting
differences between the two compartments, at least under certain conditions
(Dubbin et al., 1993; Seksek
and Bolard, 1996
). Others have
found no difference (Bright et al.,
1987
). With respect to other
small, charged ions, such as calcium, a large body of literature exists, both
supporting and refuting the idea that the nucleus can control or modulate
Ca2+ levels independently of the cytoplasm (Macdonald,
1998
; Brown et al.,
1997
; Lui et al.,
1998
; Allbritton et al.,
1994
; Meyer et al.,
1995
; Badminton et al.,
1998
).
Under our experimental conditions, we could detect no difference in pH
between the nucleus and cytoplasm during logarithmic growth. However, it
should be noted that these steady-state experiments do not rule out the
possibility that the nuclear membrane buffers changes in H+ ion
concentration, as has been suggested for Ca2+ (Al-Mohanna et al.,
1994).
With respect to vacuolar pH measurements, it is clear that this
sub-cellular compartment is homeostatically regulated in response to wide
changes in external pH, albeit at a lower set point than the surrounding
cytoplasm. Although no estimates are available in S. pombe, our
measured values are in general agreement with the literature in budding yeast,
which has yielded values of 6.0 (Carmelo et al.,
1997), 6.2 (Preston et al.,
1989
), between 5.5 and 6.0
(Plant et al., 1999
), as well
as between 6.2 and 6.4, depending on the composition of external buffers
(Greenfield et al., 1987
).
Estimates in Candida albicans yielded estimates of between 5.7 and
6.3, depending on growth form (Cassone et al.,
1983
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aerts, R. J., Durston, A. J. and Moolenaar, W. H. (1985). Cytoplasmic pH and the regulation of the Dictyostelium cell cycle. Cell 43,653 -657.[Medline]
Allbritton, N. L., Oancea E., Kuhn, M. A. and Meyer, T.
(1994). Source of nuclear calcium signals. Proc. Natl.
Acad. Sci. 91,12458
-12462.
Alfa, C., Fantes, P., Hyams, J., Mcleod, M. and Warbrick, E. (1993). Experiments with fission yeast. A laboratory course manual. Cold Spring Harbor, New York: Cold Spring Harbour Laboratory Press.
Al-Mohanna F. A., Caddy K. W. and Bolsover, S. R. (1994). The nucleus is insulated from large cytosolic calcium ion changes. Nature 367,745 -750.[Medline]
Anand, S. and Prasad, R. (1989). Rise in Intracellular pH is concurrent with `start' progression of Saccharomyces cerevisiae. J. Gen. Microbiol. 135,2173 -2179.[Medline]
Aves, S. J., Durkacz, B. W., Carr, A. and Nurse, P. (1985) Cloning, sequencing and transcriptional control of the Schizosaccharomyces pombe cdc10 `start' gene. EMBO J. 4,457 -463.[Abstract]
Badminton, M. N., Kendall, J. M., Rembold, C. M. and Campbell A. K. (1998). Current evidence suggests independent regulation of nuclear calcium. Cell Calcium. 23, 79-86.[Medline]
Barton, J. K., Den Hollander, J. A., Lee, T. M., Maclaughlin, A. and Shulman, R. G. (1980). Measurement of the internal pH of yeast spores by 31P nuclear magnetic resonance. Proc. Natl. Acad. Sci. 77,2470 -2473.[Abstract]
Basi, G., Schmid, E. and Maundrell, K. (1993). TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription start point or thiamine repressibility. Gene 123,131 -136.[Medline]
Bright, G. R., Fisher, G. W., Rogowska, J. and Taylor, D. L. (1987). Fluorescence ratio imaging microscopy: Temporal and spatial measurements of cytoplasmic pH. J. Cell Biol. 98,717 -724.[Abstract]
Brown, G. R., Kohler, M. and Berggren, P. O. (1997). Parallel changes in nuclear and cytosolic calcium in mouse pancreatic beta cells. Biochem. J. 325,771 -778.[Medline]
Busa, W. B. (1986). Mechanisms and consequences of pH-mediated cell regulation. Annu. Rev. Physiol. 48,389 -402.[Medline]
Campbell, S. D., Sprenger, F., Edgar, B. A. and O'Farrell, P. H. (1995). Drosophila Wee1 kinase rescues fission yeast from mitotic catastrophe and phosphorylates Drosophila Cdc2 in vitro. Mol. Biol. Cell. 6,1333 -1347.[Abstract]
Carmelo, V., Santos, H. and Sa-Correia, I. (1997). Effect of extracellular acidification on the activity of plasma membrane ATPase and on cytosolic and vacuolar pH of Saccharomyces cerevisiae. Biochim. Biophys. Acta 1325,63 -70.[Medline]
Carr, A. M., MacNeill, S. A., Hayles, J. and Nurse, P. (1989). Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: implications for cdc2+ protein structure and function. Mol. Gen. Genet. 218,41 -49.[Medline]
Cassone, A., Carpinella, G., Angiolella, A., Maddaluno, G. and Podo, F. (1983). 31P Nuclear Magnetic Resonance Study of Growth and Dimorphic Transition in Candida Albicans. J. Gen. Microbiol. 129,1569 -1575.[Medline]
Civan, M. M., Williams, S. R., Gadian, D. G. and Rozengurt, E. (1986). 31P NMR Analysis of intracellular pH of Swill Mouse 3T3 cells: Effects of Extracellular Na+, and K+, and mitogenic stimulation. J. Membr. Biol. 94,55 -64.[Medline]
Dubbin, P. N., Cody, S. H. and Williams, D. A. (1993). Intracellular pH mapping with SNARF-1 and confocal microscopy: pH gradients within single cultured cells. Micron 24,581 -586.
Elsliger, M. A., Wachter, R. M., Hanson, G. T., Kallio, K. and Remington S. J. (1999). Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry 38,5296 -5301.[Medline]
Fantes, P. A. and Nurse, P. (1977). Control of cell size at division in the fission yeast by a growth-modulated size control over nuclear division. Exp. Cell Res. 107,377 -386.[Medline]
Fernandez-Sarabia, M. J., McInerny, C., Harris P., Gordon, C. and Fantes, P. (1993) The cell cycle genes cdc22+ and suc22+ of the fission yeast Schizosaccharomyces pombe encode the large and small subunits of ribonucleotide reductase. Mol. Gen. Genet. 238,241 -251[Medline]
Fiechter, A., Fuhrmann, G. and Kappeli, O. (1981) Regulation of glucose metabolism in growing yeast cells. Adv. Microb. Physiol. 22,123 -183.[Medline]
Gerson, D. F. and Burton, A. C. (1976). The relation of cycling of intracellular pH to mitosis in the acellular slime mould Physarum polycephalum. J. Cell. Physiol. 91,297 -304.
Gillies, R. J. and Deamer, D. W. (1979). Intracellular pH changes during the cell cycle in Tetrahymena. J. Cell Physiol. 100,23 -32.[Medline]
Gillies, R. J., Ugurbil, K., Den Hollander, J. A. and Shulaman R. J. (1981). 31P NMR studies of intracellular pH and phosphate metabolism during cell division cycle of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 78,2125 -2129.[Abstract]
Gillies, R. J., Martinez-Zauguilan, R., Martinez, G. M., Serrano, R. and Perona, R. (1990). Tumorigenic 3T3 cells maintain an alkaline pH under physiological conditions. Proc. Nat. Acad. Sci. USA 87,7414 -7418.[Abstract]
Gonzalez, B., de Graaf, A., Renaud, M. and Sahm, H. (2000). Dynamic in vivo 31P nuclear magnetic resonance study of Saccharomyces cerevisiae in glucose-limited chemostat culture during the aerobic-anaerobic shift. Yeast 16,483 -497.[Medline]
Gorlich, D. and Mattaj, I. W. (1996). Nucleocytoplasmic transport. Science 271,1513 -1518.[Abstract]
Greenfield, N. J., Hussain, M. and Lenard, J. (1987). Effects of growth state and amines on cytoplasmic and vacuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a 31P-nuclear magnetic resonance study. Biochim. Biophys. Acta 926,205 -214.[Medline]
Grinstein, S., Cohen, S. and Rothstein, A. (1984). Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+ antiport. J. Gen. Physiol. 83,341 -369.[Abstract]
Haworth, R. S. and Fliegel, L. (1993). Intracellular pH in Schizosaccharomyces pombe - comparison with Saccharomyces cerevisiae. Mol. Cell. Biochem. 124,131 -140.[Medline]
Hiraoka, Y., Toda, T. and Yanagida, M. (1984). The NDA3 gene of fission yeast encodes ß-Tubulin: a cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39,349 -358.[Medline]
Jimenez, J., Alphey, L., Nurse, P. and Glover, D. M. (1990). Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homolog and string. EMBO J. 9,3565 -3571.[Abstract]
Johnson, J. D. and Epel, D. (1976). Intracellular pH and activation of sea urchin eggs after fertilization. Nature 262,661 -664.[Medline]
Kneen, M., Farinas, J., Li, Y. and Verkman, A. S.
(1998). Green fluorescent protein as a noninvasive intracellular
pH indicator. Biophys. J.
74,1591
-1599.
L'Allemain, G., Franchi, A., Cragoe, E. and Pouyssegur, J.
(1984). Blockade of the Na+/H+ Antiport
abolishes growth factor-induced DNA synthesis in fibroblasts. J.
Biol. Chem. 259,4313
-4319.
L'Allemain, G., Paris, S. and Pouyssegur, J.
(1985). Role of a Na+-dependent
Cl-/HCO3- exchange in regulation of intracellular pH in
fibroblasts. J. Biol. Chem.
259,5809
-5815.
Lee, M. G. and Nurse, P. (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327, 31-35.[Medline]
Lui, P. P., Kong, S. K., Fung, K. P. and Lee, C. Y. (1998). The rise of nuclear and cytosolic Ca2+ can be uncoupled in HeLa cells. Pflugers Arch. 436,371 -376.[Medline]
Llopis, J., McCaffery, J. M., Miyawaki, A., Farquhari, M. G. and
Tsien, R. Y. (1998). Measurement of cytosolic, mitochondrial,
and golgi pH in single living cells with green fluorescent proteins.
Proc. Natl. Acad. Sci. USA
95,6803
-6808.
Lucas, C. A., Gillies, R. J., Olson, K. A., Giuliano, R. M. and Sneider, J. M. (1988). Intracellular Acidification inhibits the proliferative response in BALB/c-3T3 Cells. J. Cell. Physiol. 136,161 -167.[Medline]
Macdonald, J. R. (1998). Nuclear calcium: transfer to and from the cytosol. Biol. Signals Recept. 7,137 -147.[Medline]
Madshus, I. H. (1988). Regulation of intracellular pH in eukaryotic cells. Biochem. J. 250, 1-8.[Medline]
Maniatis, T., Sambrook, J. and Fritsch, E. F. (1989). Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbour Laboratory Press.
Maundrell, K. (1993). Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123,127 -130.[Medline]
Meyer, T., Allbritton, N. L. and Oancea, E. (1995). Regulation of nuclear calcium concentration. Ciba Found. Symp. 188,252 -262.[Medline]
Miesenbock, G., De Angelis, D. A. and Rothman, J. E. (1998). Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394,192 -195.[Medline]
Mitchison, J. M. (1957). The growth of single cells I, Schizosaccharomyces pombe. Exp. Cell Res. 13,244 -262.[Medline]
Mitchison, J. M. (1990). The fission yeast Schizosaccharomyces pombe. Bioessays 12,189 -191.[Medline]
Moolenaar, W. H., Tsien, R. Y., van der Saag, P. T. and de Laat, S. W. (1983). Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature 304,645 -648.[Medline]
Moolenaar, W. H. (1986). Effects of growth factors on intracellular pH regulation. Annu. Rev. Physiol. 48,363 -376.[Medline]
Musgrove, E., Seaman, M. and Hedley, D. (1987). Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence. Exp. Cell Res. 172, 65-75.[Medline]
Navon, G., Shulman, R. G., Yamane, T., Eccleshall, T. R., Lam, K., Baronofsky, J. J. and Marmur, J. (1979). Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae. Biochemistry 18,4487 -4499.[Medline]
Nurse, P., Thuriaux, P. and Nasmyth, K. (1976). Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146,167 -178.[Medline]
Nurse, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344,503 -508[Medline]
Ober, S. S. and Pardee, A. B. (1987). Intracellular pH is increased after transformation of Chinese hamster embryo fibroblasts. Proc. Natl. Acad. Sci. USA 84,2766 -2770.[Abstract]
Pena, A., Ramirez, J., Rosas, G. and Calahorra, M. (1995). Proton pumping and the internal pH of yeast cells, measured with pyranine introduced by electroporation. J. Bacteriol. 177,1017 -1022.[Abstract]
Plant, P. J., Manolson, M. F., Grinstein, S. and Demaurex,
N. (1999). Alternative mechanisms of vacuolar acidification
in H+-ATPase-deficient yeast. J. Biol.
Chem. 274,37270
-37279.
Portillo, F. and Serrano, R. (1989). Growth control strength and active site of yeast plasma membrane ATPase studied by site-directed mutagenesis. Eur. J. Biochem. 186,501 -507.[Abstract]
Pouyssegur, J., Franchi, A., L'Allemain, G. and Paris, S. (1985). Cytoplasmic pH, a key determinant of growth factor-induced DNA synthesis in quiescent fibroblasts. FEBS Lett. 190,115 -118.[Medline]
Preston, R. A., Murphy, R. F. and Jones, E. W. (1989). Assay of vacuolar pH in yeast and identification of acidification-defective mutants. Proc. Natl. Acad. Sci. USA 86,7027 -7031.[Abstract]
Pringle, J. R., Adams, A. E. M., Drubin, D. G. and Haarer, B. K. (1991). Immunofluorescence methods for yeast. Methods Enzymol. 194,565 -626.[Medline]
Robey, R. B., Ruiz, O., Santos, A. V. P., Ma, J., Kear, F., Wang, L. J. W., Li, C. J., Bernardo, A. and Arruda, A. L. (1998). pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation. Biochemistry 37,9894 -9901.[Medline]
Rodel, C., Juptiz, T. and Schmidt, H. (1997).
Complementation of the DNA repair-deficient swi10 mutant of fission
yeast by the human ERCC1 gene. Nucleic Acids Res.
25,2823
-2827.
Russell, P. and Nurse, P. (1986). cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45,145 -153.[Medline]
Schuldiner, S. and Rozengurt, E. (1982). Na+/H+ antiport in Swiss 3T3 cells: mitogenic stimulation leads to cytoplasmic alkalinization. Proc. Natl. Acad. Sci. USA 79,7778 -7782.[Abstract]
Seksek, O. and Bolard, J. (1996). Nuclear pH
gradient in mammalian cells revealed by laser microspectrofluorimetry.
J. Cell Sci. 109,257
-262.
Shiozaki, K., Shiozaki, M. and Russell, P.
(1998). Heat stress activates fission yeast spc1/sty1 MAPK by a
MEKK-independent mechanism. Mol. Biol. Cell
9,1339
-1349.
Sychrova, H., Ramirez, J. and Pena, A. (1999). Involvement of Nha1 antiporter in regulation of intracellular pH in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 171,167 -172.[Medline]
Szankasi, P., Heyer, W. D., Schuchert, P. and Kohli, J. (1988) DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wildtype and mutant alleles including the recombination hot spot allele ade6-M26. J. Mol. Biol. 204,917 -925.[Medline]
Toone, W. M., Kuge, S., Samuels, M., Morgan, B. A., Toda, T. and
Jones, N. (1998) Regulation of the fission yeast
transcription factor Pap1 by oxidative stress: requirement for the nuclear
export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1.
Genes Dev. 12,1453
-1463.
Tsai, C., Avaledo, A., McDonald, I. and Johnson, B. (1987) Diauxic growth of the fission yeast Schizosaccharomyces pombe in mixtures of D-glucose and ethanol or acetate. Can. J. Microbiol. 33,593 -597.
Van den Hazel, H. B., Kielland-Brandt, M. C. and Winther, J. R. (1996). Biosynthesis and function of yeast vacuolar proteases. Yeast 12,1 -16.[Medline]
Volland, C., Urban-Grimal, D., Geraud, G. and Haguenauer-Tsapis,
R. (1994). Endocytosis and degradation of the yeast uracil
permease under adverse conditions. J. Biol. Chem.
269,9833
-9841.