Rapid patterning and zonal differentiation in a two-dimensional Dictyostelium cell mass: the role of pH and ammonia
1 Graduate School of Information Sciences, Tohoku University, 2-1-1
Katahira, Aoba-ku, Sendai 980-8577, Japan
2 Biological Institute, Graduate School of Science, Tohoku University, 2-1-1
Katahira, Aoba-ku, Sendai 980-8577, Japan
3 Research Institute of Electrical Communication, Tohoku University, 2-1-1
Katahira, Aoba-ku, Sendai 980-8577, Japan
* Author for correspondence at present address: Princeton University, Department of Molecular Biology, Princeton, NJ 08544, USA (e-mail: ssawai{at}molbio.princeton.edu)
Accepted 20 May 2002
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Summary |
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Key words: oxygen, pH, ammonia, H+-ATPase, reaction-diffusion, Dictyostelium discoideum
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Introduction |
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Several lines of evidence suggest that the initial rapid patterning itself
is not merely due to a threshold response to oxygen, but to a
reaction-diffusion mechanism independent of the well-studied cAMP relaying
system (Sawada et al., 1998;
Bonner et al., 1998
). The
patterning has some required properties for Turing instability, which so far
has only been realized in physico-chemical systems
(Sawai et al., 2000
). If so,
what are the diffusive molecules involved and why do they evoke rapid cell
shape change? Apart from interest in the `generic' patterning mechanism
(Newman and Comper, 1990
), our
recent findings that prespore cells initially appear in an outer region and
prestalk cells in an inner region (Hirano
et al., 2000
) were contradictory to the earlier prediction of
Bonner and colleagues (Bonner et al.,
1998
). According to their theory
(Bonner et al., 1998
), the main
environmental factor which gives preference to the cell-type fate is oxygen
(Sternfeld, 1988
); thus one
would expect prestalk cells to appear in the outer region facing the air. If
not oxygen, what biases the fate of the cells in the confined 2-D culture?
In this report, we sought to answer the above questions by looking at the physiological aspects of the initial patterning. Temperature dependence was studied to determine the activation energy of the kinetics as well as its effect on zonal differentiation. We then show that rapid alteration in the cytosolic pH (pHi) and ammonia secretion take place during the patterning. The observed zonal-differentiation pattern supports the proposed relationship between intracellular pH and cell-type differentiation of Dictyostelium cells based on monolayer and other in vitro assays. A possible reaction scheme of the rapid patterning and its relationship to cell differentiation in a confined culture is discussed.
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Materials and methods |
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2-D culturing
A cell mass confined between two glass plates was prepared as previously
described (Sawada et al.,
1998). Cells were pelleted by centrifugation and the supernatant
was removed. Cells were carefully collected from the tube with a Pasteur
pipette and placed on a microscope slide. Two spacers of approximately 100
µm thickness were placed beside the cell mass. A coverslip was immediately
laid on top of the cell mass and the spacers; this time is denoted as
t=0. The culture was kept on a plastic plate together with wet paper
towels to maintain the humidity above 95%. Measurements were performed under
an Olympus IMT-2 microscope equipped with a Sony CCD-IRIS camera. For some of
the measurements, time-lapse recording and video capturing were employed. A
CCD camera was connected to a Hi8 VISCA system controlled by a Macintosh
computer running NIH Image software.
Fluorescent labeling
For loading of fluorescein conjugated with dextran
(Mr=1x105; Molecular Probes), cells were
washed twice with PB (20 mmol l-1 sodium/potassium phosphate
buffer, pH 6.5) and suspended at a density of 5.0x107 cells
ml-1 in the same buffer containing 1 mg ml-1 of the
dextran. Loading of the fluorescent probe was achieved by applying, at 5 s
intervals, two exponentially decaying pulses of 2kV cm-1 with a
time constant of 0.7 ms using a simple circuit described by Yumura et al.
(1995) at 2.2 µF
capacitance. Electroporated cells were immediately suspended in a healing
solution (PB, pH 6.5, containing 2 mmol l-1 MgCl2 and
0.2 mmol l-1 CaCl2)
(van Haastert et al., 1989
)
for 10-15 min on ice and washed twice with PB.
Temperature control
Temperature dependence of the pattern size was measured by constructing a
transparent vessel which let a thin layer of water (approximately 1 mm) run
beneath a glass plate where the sample was kept. The temperature of the water
was controlled by a circulator (Coolnics CTE-22W, Komatsu) from 2-40°C.
The temperature of the sample was read by a thin metal sensor placed on the
sample plate. Conical tubes containing pelleted cells were placed on ice and
used immediately to prepare a sample in the vessel. Fixation and X-gal
staining of transformed cells bearing the reporter lacZ gene under
the control of cell type-specific promoters were performed as described
previously (Hirano et al.,
2000).
Proton pump inhibition, weak acid and base loading
Following the method described by Inouye
(1988), washed cells were
suspended for 5-10 min either in PB (pH 6.0) or BSS containing 20 µmol
diethystilbestrol (DES) or miconazole. For weak acid treatment, cells were
suspended in PB (pH 6.0) containing 20 mmol propionic acid sodium salt or
5,5-dimethyl-2,4-oxazolidinedione (DMO). Similarly 0-20 mmol-1
NH4Cl or methylamine was added to PB (pH 8.0). Cells were
immediately pelleted by centrifugation without washing and used in the 2-D
culture chamber to observe patterning. DES, miconazole, propionate, DMO, and
methylamine were purchased from Sigma.
Detection of extracellular pH
Bromocresol Purple was added to the cell suspension (0.02-0.04 % in BSS)
just before preparing the pellet for a 2-D culture. For detection of ammonia,
a drop of the cell-free Bromocresol Purple solution was placed approximately 1
cm away from the cell mass to detect any change in the external pH (pHe).
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Results |
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The effect of low temperature on zonal differentiation was also studied. Fig. 1D-G shows the expression pattern of the prespore-specific D19 and prestalk-specific ecmB genes observed under our experimental conditions at 22 °C and 10 °C. A region where D19 gene expression is first observed always appears at the outermost domain and is relatively longer in width when the outer zone is expanded at the low temperature. In contrast, ecmB expression is almost completely suppressed at 10 °C.
pH effects
Relative change in pHi was observed using fluorescein, whose fluorescence
decreases as the pH is lowered (Haugland,
1996). As shown in Fig.
2, the pHi of cells in the inner zone is relatively lower than
that of the outer zone cells, with a sharp transition at their border. The
time required for such a pHi gradient to become established is almost the same
as that of the appearance of the dark and bright patterns observed by
transmitted light. From the difference in the fluorescence intensity, if one
assumes the highest value of pHi to be around 7.5, there could be a difference
of more than 0.5 pH units between the two zones.
|
We detected changes in the extracellular pH by adding Bromocresol Purple (pH 5.2, yellow; pH 6.8, purple) to the suspension just before preparing a pellet. As shown in Fig. 3A, we observed alkalinization of the extracellular medium in the outer zone, whereas that of the inner zone remained acidic. The change in color occurred simultaneously with the rapid emergence of the pattern itself. When a drop of the same Bromocresol Purple solution was placed separately from the sample, we could also see the same change in color from yellow to purple (Fig. 3B). This indicates that at least one of the diffusive molecules responsible for the rise in extracellular pH is volatile, and was possibly ammonia. We did not observe the staining pattern when cells were incubated for 10 min with Bromocresol Purple and then washed before preparing the 2 D culture. Intake of the dye is minimized under our experimental conditions, and the color mainly represents the pH of the extracellular space. The arrow in Fig. 3A shows that the change in color is observed where there are no cells.
|
Effect of weak acid, proton pump inhibitors and weak base
Fig. 4 shows the effect of
proton pump inhibitors. When cells were pre-incubated with either 10
µmoll-1 DES or 10 µmoll-1 miconazole, the
patterning became almost undetectable. The effect was almost negligible with
inhibitors at 5 µmoll-1, and the distinction between the two
zones was the same as in the controls. Preincubation at concentrations above
10 µmoll-1 (50 µmoll-1) had a stronger
pattern-erasing effect. 0.5% ethanol, used as a solvent, had no effect on the
rapid patterning when added alone. The effective concentrations of these
inhibitors are close to those reported to inhibit the plasma membrane proton
pump in Dictyostelium cells and to lower the pHi
(Inouye, 1989
;
van Duijn and Vogelzang,
1989
).
|
When freshly starved cells were preincubated with 10-20 mmoll-1 sodium propionate at pHe 6.0 before sample preparation, there was a concentration-dependent delay in the onset of the patterning (Fig. 5A). When the same experiment was conducted at pHe 8.0, there was no noticeable effect on the patterning, indicating that the active molecule is the neutral form of propionate, which is more membrane permeable than its ionized form. The same effect was observed when DMO was used instead of propionate (Fig. 5C).
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When freshly starved cells were pre-incubated with 5-20 mmoll-1 ammonia at pHe 8.0, patterning occurred very rapidly, and was almost complete within 1 min (Fig. 5B). The same treatment at pHe 6.0 had no effect, indicating again that NH3 rather than NH4+ is the active species. The same effect was observed when methylamine was used instead of ammonia (Fig. 5C).
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Discussion |
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While D19 expression remained constant, strong ecmB
expression characteristic of 2-D culturing was suppressed at low temperatures.
These results are similar to the changes observed under high O2
environments (Hirano et al.,
2000). It should be noted that in both high oxygen and low
temperature, depth of the weak-base-rich outer zone is increased. This change
in ecmB expression is consistent with the idea that a decrease in
ammonia plays a part in switching from the slug stage to the fruiting body
formation (Singleton et al.,
1998
).
Cytosolic pH
We present evidence that rapid patterning is closely linked to changes in
pHi in Dictyostelium. It is well known that the pHi of
Dictyostelium cells is lowered by incubation with weak acid such as
propionate and DMO, or by plasma membrane proton pump inhibitors such as DES
and miconazole (Inouye, 1989;
van Duijn and Vogelzang,
1989
). In our present study, cells in the outer zone, which are
darker and more motile than those in the inner zone, have a relatively high
pHi. DES and miconazole at low concentrations known to inhibit plasma membrane
proton pump cause the pattern to disappear, suggesting that pHi regulation is
an essential part of the patterning response. A previous report of pattern
inhibition by azide (Hirano et al.,
2000
) could also be explained not by energy depletion but by
inhibition of the plasma membrane proton pump activity, as reported by van
Duijn and his colleagues (van Duijn et
al., 1990
). Change in timing of the patterning by weak acid and
weak base should be caused by an alteration of the initial pHi and also in the
buffering capacity of the cells.
The observed pHi gradient is similar to that reported by Azhar and
Nanjundiah (1996) using
fluorescence of Neutral Red, except that they found that acidification was
more localized around the border between the two zones. The reason for this
difference could be due both to the fact that Neutral Red reflects the pH of
acidic compartments rather than the cytosol, and also that there is a
difference in the oxygen gradient present in their 1-D experimental design,
which permits a secondary peak of weak base to appear in the inner region.
The fact that motile cells in the outer zone have higher pHi is consistent
with our current knowledge of pHi and cell motility of Dictyostelium
cells (van Duijn and Inouye,
1991). In contrast, cells in the inner zone have a lower pHi and
are less motile. An actin-binding protein, hisactophilin, is known to bind
along actin filaments only when the pH is <7.2
(Stoekelhuber et al., 1996
).
Thus the difference in levels of transmitted light between the inner and outer
zones could reflect an alteration in cortical actin assemblies. It would still
be an oversimplification, however, to attribute cell-shape differences only to
a pH change. Other effects, such as tyrosine phosphorylation of actin, are
also known to occur under conditions of low oxygen or DNP treatment
(Jungbluth et al., 1994
) and
should be carefully taken into account in future studies.
It may also be possible that patterning is more directly related to the
plasma membrane potential than to pH or other ions, since the DES- and
miconazole-sensitive plasma membrane H+-ATPase
(Pogge-von Strandmann et al.,
1984) is the major generator of the membrane potential in
Dictyostelium (van Duijn and
Vogelzang, 1989
). However, TMRM (tetramethyl rhodamine methyl
ester), a dye that distributes across the cell membrane according to the
Nerntian potential (Ehrenberg et al.,
1988
), stains not only the mitochondria but also the cytosol of
inner-zone cells much more strongly than those of the outer-zone cells
(Hirano et al., 2000
). This
implies that the plasma membrane of the inner-zone cells is somewhat
hyperpolarized compared to that of outer-zone cells and is quite the opposite
of what one would expect if the patterning were determined merely by
inhibition of plasma membrane proton pumps in the inner-zone cells. In this
light, it would be interesting to see whether the plasma membrane proton pump
encoding gene patB, which is known to be highly expressed under
acidic conditions (Coukell et al.,
1997
), is expressed in the inner-zone cells.
A reaction scheme
As we saw with weak acid/base and proton pump inhibition experiments,
altering either pH or intracellular buffering capacity has a strong influence
on the patterning. It is known that Dictyostelium development relies
on catabolism of about 50% of its protein and RNA. As a result, a large amount
of TCA-cycle intermediates and related metabolites
(Kelly et al., 1979) and
ammonia (Schindler and Sussman,
1977
) are released from the cells. Under 2-D culturing conditions,
outward proton pumping by the plasma membrane H-ATPase may be partially
blocked by the low oxygen levels. It is known that under such conditions,
where ionic regulation is suppressed, cellular pH depends more on the
acidbase balance of metabolites accumulated in the extracellular and
intracellular space (Pörtner,
1993
). Thus secreted organic acids and ammonia could play
important roles in the rapid patterning by causing pHi changes.
Another explanation for the patterning is that cells respond to a threshold
level of oxygen (Bonner et al.,
1998). We have calculated the oxygen level in the 2-D cell mass
based on the reported oxygen consumption rate of Dictyostelium cells
(Sternfeld and David, 1981
;
Krill and Town, 1988
) and find
it difficult to explain the known oxygen dependency of outerzone size or other
inherently nonlinear patterning properties
(Bonner et al., 1998
;
Sawai et al., 2000
).
Two earlier studies suggest a strong correlation between oxygen and ammonia
production. Bonner et al.
(1990) reported a rapid
bleaching of Neutral-Red staining of the posterior region of a young slug
which occurs within 10 min when it is submerged under mineral oil. This could
be explained by stabilization of an ammonia gradient triggered by low oxygen.
Sternfeld (1988
) observed that
when a slug is tranferred to 100% O2 environment, Neutral-Red
staining of the anterior region is also bleached due to higher ammonia
production by prestalk cells. Although the pattern of bleaching is different,
these findings suggest that altering the oxygen concentration has a
significant influence on ammonia production and hence acidbase balance
of the Dictyostelium cells.
We propose that there are two major diffusive players in the patterning,
(1) Y, a fast-diffusing ammonia or other weak base that raises the
pHi and/or the pH of other intracellular compartments, and (2) X, a
weak acid or its precursor produced as a result of protein degradation. A
possible scheme is shown in Fig.
6A, in which Y acts as a substrate for further generation
of X. This is the so-called substrate-depletion type reaction, which
is well known to generate self-organized patterns when coupled with diffusion
(Nicolis and Prigogine, 1977;
Meinhardt, 1982
;
Harrison, 1993
). Since
X acts to decrease pHi, and Y is a molecule with the
opposite effect, it is easy to understand why such a sudden drop in pHi was
observed at the border between the outer and the inner zone
(Fig. 6B). In the scheme,
oxygen has a role of giving polarity to the patterning. Higher oxygen implies
more production of ammonia (Y), which means that the peak of
Y is always at the outer zone edge.
|
The scheme resembles the weak-acid and weak-base model
(MacWilliams and Bonner, 1979;
Inouye, 1990
). However, the
current model predicts X and Y to have peaks at different
locations, which are not achievable by those previous models based on
activatorinhibitor type equations. The main difference is that ammonia
is consumed for the production of X in our model. Such a positive
role for ammonia has been proposed by Cotter et al.
(1992
) as a sourcesink
model to explain the ammonia gradient within a slug. Given the role of ammonia
as a substrate, it is natural that a higher initial content of ammonia
hastened the patterning. Similarly, differentiating cells show much faster
patterning compared to freshly starved cells (S. Sawai, unpublished
observation). This may also be due to the higher content of ammonia or higher
activity of ammonia-regulating enzymes such as AMP-deaminase
(Jahngen and Rossomando, 1986
)
and NAD-dependent glutamate dehydrogenase
(Pamula and Wheldrake, 1992
)
in the cells at the later stages of development.
Cell differentiation
The pH gradient presented in this paper could partially explain the zonal
differentiation pattern reported earlier
(Hirano et al., 2000). In the
2-D cultures, cells at the outermost region of the outer zone, where the pHi
is the highest, show prespore D19 gene expression, followed by
prestalk ecmB and then ecmA expressed just near the border
(Fig. 6Bii). It should be noted
that our findings are quite the opposite to what Bonner and his colleagues had
first postulated based on their Neutral-Red staining results and oxygen alone
as a factor giving preference to differentiation. Conditions that decrease pHi
favor prestalk and stalk cell differentiation (Gross et al.,
1983
,
1988
;
Kubohara and Okamoto, 1994
;
Dominov and Town, 1986
;
Wang et al., 1990
). It is also
known that incubation with weak bases such as ammonia and methylamine favors
prespore cell differentiation (Gross et
al., 1983
; Neave et al.,
1983
; Davies et al.,
1993
). The slope of the pHi gradient does not seem to differ so
much when the oxygen concentration is high, thus pHi may not explain why we
observed more ecmA expression in the inner zone when oxygen was
higher (Hirano et al., 2000
).
Low pHi may impose only a bias toward the prestalk pathway and a more precise
mechanism mediated by cAMP, DIF and ammonia must be involved in cell-type
proportion regulation, even under the present culturing conditions.
The present findings provide clues as to what may take place in a normal
aggregate developing in 3-D when oxygen becomes scarce. Oxygen depletion is a
common phenomenon in soil, especially when the pores become filled with water
and gas diffusion becomes limited
(Baumgartl et al., 1994;
Cooper and van Gundy, 1971
;
Drew, 1992
). Broad oxygen
tolerance is reported in the soil nematode
(van Voorhies and Ward, 2000
),
which shares a natural habitat with Dictyostelium
(Kessin et al., 1996
).
Although Dictyostelium development takes place in three dimensions,
it is possible that situations similar to the 2-D culture, i.e. conditions of
limited oxygen, as suggested by Sandona et al.
(1995
), and space, are
encountered by developing Dictyostelium cells in soil. At present,
there are two different views as to how the early cell differentiation takes
place in Dictyostelium; one is a position-dependent mechanism
(Maeda, 1993
;
Early et al., 1995
) and the
other is a cell-autonomous mechanism (Kay
et al., 1999
; Thompson and
Kay, 2000
). The present findings and the model suggest, at least
under confined conditions, that an oxygen gradient within a cell aggregate is
converted to an ammonia or pHi gradient. As has been postulated in many
hypoxia-tolerant organisms, the pH response may reflect
Dictyostelium's defense mechanism to cope with oxygen depletion.
Since cytoplasmic acidification is reported to facilitate induction of
prestalk gene expression by DIF-1 (Wang et
al., 1990
), it may be that Dictyostelium development is
well adapted to hypoxia.
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Acknowledgments |
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