Rapid patterning and zonal differentiation in a two-dimensional Dictyostelium cell mass: the role of pH and ammonia
Satoshi Sawai1,*,
Takashi Hirano2,
Yasuo Maeda2 and
Yasuji Sawada3
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
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Fig. 1. (A,B) Dependence of the outer zone depth on temperature in 2-D cultures. a,
outer zone; b, inner zone. The dark outermost edge is a meniscus. Freshly
starved NC-4 cells at (A) 30°C, 10 min after preparation, and (B)
4.5°C, 15 min after preparation. (C) Freshly starved NC-4 and Ax-2 cells
were two-dimensionally cultured in an isothermal vessel. Each plot represents
a 1-2 h time average of the outer zone depth Lout. Plots
are fitted by
Lout  Texp(E/2RT),
where T is absolute temperature (K) and R is the gas constant,
giving activation energy of E=65 kJ mol-1, E=69
kJ mol-1 for Ax-2 and NC-4 cells, respectively. Plots at 40°C
have been omitted for the curve fitting. (D—G) Temperature dependence on
cell differentiation. Earlymound stage cells were subjected to 2-D culture.
Cells at the outermost region of the outer zone show D19 expression
at both 22°C, t=4 h (D) and 10°C, t=4 h (E). The
ecmB expression observed near the border at 22°C (t=2 h)
(F) is suppressed at 10°C (t=3 h) (G). Scale bars, 200 µm
(A,B); 100 µm (C,D).
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Fig. 2. Cytosolic pH change and patterning. (A,B) Cells were grown in atmospheric
oxygen or (C,D) 100 % O2. (A,C) Fluorescence intensity obtained by
confocal microscopy; excitation at 488 nm and 510-550 nm band pass filter.
(B,D) Transmitted light. (E,F) Fluorescence intensity averaged over the
vertical direction in the indicated boxes. Low fluorescence intensity in the
inner-zone cells suggests acidification of their cytosolic pH.
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Fig. 3. Extracellular pH change demonstrated by Bromocresol Purple (pH 5.2, yellow;
pH 6.8, purple). (A) Alkalinization of extracellular space in the outer zone,
t=1 h, 7 min. The arrow shows a position partially deprived of cells
to show the change in color more clearly. (B,C) A drop of Bromocresol Purple
solution (left) kept at a distance from a 2-D cell mass (right) also changes
color after (B) t=15 min and (C) t=2h, 44 min. Scale bars,
100 µm (A); 1 mm (B,C).
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Fig. 4. Effect of proton pump inhibitors. Ax-2 cells were preincubated with (A) 5
µmoll-1 or (B) 10 µmoll-1 DES, and (C) 5
µmoll-1 or (D) 10 µmoll-1 miconazole, prior to 2-D
culturing. Photographs were taken at t=30 min (A,B) and t=10
min (C,D). Scale bar, 200 mm.
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Fig. 5. Freshly starved Ax-2 cells pre-incubated for 15 min in weak acid or weak
base show an altered timing of rapid patterning. (A) 0-20 mmoll-1
sodium propionate added to PB delays the patterning in a
concentration-dependent manner at pH 6, but no effect was seen at pH 8. (B)
The pattern becomes visible at t=1.5 min when cells are preincubated
in 5 mmoll-1 NH4Cl at pH 8, whereas in the control it
only becomes visible at t=3-5 min. The same effect is seen in 20
mmoll-1 NH4Cl at pH 8 but not at pH 6. (C) Similarly,
the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) delays the patterning,
and the weak base methylamine hastens it. Scale bar, 400 µm.
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Fig. 6. A diagram of the proposed reaction scheme. (A) Low oxygen acts as a trigger
to lower pHi, which enhances protein degradation. As a result, weak acids and
other related metabolites (X) increase, which further lowers pHi. At
the same time, ammonia (Y) is produced as the final end product.
Ammonia, being a small neutral molecule, could permeate the cell membrane
easily compared to weak acids, resulting in a large difference in their
diffusion coefficient, which is a required condition for the Turing
instability (Nicolis and Prigogine,
1977 ). When the pH is low, ammonia levels will decrease by
protonation. Y is consumed in an oxygen-dependent fashion for the
production of X by transamination, thus realizing a
substrate-depletion type reaction
(Meinhardt, 1982). (B)
Reaction and diffusion of X and Y creates a stationary
gradient with each peak positioned off-phase from each other (Bi). Gradients
of X and Y influence pHi and, furthermore, cell
differentiation (Bii). The positions of D19, ecmA and ecmB
gene expression are also indicated (see text for details).
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© The Company of Biologists Ltd 2002
