Fig. 1. Phluorin expression in S. pombe. (A) Wild-type cells carrying
empty pREP1 or pREP1-phluorin vectors were grown to early logarithmic phase in
EMM pH 5.5 and examined. Images were acquired using Nomarski optics or dual
channel fluorescence microscopy. Emission from the low excitation channel was
arbitrarily given a false colour of blue, whereas emission from the high
excitation channel was given a false colour of green. The final colour of the
image is the combination of the two channels. (B) FACS analysis of
phluorin-expressing cells. (a) Wild-type carrying pREP1-phluorin. (b)
cdc2-1w carrying pREP1-phluorin. (c) Wildtype carrying empty pREP1.
(d) Wild-type cells grown in rich media to logarithmic phase (2C control). (e)
cdc10-129 cells arrested in G1 (1C control). (C) Phluorin
standard curve. Phluorin-expressing wild-type cells were permeabilized in
buffers of the indicated pH and photographed at the two excitation
wavelengths. The means of the calculated ratios of emission intensity
(low/high) are plotted for 40 cells at each pH. Error bars indicate standard
deviations. False colour images for each pH are shown to the right of the
figure. (D) Phluorin-expressing wild-type cells were grown in EMM pH 5.5 to
early logarithmic phase and examined. Examples of cells in different phases of
the cell cycle along with their respective cell lengths are indicated: (a)
short uni-nucleate (late S/early G2); (b) long uni-nucleate (late
G2); (c) non-septated binucleate (late M/early G1); (d)
septated binucleate (late G1/early S). Note that cells in late
G2 to septation are in the constant length phase of the cell cycle
(Mitchison, 1957). (E)
pHi as a function of emission intensity. Phluorin-expressing
wild-type cells were grown to early logarithmic phase in EMM pH 3.5 (
),
EMM pH 4.5 (
), EMM pH 5.5 (
) or EMM pH 6.5 ([UNK]) and examined.
The plotted results are normalized to the most weakly expressing cell in each
data set. n ranges from 60-120. (F) pHi as a function of
external pH. Phluorin-expressing wild-type cells were grown to early
logarithmic phase in EMM of the indicated pH and examined. The plotted results
are the average of three independent replicates. In each replicate, 20-40
cells were sampled for every external pH value tested. Error bars indicate
s.d. (G) Phluorin-expressing wild-type cells were grown to early logarithmic
phase in EMM pH 5.5 and mounted in the same medium in the flow chamber. After
a 30-60 minute equilibration time, medium was switched (t=0) to EMM (adjusted
to a final pH of 5.5) containing either 15 mM acetic acid () or 200 mM
bicarbonate (). Images were acquired at the indicated times. 20 cells
were sampled per time point. Error bars indicate s.d.