FEMS MicrobiologyLetters 109 (1993) 7-12
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
FEMSLE 05395
Decreased endo-lysosomal acidification capacity
in methylene diphosphonate-resistant mutants
of Dictyostelium discoideum
Franqoise Br6not and Michel Satre
Laboratoire de Biologie Cellulaire (UA 1130 du CNRS), D~partement de Biologie Mol~culaire et Structurale,
Groupement CEA-CNRS-INSERM-UJF, Centre d'Etudes Nucl~aires, Grenoble, France
(Received 9 February 1993; accepted 10 February 1993)
Abstract: The in vivo capacity for endo-lysosomal acidification has been monitored in Dictyostelium discoideurn amoebae with
acridine orange, a fluore~ent weak base dye commonly used to probe transmembrane pH gradients. In the presence of aerobic
amoebae, the initial rate of fluorescence quenching was found to be proportional to cell density between 5 × 105 and 2.5 × 10 6 cells
ml- i and independent of acridine orange concentration in the 1.5 to 7.5 v.M range. The dye response was sensitive to agents that
perturb endo-lysosomal acidification such as NaN 3, nigericin or imidazole. Several mutant cell lines whose growth was resistant to
methylene diphosphonate were found to be partially deficient in the acridine orange quenching test, suggesting that endo-lyso~mal
acidification was altered in these mutants.
Key words: Dictyostelium diacoideum; Amoebae; Acidification; Acridine orange; Endocytosis
Introduction
In eukaryotic cells, endocytosis is the biological system leading to i n t e r n a l i s a t i o n of extracellular material in vesicles derived initially from inv a g i n a t i o n s of the plasma m e m b r a n e [1-3]. Both
phagocytosis a n d fluid-phase endocytosis (pinocytosis) are highly active in a m o e b a e of the cellular slime m o u l d Dictyostelium discoideum. In this
Correspondence to: M. Satre, Laboratoire de Biologie Cellulaire, DBMS/BC, CEN-G, 85X, 38(141 Grenoble Cedex,
France.
organism, endocytosis is the primary process by
which n u t r i e n t s are t a k e n u p [4-8].
W e have a d o p t e d a genetic a p p r o a c h to investigate the endocytic pathway in Dictyostelium, by
isolating defective m u t a n t s [9-11]. O n e screening
strategy we have developed relies on the use of
p y r o p h o s p h a t e analogs, such as m e t h y l e n e dip h o s p h o n a t e (MDP). T h e s e c o m p o u n d s are cytotoxic towards Dictyostelium a n d they e n t e r into
a m o e b a e only by the fluid-phase endocytosis
pathway [12,13]. M u t a n t cell lines that have acq u i r e d resistance to 7.5 m M M D P had previously
b e e n isolated a n d three of them, H G R 5 , H G R 8
a n d H G R 9 , were characterised in more detail.
Cell fractionation studies and 31p N M R measurements of pH showed that they are defective in
endo-lysosomal acidification [10,14]. Low internal
pH of endo-lysosomal compartments plays a critical role in the regulation of several endocytic
aspects, such as receptor-ligand dissociation,
vesicular trafficking, co- or counter-transport of
solutes and intracellular hydrolysis of macromolecules [15-17]. In Dictyostelium amoebae,
endo-lysosomal compartments are acidified by association with acidosomes, organelles rich in vacuolar type H+-ATPase [18,19].
Various weak bases of the acridine dye family
such as acridine orange (AO) are commonly used
to monitor pH gradients across membranes and
to visualise acidic compartments in intact cells
[20-22]. These molecules equilibrate freely across
membranes in their unprotonated form but their
protonated form has a reduced membrane permeability. If a ApH is established, they will be
entrapped on the side of the membrane where
the pH is the most acidic. The accumulation of
dye leads to the quenching of its fluorescence and
thus reports d p H . In this work, we have developed a simple assay with living amoebae using
AO as a tool to screen mutants for their acidification capacity. Six new mutants were shown to
have a reduced response thus suggesting a defec-
tive acidification of their endo-lysosomal pathway.
Materials and Methods
Culture conditions
Dictyostelium discoideum, strain AX2 (ATCC
24397), was grown at 22°C in axenic medium [23]
containing 18 g I-1 maltose and 0.25 g l - t dihydrostreptomycin. Cell numbers were determined
with a Coulter Counter ZM. Amoebae were harvested in their exponential phase of growth (between 5 × 106 and 1 X 107 cells ml -~) by centrifugation and immediately used for experiments.
MDP-resistant mutants isolated as detailed previously [10] were maintained in the same medium
containing 7.5 mM MDP. MDP was suppressed
for at least three generations before experiments.
Acridine orange acidification assay
Dictyostelium amoebae (1 x 10 6 to 1 )<
10 7
cells) were collected by centrifugation, suspended
in 0.1 ml 0.1 M KCI, 5 mM MgSO4, 0.75 mM
EGTA, 5 mM Hepes buffer, pH 7.0 and injected
in 1.9 ml of the same medium containing 1.5 to 15
/zM AO. The fluorescence was monitored at 22°C
at ;tcx = 490 nm and '~e,, = 530 nm [24] using a
Cells
Cells
f
Ni, l
Fig. 1. Changes in AO fluorescence during incubation with Dictyostelium amoebae. Cells (2.5x 10~ ml i) were incubated as
described under Materials and Methods with 3 ~M AO. The evolution of AO fluorescence is shown as a function of time: (a,c)
strain AX2; (b) strain AX2+I mM NAN3;(d) strain HGR7. Addition of 20 mM imidazole (Ira) or 20 ~,M nigericin (Nig) is
indicated by the arrows.
Hitachi F-2000 fluorimeter. Intensity data were
collected at 2 s intervals, with a gentle stirring to
avoid cell settling and to maintain aerobic conditions.
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Materials
Acridine orange was purchased from EastmanKodak (Rochester, NY). Other biochemicals were
from Sigma Chimie (Saint-Quentin-Fallavier,
France).
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Results and D i s c u s s i o n
Acridine orange fluorescence response during incubation with Dictyostelium AX2 amoebae
As shown in Fig. la, Dictyostelium AX2 amoebae were added to a medium containing A O and
fluorescence was measured continuously. Two
distinct kinetic phases were observed. First, a
very rapid drop in fluorescence was recorded
(tn/2 < 4 s). It was followed by a second phase
characterised by a progressive fluorescence decrease whose rate slowed in the course of time.
The initial fluorescence drop was insensitive to
cellular A T P depletion by addition of NaN 3, an
inhibitor of mitochondrial F 1F0-ATP synthase or
to nigericin, a K + / H ÷ ionophore that dissipated
A p H (not shown). This fluorescence decrease was
due in part to a dilution effect upon addition of
cells and possibly also to the binding of a fraction
of the dye to cellular membranes [20]. The second
phase of fluorescence quenching is likely to be
relevant to A O accumulation in Dictyostelium
intracellular acidic compartments as 1) it was
inhibited by 1 mM NaN.~ (Fig. lb) and 2) fluorescence intensity was rapidly restored to its initial
level by the addition of 2 0 / x M nigericin (Fig. la)
or in presence of 20 mM imidazole (Fig. lc) or 20
mM NH4CI (not shown). Therefore, the rate of
linear fluorescence decrease could correspond to
the existing A p H between cytosol and endolysosomal compartments as discussed previously in
3T3 cells [24,25].
Effect of cellular density and acridine orange concentration
We have examined the fluorescence response
as a function of both cell and dye concentrations.
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Cells/ml (x 1 0 6)
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[Acridine Orange] IM
Fig. 2. Effect of cellular density and AO concentration on the
initial rate of AO fluorescence decrease. (A) Dictyostelium
AX2 amoebae (1 x l0 s to 5 x 10 6 m l - l) were incubated with 3
/J.M AO (-) or 6 /.tM AO (o). (B) Dictyostelium AX2 amoebae (2.5 × l0 6 ml-- i) were incubated in the presence of various AO concentrations (from 1.5 to 15 p.M). In both sets of
experiments, the initial rate of AO fluorescence quenching
was determined and expressed as percentage of initial fluorescence ( A F / F o) m i n - t (see legend to Fig. l). Error bars are
SD (at least three independent experiments). Data points
without error bars correspond to single experiments.
The initial quenching rate / t F / F 0 / m i n , where F 0
is the initial fluorescence (see Fig. 1), was found
to be proportional to cell density at least up to
2.5 x 10 6 amoebae ml-1 (Fig. 2A) and to remain
almost constant when A O concentrations varied
between 1.5 and 7.5 ~ M (Fig. 2B). In our experimental conditions, the best compromise between
a strong fluorescence signal and turbidity of the
cell suspension was obtained with 2.5 x 10 6
amoebae ml-1 and 3 / ~ M AO.
Acridine orange fluorescence response of methylene
diphosphonate resistant Dictyostelium mutants
A O response somehow reflected endo-lysosomal acidification capacity of Dictyostelium
amoebae and as such appeared as a useful and
simple assay to screen several mutant strains. As
shown in Fig. l c - d , we have compared A O
quenching obtained with amoebae from parent
strain AX2 and from strain H G R 7 , one of the
MDP-resistant mutants isolated previously [10].
H G R 7 exhibited a decreased capacity of endolysosomal acidification and both the rate of A O
10
Acknowledgement
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The authors wish to thank Dr. Eamonn Rooney
for helpful comments and careful reading of the
manuscript.
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References
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Fig. 3. Rate of AO fluorescence decrease during incubation
with Dictyostelium amoebae from parent strain AX2 and from
MDP-resistant mutants. Cells from strains AX2, HGR1 and
HGR3 to 10 (2.5×106 ml -]) were incubated as described
under Materials and Methods with 3/~M AO. Initial rates of
AO fluorescence quenching (AF/Fo)min -t were determined. Results are the means_+SD from at least three independent experiments.
quenching and the amplitude of quenching were
50% reduced in this mutant. As observed with
AX2 amoebae, AO fluorescence fully recovered
on addition of 20 mM imidazole (Fig. ld). Previously characterised acidification defective mutants, HGR5, H G R 8 and H G R 9 [10,14], showed
the expected reduced rate of AO fluorescence
quenching (see Fig. 3).
Using the AO assay, the capacity of acidification of the endocytic compartments has been
investigated in several independently isolated
MDP-resistant mutants [10]. The results are presented in Fig. 3. All the mutants showed a more
or less strongly marked deficiency in their AO
fluorescence quenching capacity ( = 40 to 80%
the AX2 value). Among them, HGR3 was the
least affected although it should be pointed that
its degree of MDP-resistance was similar to that
in the other mutants. With all the mutant strains,
it was checked that addition of 20 mM imidazole
cancelled the fluorescence decrease. To explain
the observed prevalence of acidification defective
mutants among diphosphonate-resistant strains,
one can postulate that these compounds use a
lysosomal H+-diphosphonate symport to gain access to the cytosol where they react with aminoacyl-tRNA synthetases [12,13]. A reduced ApH
would thus limit the entry of diphosphonate
molecules in the cytosol.
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