Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling
Mirkka Koivusalo, Christopher Welch, Hisayoshi Hayashi, Cameron C. Scott, Moshe Kim, Todd Alexander, Nicolas Touret,
Klaus M. Hahn, and Sergio Grinstein
Vol. 188 No. 4, February 22, 2010. Pages 547–563.
The orientation of the schematic in Fig. 2 B was erroneously reversed in this article. The correct panel appears below.
Figure 2. (B) Top: schematic of the structure of membrane-targeted SEpHluorin/mCherry
chimaera used to measure pHsm. Bottom: confocal images of SEpHluorin (left) and mCherry
fluorescence (right) in A431 cells. Bar, 10 µm.
T H E J O U R N A L O F C E L L B I O L O G Y
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The Rockefeller University Press $30.00
J. Cell Biol. Vol. 188 No. 4 547–563
Correspondence to Sergio Grinstein: firstname.lastname@example.org
Abbreviations used in this paper: CRIB, Cdc42/Rac-interacting binding; DIC,
differential interference contrast; FBE, free barbed end; GEF, guanine nucleotide
exchange factor; Grb2, growth factor receptor-bound protein 2; HOE-694,
(3-methylsulphonyl-4-piperidinobenzoyl)guanidine methanesulphonate; NHE,
Na+/H+ exchanger; NMG, N-methylglucamine; PAK, p21-activated kinase;
PBD, p21-binding domain of PAK; PI3K, phosphatidylinositol-3-kinase; PIP3,
phosphatidylinositol-(3,4,5)-trisphosphate; pHc, cytosolic pH; pHsm, submembra-
nous pH; SNARF-5F, seminaphthorhodafluor dye-5; SEpHluorin, SuperEcliptic
pHluorin; TMR, tetramethylrhodamine.
Macropinocytosis is the most effective way for cells to ingest large
amounts of extracellular fluid. In some cell types macropinocytosis
is a constitutive process: immature dendritic cells use it to sample
soluble antigens (Sallusto et al., 1995) and Dictyostelium amoeba
for nutrient uptake (Cardelli, 2001). Constitutive macropinocytosis
is also observed in fibroblasts transformed with oncogenic v-Src or
K-Ras (Amyere et al., 2000, 2002). Alternatively, macropino-
cytosis can be transiently induced by growth factors, such as
epidermal growth factor or macrophage colony–stimulating factor
(Racoosin and Swanson, 1989; West et al. 2000).
The remodelling of the cytoskeleton that leads to macro-
pinocytosis requires phosphatidylinositol-3-kinase (PI3K)
activity at the plasma membrane (Araki et al., 1996; Rupper
et al., 2001; Lindmo and Stenmark, 2006). Although the entire
signaling sequence is incompletely understood, the GTPases
Rac1 (West et al., 2000) and Cdc42 (Garrett et al., 2000), as
well as p21-activated kinase 1 (PAK1; Dharmawardhane
et al., 2000), are involved in actin polymerization, and CtBP1/
BARS is required for macropinosome closure (Liberali et al.,
2008). The activation of PI3K and the engagement of Rho
family GTPases are common to a variety of actin-dependent
processes such as phagocytosis and chemotaxis. Thus, treat-
ment with inhibitors like wortmannin and Clostridium diffi-
cile toxin B effectively blocks these processes, as well as
relationship between Na+/H+ exchange and macropino-
some formation remains obscure. In A431 cells, stimulation
by EGF simultaneously activated macropinocytosis and
Na+/H+ exchange, elevating cytosolic pH and stimulating
Na+ influx. Remarkably, although inhibition of Na+/H+
exchange by amiloride or HOE-694 obliterated macropino-
cytosis, neither cytosolic alkalinization nor Na+ influx were
required. Instead, using novel probes of submembranous pH,
acropinocytosis is differentiated from other types
of endocytosis by its unique susceptibility to in-
hibitors of Na+/H+ exchange. Yet, the functional
we detected the accumulation of metabolically generated
acid at sites of macropinocytosis, an effect counteracted by
Na+/H+ exchange and greatly magnified when amiloride or
HOE-694 were present. The acidification observed in the
presence of the inhibitors did not alter receptor engage-
ment or phosphorylation, nor did it significantly depress
phosphatidylinositol-3-kinase stimulation. However, activa-
tion of the GTPases that promote actin remodelling was
found to be exquisitely sensitive to the submembranous pH.
This sensitivity confers to macropinocytosis its unique sus-
ceptibility to inhibitors of Na+/H+ exchange.
Amiloride inhibits macropinocytosis by lowering
submembranous pH and preventing Rac1 and
Mirkka Koivusalo,1 Christopher Welch,2 Hisayoshi Hayashi,1,3 Cameron C. Scott,1,4 Moshe Kim,1 Todd Alexander,1,5
Nicolas Touret,1,6 Klaus M. Hahn,2 and Sergio Grinstein1
1Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
2Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
3Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan
4Department of Biochemistry, University of Geneva - Sciences II 30, CH-1211 Geneva, Switzerland
5Department of Pediatrics and Physiology and 6Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
© 2010 Koivusalo et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
on March 18, 2013
Published February 15, 2010
Supplemental Material can be found at:
JCB • VOLUME 188 • NUMBER 4 • 2010 548
Inhibition of macropinocytosis by
A431 cells, which have been used extensively to study
macropinocytosis, were chosen to investigate the mecha-
nism of action of amiloride and its analogues. As reported
previously (West et al., 1989; Araki et al., 2007; Liberali
et al., 2008), addition of EGF to serum-depleted A431 cells
led to extensive membrane ruffling and uptake of extracellu-
lar medium, visualized as trapping of the fluid-phase marker
tetramethylrhodamine (TMR)-dextran (Fig. 1 A). The ruffling,
which was apparent by differential interference contrast (DIC)
microscopy (Video 1), was associated with extensive actin re-
cruitment, revealed by staining with labeled phalloidin. These
effects were most noticeable in the cells at the periphery of
the subconfluent islands (Fig. 1 A). The increases in fluid
phase uptake and actin polymerization were obliterated by
pretreatment with either latrunculin B or with the PI3K inhibi-
tor LY294002, consistent with mediation by macropinocytosis
(Fig. 1 A).
As illustrated in Fig. 1 A, the prototypical NHE inhibitor
amiloride effectively inhibited EGF-induced fluid phase up-
take and actin polymerization. Because at the concentrations
used to inhibit Na+/H+ exchange amiloride has been reported
to affect several other pathways (Alvarez de la Rosa et al.,
2000; Masereel et al., 2003), we also tested HOE-694, a more
selective NHE antagonist. As shown in Fig. 1, A and B, 10 µM
HOE-694 greatly depressed macropinocytic activity. Parallel
experiments verified that, at this concentration, HOE-694
eliminated Na+/H+ exchange. NHE activity was measured as
the rate of Na+-induced recovery of the cytosolic pH (pHc)
from an acid load. Ratiometric determinations of pHc using
seminaphthorhodafluor dye-5 (SNARF-5F) demonstrated that
when Na+ was reintroduced to the medium the cells recovered
rapidly from a cytosolic acidification imposed by an ammo-
nium prepulse. In the presence of 10 µM HOE-694, however,
this response was completely eliminated (Fig. 1 C). At the
submicromolar doses found to inhibit exchange in A431 cells
(Fig. 1 D) HOE-694 selectively inhibits NHE1, with negligi-
ble effects on other isoforms (Counillon et al., 1993). Fig. 1,
C and D therefore suggest that NHE1 is the main, if not the
sole isoform active in the plasma membrane of A431 cells. For
this reason, and to minimize off-target effects, HOE-694 was
the inhibitor of choice in subsequent experiments.
Changes in pHc during macropinocytosis
EGF is known to stimulate Na+/H+ exchange and is capable of
elevating pHc (Moolenaar et al., 1983; Rothenberg et al., 1983;
Yanaka et al., 2002). The resulting alkalinization has been im-
plicated in the initiation of the proliferative effects of EGF
(L’Allemain et al., 1984; L’Allemain and Pouyssegur, 1986)
and may similarly be required for macropinocytosis. This
notion was tested by measuring the pHc changes elicited by
the growth factor in the presence and absence of HOE-694.
As shown in Fig. 2 A, A431 cells stimulated with EGF underwent
a rapid and sizable (≥0.3 unit) alkalinization. In contrast, a net
macropinocytosis. In contrast, macropinosome formation ap-
pears to be uniquely susceptible to inhibition by amiloride
and its analogues, and this property has been extensively
used as an identifying feature of macropinocytosis (West
et al., 1989; Veithen et al., 1996; Meier et al., 2002). Amiloride,
a guanidinium-containing pyrazine derivative, has been used
extensively as an inhibitor of Na+/H+ exchangers (NHEs;
Grinstein et al., 1989; Orlowski and Grinstein, 2004).
However, amiloride is not a universal nor a specific inhibi-
tor of NHE: the affinity of the different NHE isoforms for
amiloride varies greatly and, importantly, the drug also in-
hibits conductive Na+ channels and Na+/Ca2+ exchangers
(Alvarez de la Rosa et al., 2000; Masereel et al., 2003). To
increase the potency and selectivity of NHE inhibitors sev-
eral amiloride analogues have been synthesized, including
ethylisopropylamiloride (EIPA; Masereel et al., 2003) and
sulphonate (HOE-694), which is specific for the NHE1 iso-
form (Counillon et al., 1993).
How amiloride inhibits macropinocytosis remains un-
known. To the extent that EIPA also blocks macropinocytosis,
NHEs are likely to play a role in the process (Cosson et al.,
1989; West et al., 1989), but the mechanism linking ion ex-
change and vacuole formation is not apparent. Three possible
mechanisms can be contemplated: (1) uptake of Na+ by the
exchangers may increase the intracellular solute concentra-
tion, driving osmotically obliged water and causing swelling
that would favor the protrusion of macropinocytic pseudo-
pods. Though the stoichiometric exchange of Na+ for H+ is os-
motically neutral, extruded H+ are replaced from intracellular
buffers, resulting in a net osmotic gain; (2) NHE could be act-
ing indirectly by altering the cytosolic concentration of cal-
cium, which has been suggested to regulate macropinocytosis
(Falcone et al., 2006). Na+ delivered intracellularly in ex-
change for H+ can promote the uptake of calcium via Na+/Ca2+
exchange; (3) the effect of NHE on macropinocytosis may
be mediated by changes in cytosolic pH. Stimulation of NHE
by hormones or growth promoters has been shown to alkalinize
the cytosol (Rothenberg et al., 1983; L’Allemain et al., 1984;
Grinstein et al., 1985; Van Obberghen-Schilling et al., 1985).
Conversely, inhibition of the antiporters impairs the ability
of cells to eliminate H+ generated metabolically and can
cause acidification (L’Allemain et al., 1984, 1985; Grinstein
et al., 1985; Liaw et al., 1998). The changes in pH resulting
from modulation of NHE activity could conceivably alter
the signaling and/or cytoskeleton rearrangements required
We investigated the functional relationship between
macropinocytosis and Na+/H+ exchange. Macropinocytosis
was induced in A431 cells by EGF, and NHE activity was
modulated pharmacologically and by ion substitution. More-
over, we measured the bulk cytosolic pH and the pH of the
inner aspect of the plasma membrane during the course of
macropinocytosis. Our results indicate that NHE1 activity is
required to attain a critical H+ concentration in the immediate
vicinity of the plasma membrane that promotes actin polymer-
ization during macropinocytosis.
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549Amiloride inhibits macropinocytosis by lowering pH • Koivusalo et al.
Measurements of the bulk cytosolic pH, such as those
described above using SNARF-5F, may not accurately reflect
the H+ concentration in the vicinity of the membrane where the
receptors become activated and ruffling is initiated. To more
precisely determine the submembranous pH (pHsm) we generated
a genetically encoded ratiometric pH probe, shown schemati-
cally in Fig. 2 B, which was targeted to the inner aspect of plasma-
lemma. When expressed in A431 cells the Lyn-SuperEcliptic (SE)
acidification was observed when cells were treated with EGF
in the presence of maximally inhibitory doses of HOE-694. The
rapid acidification likely results from the generation of acid
equivalents by metabolic pathways stimulated by the growth
factor. This burst of acid generation is normally not apparent
because it is outstripped by the vigorous H+ extrusion mediated
by Na+/H+ exchange and is only detectable when unmasked by
inhibition of NHE1.
Figure 1. Effect of inhibitors on macropinocytosis and NHE activity. (A) DIC (left) and TMR-dextran epifluorescence images (middle) of islands of A431
cells incubated in the absence (Untreated) or presence of EGF as detailed in Materials and methods. Arrowheads point to dextran-filled macropinosomes.
After determination of macropinocytosis, cells were fixed and stained with rhodamine-phalloidin to visualize actin (left). Arrowheads point to the aspect of
the cell not in contact with neighboring cells. Bar, 10 µm. (B) Quantification of macropinocytosis in control and HOE-694-treated cells. Data are means ±
SE of ≥5 separate experiments. (C) Effect of 10 µM HOE-694 on Na+-induced recovery of pHc after an acid load. NHE activity initiated where indicated
by reintroduction of Na+. Results are representative of 3–4 similar experiments. (D) Concentration dependence of the effect of HOE-694. NHE activity was
measured as in C and rates were calculated from the slopes from Na+-induced pHc recovery curves. Data are means ± SE of three experiments. Where
missing, error bars are smaller than symbol.
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JCB • VOLUME 188 • NUMBER 4 • 2010 550
H+ in the submembranous space. Together, these measurements
not only confirm the burst of metabolic acid generation, but
in addition reveal that its effects are more pronounced in the
immediate vicinity of the membrane, where macropinocytic
Macropinocytosis under Na+-free conditions
To confirm that amiloride and HOE-694 inhibit macropinocytosis
by impairing Na+/H+ exchange, we performed experiments in
media devoid of Na+. As shown in Fig. 3, A–C, omission of Na+
resulted in a drastic reduction in macropinocytic efficiency, in
accordance with previous findings (West et al., 1989), regard-
less of whether the substituent was K+ or N-methylglucamine
(NMG+). Neither of these cations is transported by NHE1 and,
as a result, the alkalinization induced by EGF in physiological
media is absent when Na+ is omitted (Fig. 3 C). Instead, a sharp
acidification is recorded, resembling the effects of maximal
doses of HOE-694 (Figs. 2 A and 3 C).
pHluorin/mCherry probe was found predominantly at the plasma
membrane (Fig. 2 B). That the chimera is a suitable indicator of
pH was verified by in situ calibrations using ionophores to clamp
the intracellular pH (see Materials and methods); the SEpHluorin to
mCherry fluorescence ratio varied nearly linearly with pH in the
6.8–7.8 range (Fig. 2 C), in accordance with the pKa = 7.2 reported
for SEpHluorin (Sankaranarayanan et al., 2000).
Next, we examined the effect of EGF and of maximally
inhibitory concentrations of HOE-694 on pHsm. Although the
overall pattern of responsiveness was similar, the changes re-
ported by the submembranous chimera were more profound:
whereas in stimulated cells the NHE inhibitor produced a net pHc
decrease of 0.5 pH units, pHsm dropped by as much as 0.7 pH
units (Fig. 2 D). A soluble form of the SEpHluorin/mCherry
probe lacking the membrane-targeting domain yielded results
that were similar to those obtained with SNARF-5F (Fig. 2 D),
implying that the larger response detected by Lyn-SEpHluorin/
mCherry is a valid measure of the localized accumulation of
Figure 2. Effect of HOE-694 on EGF-induced changes in pH. (A) SNARF-5F fluorescence ratio measurement of pHc. Where indicated by arrow A431 cells
were stimulated with EGF in the absence (Control) or presence of HOE-694. Data are means ± SE of 3–6 experiments. (B) Top: schematic of the structure of
membrane-targeted SEpHluorin/mCherry chimaera used to measure pHsm. Bottom: confocal images of SEpHluorin (left) and mCherry fluorescence (right) in
A431 cells. Bar, 10 µm. (C) Representative pHsm calibration curve. Cells transfected with membrane-targeted SEpHluorin/mCherry were incubated in the
presence of K+/nigericin buffers of predetermined pH. Fluorescence intensities were measured and the ratio of SEpHluorin/mCherry fluorescence is plot-
ted as a function of pH. (D) Comparison of pHc (SNARF5-F and soluble SEpHluorin/mCherry) vs. pHsm (membrane-targeted SEpHluorin/mCherry) in cells
treated with EGF for 10 min in Na+ medium in the presence and absence of HOE-694 (10 µM). Data are means ± SE of 3–5 experiments. *, P < 0.05.
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551Amiloride inhibits macropinocytosis by lowering pH • Koivusalo et al.
the development of an acidification that may be deleterious
pH dependence of macropinocytosis
The preceding experiments suggested that, in the absence of
Na+/H+ exchange, macropinocytosis may be impaired by the
accumulation of H+ (equivalents) generated metabolically after
engagement of EGF receptors. To validate this notion we mea-
sured the intracellular pH dependence of macropinocytosis. The
uptake of TMR-dextran in response to EGF was quantified in cells
where pHc was clamped at the desired level using nigericin/K+
(Fig. 4). Maintaining pH at a level comparable to that attained when
cells are stimulated in physiological media enabled the cells
to respond to EGF with robust macropinocytosis, despite the
absence of Na+. Normal macropinocytosis was also observed
when pHc was clamped near the resting level recorded in un-
stimulated cells (7.5–7.6). Remarkably, TMR-dextran uptake
The preceding experiments confirm that Na+/H+ exchange
is required for macropinocytosis, but these and previous data
(Cosson et al., 1989; West et al., 1989) cannot define whether
entry of Na+ or extrusion of H+ is the critical event. This was ad-
dressed using nigericin, an electroneutral K+/H+ exchanger. As
shown in Fig. 3 C, when added in the presence of 140 mM
extracellular K+ to balance the osmolarity when omitting Na+,
the ionophore effectively neutralized the metabolic acidification
triggered by EGF. Importantly, the ability of EGF to induce
TMR-dextran uptake was restored by nigericin, implying that
extrusion of H+, and not the entry of Na+, per se, is the key
requirement for macropinosome formation.
The experiments in Fig. 3 also imply that the alkaliniza-
tion mediated by NHE1 that normally accompanies stimula-
tion by EGF is not absolutely required for macropinocytosis
because the latter persists when pHc is clamped with nigericin/K+.
Instead, it is more likely that NHE activity is required to prevent
Figure 3. Effect of Na+ omission on macropinocytosis and pHc. (A) Epifluorescence images of islands of A431 cells after 10 min incubation with TMR-
dextran and EGF in the indicated Na+-free media. To clamp pHc (bottom panel), cells were preincubated in K+/nigericin. Bar = 10 µm. (B) Quantification of
macropinocytosis in Na+-free solutions. Data are means ± SE of 3–4 experiments. Data were compared with controls in Na+-rich medium (as in Fig. 1 B) and
significance calculated using Student’s t test; ***, P < 0.001. (C) Measurement of pHc using SNARF-5F in cells treated with EGF in the indicated buffers.
Data are means ± SE of three experiments, each measuring 10 cells.
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JCB • VOLUME 188 • NUMBER 4 • 2010 552
than NHE activity itself or the associated Na+ gain, is required
In contrast to the exquisite sensitivity of macropinocytosis
to acidification, clathrin-mediated endocytosis was virtually un-
affected by modest changes in pHc and was inhibited only after
marked cytosolic acidification (Fig. 4, A and B). This was de-
termined by measuring the uptake of Alexa 546–conjugated
transferrin (Tfn-A546) in cells where pHc was clamped with
dropped acutely as pHc was decreased progressively. Even
comparatively modest changes in pH produced marked, highly
significant decreases in macropinocytic efficiency (Fig. 4 B)
and virtually complete inhibition was noted at pH 6.8 (Fig. 4,
A and B). Of note, when pHc was clamped at physiological
values the presence of 10 µM HOE-694 was without effect on
macropinocytosis (not depicted). This rules out off-target effects
of the inhibitor and confirms that pH maintenance, rather
Figure 4. Measurement of macropinocytosis and endocytosis in pHc-clamped cells. (A) The cytosolic pH of A431 cells was clamped at 7.8 or 6.8 with
K+/nigericin and TMR-dextran and EGF were added to measure macropinocytosis. After washing, red fluorescence images were acquired. To measure
endocytosis, cells were pHc-clamped, incubated with Tfn-A546 for 15 min, acid-washed, fixed, and imaged. Bar, 10 µm. (B) Effect of pH on macropinocyto-
sis and on clathrin-mediated endocytosis. Cells were subjected to pHc clamping and macropinocytosis quantified as in Fig. 1; clathrin-mediated endocytosis
was assessed as Tfn-A546 uptake. Data are means ± SE of 3–4 experiments. Data were normalized to pHc 7.8 and the significance of the differences
calculated using Student’s t test comparing values within a dataset to pHc 7.8; ***, P < 0.001.
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553Amiloride inhibits macropinocytosis by lowering pH • Koivusalo et al.
clamped at 6.8. The source of the increased binding noted at pH 7.8
is unclear. Nevertheless, altered localization of the kinase is not the
explanation for the impaired macropinocytosis in acidified cells.
The activation of the kinase was assessed next measuring
the phosphorylation of Akt, which is recruited to the membrane
by phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), the prod-
uct of class I PI3K. Akt becomes phosphorylated at the mem-
brane by PDK1 and 2, which are themselves PIP3-activated
kinases (Cantley, 2002; Song et al., 2005). As illustrated in Fig. 6 E,
Akt undergoes a marked phosphorylation at Ser473 when cells
are stimulated with EGF and this effect is unaltered by HOE-694
nigericin/K+. The uptake of Tfn-A546 was largely unaffected
at pH 6.8 and much more acidic values had to be reached before
a sizable inhibition was detected, in good agreement with ear-
lier data (Sandvig et al., 1987). These findings imply that the
inhibition of macropinocytosis seen after a modest acidifica-
tion was not caused by generalized deleterious effects and
provide convenient means for discerning between endocytosis
pH sensitivity of the signals leading
Dynamic assessment of the behavior of pHc-clamped cells by
DIC microscopy revealed that the extension of membrane ruf-
fles, rather than their closure to form macropinosomes, was
affected by moderate acidification (Video 1). This suggested
that an early step in the signaling cascade was impaired by
pH. As shown in Fig. 5, phosphorylation of its receptor was
robustly stimulated by EGF and this effect persisted in the
presence of HOE-694 or in the absence of Na+. Some inhibi-
tion was noted when NHE1 activity was impaired, but this
modest (≈30%) decrease was considerably smaller than the
effect on TMR-dextran uptake (Fig. 5 B) and therefore un-
likely to account for the inhibition of macropinocytosis. This
conclusion was supported by experiments where receptor
phosphorylation was studied in cells where pHc was clamped
in the absence of Na+. Under these conditions, only small de-
creases in phosphorylation were recorded between pH 7.8 and
6.8, whereas macropinocytosis underwent a sharp monotonic
decline (Fig. 5 C). Importantly, TMR-dextran uptake declined
by >80% between pH 7.4 and 6.8, without discernible change
in the extent of receptor phosphorylation. This implies that
downstream signaling events must be responsible for most of
the pH dependence of macropinocytosis.
Next, we measured the effect of pHc on the association
of the adaptor Grb2 (growth factor receptor-bound protein 2;
Liu and Rohrschneider, 2002) with the stimulated receptor by
transfecting A431 cells with a fluorescent version of the SH2
domain of Grb2 (Grb2-SH2-YFP). Before stimulation Grb2-
SH2-YFP had a cytosolic distribution, but upon EGF addition
a fraction redistributed to the plasma membrane, in particular
to regions undergoing ruffling (Fig. 6 A). Re-localization of
Grb2-SH2-YFP upon EGF stimulation was also observed
when Na+ was replaced by NMG+, though partial inhibition
was noted. More importantly, recruitment of the adaptor to the
membrane was essentially identical when pHc was clamped
at pH 7.8 and 6.8 (Fig. 6, A and B). Defective recruitment
of Grb2 is therefore unlikely to account for the pH-induced
inhibition of macropinocytosis.
The recruitment and activation of PI3K (Cantley, 2002;
Hawkins et al., 2006) were studied next. Cells were transfected
with a tagged version of the p85 subunit of the kinase and
its distribution was assessed by confocal microscopy (Fig. 6,
C and D). The regulatory subunit, which was largely cytosolic in
unstimulated cells, redistributed to the plasma membrane upon
stimulation with EGF. The extent of the recruitment was com-
parable in cells stimulated in the presence and absence of Na+
(Fig. 6 D) and was not significantly reduced even when pHc was
Figure 5. Effect of NHE inhibition and of cytosolic pH on EGF receptor
autophosphorylation. (A) Immunoblot analysis of tyrosine phosphorylation
(P-Tyr) of EGF-R (Mw 170 kD) in A431 cells incubated for 5 min with or
without EGF in Na+-rich buffer, with HOE-694 in Na+-rich buffer or in
NMG+-rich buffer. Blot is representative of four experiments. (B) Quantita-
tion of the effect of HOE-694 or NMG+ on EGF-R autophosphorylation,
obtained by scanning immunoblots like the one in A (black bars). Data are
means ± SE of 4–7 experiments. The effect of the same agents/conditions
on macropinocytosis is shown for comparison (open bars). (C) Quantifi-
cation of EGF-R phosphorylation in cells stimulated in Na+-rich medium
or clamped with nigericin/K+ at the indicated pH (black bars). Data are
means ± SE of 3–4 experiments. Data were normalized to controls in
Na+-rich medium; normalized macropinocytosis is shown for comparison
(open bars). ***, P < 0.001.
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JCB • VOLUME 188 • NUMBER 4 • 2010 554
Figure 6. EGF-induced recruitment of Grb2 and p85 to the membrane and Akt phosphorylation: effect of Na+ omission and pH. (A) Confocal images of
A431 cells transfected with Grb2-SH2-YFP acquired before and after treatment for 5 min with EGF, while clamping pHc at the indicated values with nigericin/K+.
(B) Quantification of Grb2-SH2-YFP recruitment to the membrane in response to EGF. Data are means ± SE of 3–4 experiments. (C) Confocal images of cells
transfected with p85-YFP acquired before and after treatment for 5 min with EGF, while clamping pHc at the indicated values using nigericin/K+. Arrowheads in
A and C point to membrane surface not in contact with neighboring cells. Bar, 10 µm. (D) Quantification of the extent of p85-YFP recruitment to the membrane
in response to EGF. Data are means ± SE of three separate experiments. (E) Immunoblot of Akt Ser473 phosphorylation (P-Akt) in cells incubated for 5 min with
EGF in Na+-rich buffer with or without HOE-694 or in NMG+-rich buffer. GAPDH was probed in the same blots to ensure comparable loading. Blot is representa-
tive of 3–4 similar experiments. (F) Quantification of the effect of NHE inhibitors, Na+ omission, and pHc clamping on EGF-induced Akt phosphorylation. Data
are means ± SE of ≥3 experiments of each type. *, P < 0.05; ***, P < 0.001.
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Amiloride inhibits macropinocytosis by lowering pH • Koivusalo et al.
in the cytosol with little association with the plasma membrane,
indicative of a modest tonic activation of Rac1/Cdc42. Upon
addition of EGF, however, PBD-YFP accumulated at the ruffling
plasma membrane; similar results were obtained in cells bathed
in Na+-rich buffer or pHc clamped in K+-rich buffer at 7.8 (Fig. 7,
A and B). In sharp contrast, the EGF-induced redistribution
of PBD-YFP to the membrane was virtually eliminated when
cells were stimulated in Na+-free, NMG+-rich buffer. Failure of
the construct to relocalize was attributed to the acidification
unmasked by omission of Na+ because similar results were ob-
tained when pHc was clamped in K+-rich buffer at 6.8 (Fig. 7,
A and B). These results imply that Rac1/Cdc42 activation is
impaired by decreased cytosolic pH.
To assess whether decreased pHc preferentially affects Rac1
or Cdc42, we used two different methods. We initially performed
a biochemical assay, sedimenting the active form of the GTPases
using immobilized PBD-GST, followed by immunoblotting with
or by omission of Na+. Moreover, a similar degree of phosphory-
lation was observed when cells were clamped at pH 7.8 and
6.8 (Fig. 6 F). Jointly, these observations indicate that activa-
tion of PI3K is not the step responsible for the pH dependence
PIP3 also serves to target to the membrane and to stimulate
guanine nucleotide exchange factors (GEFs) that activate Rho
family GTPases (Lemmon et al., 2002; Lindmo and Stenmark,
2006). GEFs like Vav2 and Tiam1 transduce the signals of the
PI3K to Rac1 and Cdc42, contributing to membrane ruffling
and macropinocytosis (Marcoux and Vuori, 2003; Ridley, 2006;
Garrett et al., 2007; Ray et al., 2007). Therefore, we next mea-
sured the activation of the GTPases using a fusion protein con-
sisting of the p21-binding domain of PAK fused to YFP
(PBD-YFP). This construct binds to the active (GTP-bound) form
of Rac1 and to a lesser extent Cdc42 (Srinivasan et al., 2003).
In unstimulated cells PBD-YFP was distributed predominantly
Figure 7. EGF-induced recruitment of PBD-YFP and Arp3-GFP to the membrane: effect of Na+ omission and pH. (A) Confocal images of A431 cells trans-
fected with PBD-YFP acquired before and after treatment for 5 min with EGF, while clamping pHc at indicated values using nigericin/K+. Cells with low levels
of expression were selected for this experiment. Arrowheads point to membrane surface not in contact with neighboring cells. Bar, 10 µm. (B) Quantification
of PBD-YFP recruitment to the membrane in response to EGF. Data are means ± SE of 3–4 separate experiments. (C) Confocal images of cells transfected
with Arp3-GFP acquired before and after treatment for 5 min with EGF, while clamping pHc. (D) Quantification of the extent of Arp3-GFP recruitment to the
plasma membrane in response to EGF. Data are means ± SE of 3–4 separate experiments. ***, P < 0.001.
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JCB • VOLUME 188 • NUMBER 4 • 2010 556
Figure 8. Effect of pH on activation of Rac1 and Cdc42. (A) Analysis of activated Rac1 and Cdc42 before and after treatment for 5 min with EGF, while
clamping pHc at the indicated values. Active Rac1 and Cdc42 were pulled down using GST-PBD–coated beads. (B) Quantification of the effect of pHc-clamping
on EGF-induced Rac1 and Cdc42 activation analyzed by GST-PBD pull-down. Data are means ± SE of three experiments. Data were compared between pHc
7.8 and pHc 6.8; *, P < 0.05. (C) Assessment of Rac1 activation by FRET imaging of genetically encoded biosensors. Activation of Rac1 was measured by
FRET as detailed in Materials and methods before and after treatment with EGF, while clamping pH at pHc 6.6 or pHc 7.8. Dashed lines in whole-cell images
(middle) align with the direction of protrusion and indicate the area selected for kymography and line-scan analysis (right). Bar, 10 µm. (D) Quantification
of Rac1 and Cdc42 activation analyzed by FRET using line-scan analysis of the regions studied by kymography as in A. Data are means ± SE of three ex-
periments analyzing 6–8 cells in each; ***, P < 0.001. (E) Effect of overexpression or PBD-YPet or CBD-YPet on macropinocytosis. Confocal images of cells
transfected with PBD-YPet or CBD-YPet (left) incubated with EGF and TMR-Dextran (right) for 10 min in Na+-rich medium to assess macropinocytosis. Dashed
lines indicate outlines of cells. Bar, 10 µm. (F) Quantification of macropinocytosis in untransfected or in highly transfected cells by measuring TMR-Dextran
fluorescence intensity (right). Data are means ± SE of three experiments. Data were compared between untransfected and transfected cells; ***, P < 0.001.
on March 18, 2013
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557Amiloride inhibits macropinocytosis by lowering pH • Koivusalo et al.
mediated by Arp2/3 (Condeelis, 2001) or formins (Goode and
Eck, 2007). In addition, FBEs can be generated in stimulated
cells by the actin-binding protein cofilin, a process that occurs
independently of the Rho family GTPases (Condeelis, 2001;
Mouneimne et al., 2004; van Rheenen et al., 2007). Although
free cofilin induces severing of actin filaments and generation
of FBEs, cofilin is inactive when phosphorylated or when
bound to PI(4,5)P2 (Condeelis, 2001; Ridley, 2006). Release
from PI(4,5)P2 can occur as a result of hydrolysis of the
phosphoinositide, but also because of changes in pH. Frantz
et al. (2008) recently demonstrated that cofilin is released
from PI(4,5)P2 at alkaline pH, and provided evidence that
this contributes to PDGF-induced cell migration. The con-
verse reaction, i.e., the persistent attachment of cofilin to
PI(4,5)P2 at more acidic pH, may well explain the inhibitory
effect of amiloride on macropinocytosis. We therefore ana-
lyzed the role of cofilin in our system.
We studied whether cofilin is activated by dephosphorylation
during macropinocytosis. As illustrated in Fig. 9 A, the level of
phospho-cofilin in A431 cells in fact increased in response to EGF
stimulation, as shown earlier in other cells (Mouneimne et al.,
2004; Song et al., 2006). Thus, dephosphorylation does not con-
tribute to cofilin activation in macropinocytosis. Of note, the
level of phospho-cofilin was the same in cells clamped at pHc
7.8 or 6.8, implying that pH had little effect on phosphorylation
We next considered whether cofilin was released by hydro-
lysis of PI(4,5)P2, as found in migrating carcinoma cells
(Mouneimne et al., 2004; van Rheenen et al., 2007). To this end,
we analyzed the fate of the phosphoinositide during macropino-
cytosis using PLC-PH-GFP, a PI(4,5)P2-specific probe. As
shown in Fig. 9 B, PLC-PH-GFP was present at the membrane
before stimulation and, importantly, persisted in the ruffles and
even in nascent macropinosomes—identified by trapped rhoda-
mine dextran—disappearing only seconds to minutes after sealing,
in accordance with previous data (Araki et al., 2007). Quantifi-
cation of the density of the probe (Fig. 9 C) confirmed that
PI(4,5)P2 did not decrease significantly at the early stages of the
process, when actin polymerization is induced. Therefore, re-
lease of cofilin as a result of PI(4,5)P2 hydrolysis is unlikely to
contribute importantly to actin polymerization.
Even if PI(4,5)P2 remains unaltered, its interaction with
cofilin can be weakened by changes in pH (Frantz et al., 2008).
We therefore tested whether EGF-induced formation of FBEs,
a hallmark of cofilin activation, requires cytosolic alkaliniza-
tion. As shown in Fig. 9, D and E, the induction of FBEs by
EGF could be readily detected in A431 cells. Remarkably, the
generation of FBEs persisted when pH was clamped before
stimulation at either pH 7.8 or 7.6. Note that elevation of the
pH alone, in the absence of EGF, had no discernible effect on
FBE formation, implying that alkalinization within the range
induced by EGF was insufficient to promote cofilin-induced
actin polymerization. Together, these results suggest that an
increase in free cytosolic cofilin is not critical to the generation
of FBEs or to actin polymerization during macropinocytosis.
Accordingly, analysis of the localization of either endogenous
or GFP-tagged cofilin indicated that the vast majority of the
Rac1- or Cdc42-specific antibodies. In cells clamped at pH 7.8,
both Rac1 and Cdc42 were stimulated by EGF (Fig. 8, A and B),
as found earlier (Kurokawa et al., 2004). At pHc 6.8, however, the
activation of both GTPases was depressed. The effect was more
apparent for Rac1, which is stimulated more robustly at pH 7.8.
We also analyzed the spatio-temporal dynamics of Rac1
and Cdc42 activation using FRET biosensors (Fig. 8, C and D).
A clone of A431 cells that is more amenable to transfection was
used for these experiments, which require simultaneous expres-
sion of two constructs. This clone also responded to EGF with
ruffling and macropinocytosis and the response was largely sup-
pressed at pH 6.6. As shown in Fig. 8 C and Video 2, treatment
with EGF induced localized activation of Rac1 at the ruffles and
similar, though less robust responses were recorded for Cdc42
(not depicted). When the cytosol was acidified, however, the
responses of both GTPases were largely obliterated (Fig. 8,
C and D). Thus, the FRET analysis is consistent with the bio-
chemical data, indicating that Rac1 and to a lesser extent Cdc42
are activated by EGF and that both GTPases are sensitive to
moderate cytosolic acidification.
The preceding results indicate that Rac1 and Cdc42 are
stimulated by EGF, but do not directly link their activity to ruf-
fling and macropinocytosis. A causal relationship was estab-
lished, taking advantage of the ability of the PBD domain of
PAK and the Cdc42/Rac-interacting binding (CRIB) domain of
WASP to bind to active Rac1 and Cdc42, respectively. When
expressed at low levels, these domains serve as reliable probes
of GTPase activation, but when overexpressed they can scav-
enge away a major fraction of Rac1 or Cdc42 and thereby in-
duce functional inhibition. As shown in Fig. 8, E and F, deliberate
overexpression of either PBD-Ypet or CBD-YPet, the PAK-
PBD and WASP-CRIB domain constructs, caused inhibition of
EGF-induced dextran uptake. Thus, involvement of both Rac1
and Cdc42 is required for optimal macropinocytosis.
Activated Rac1/Cdc42 stimulate WASP and SCAR/
WAVE, which induce actin polymerization via the Arp2/3 com-
plex (Ridley, 2006; Swanson, 2008). Based on the preceding
results, we anticipated that recruitment of Arp2/3 to the mem-
brane during macropinocytosis would also be highly sensitive
to pHc. This prediction was validated in cells transfected with
Arp3-GFP. This indicator was largely cytosolic in unstimulated
cells (Fig. 7 C). Addition of EGF prompted a distinct relocaliza-
tion of Arp3-GFP to the plasma membrane, but this response
was only observed in Na+-rich buffer or when pHc was clamped
at 7.8 using nigericin/K+. When Na+ was replaced by NMG+ or
when pHc was maintained at 6.8, Arp3-GFP remained cytosolic
(Fig. 7, C and D). Jointly, these results indicate that activation of
the small GTPases Rac1 and Cdc42, and of their downstream
effectors that lead to recruitment of Arp2/3 and actin is greatly
impaired by a decrease in cytosolic pH, likely accounting for
the inhibition of macropinocytosis observed when Na+/H+
exchange is blocked.
Role of cofilin
Actin polymerization at sites of membrane protrusion requires
elongation of filaments at free barbed ends (FBEs). After acti-
vation of small GTPases, actin polymerization is most often
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JCB • VOLUME 188 • NUMBER 4 • 2010 558
above its resting value. In support of this notion, amiloride and
its analogues were reported to preclude the alkalinization and
in parallel inhibit cellular proliferation (L’Allemain et al., 1984,
1985; Van Obberghen-Schilling et al., 1985).
Amiloride and HOE-694 also effectively inhibit macropino-
cytosis (West et al., 1989; Sallusto et al., 1995; Veithen et al., 1996;
Meier et al., 2002). Extending the rationale applied to cellular
proliferation, it can be postulated that cytosolic alkalosis signals, or
is permissive to macropinosome formation. An alternative possibil-
ity is that the net osmotic gain associated with Na+/H+ exchange
drives water influx and swelling of the advancing lamellipodia.
Although appealing, these possibilities are not consistent with
our data: EGF activated macropinocytosis under conditions where
pHc was maintained at or even slightly below the resting (unstimu-
lated) level. Moreover, macropinocytosis persisted in the absence
of Na+, e.g., when nigericin/K+ were used to clamp pHc.
These observations raise the possibility that amiloride
analogues may be exerting off-target, nonspecific effects. Indeed,
protein is cytosolic (Fig. S1) and this distribution was unaltered
by EGF stimulation.
Because we failed to implicate cofilin in FBE generation,
we tested whether Rho family GTPases were instead involved,
possibly through the activation of Arp2/3 and/or formins.
Indeed, C. difficile toxin B, an inhibitor of Rho GTPases, oblit-
erated the induction of FBEs by EGF (Fig. 9 E).
EGF is a potent activator of macropinocytosis. Concomitantly,
EGF also stimulates Na+/H+ exchange via NHE1. Stimulation
of NHE1 by growth promoters, including EGF, has been repeat-
edly found to induce cytosolic alkalinization, particularly when
bicarbonate is omitted (Rothenberg et al., 1983; Ganz et al.,
1989; Yanaka et al., 2002). These observations prompted the
widely held view that the stimulatory effects of the growth
factors were mediated by, or at least required, an increase of pHc
Figure 9. Cofilin phosphorylation, PI(4,5)P2 hydrolysis, and actin FBE formation. (A) Analysis of cofilin phosphorylation from lysates of A431 cells before
and after treatment for 1 or 3 min with EGF in Na+-rich buffer. Blot is representative of three experiments. (B) Assessment of PI(4,5)P2 hydrolysis during EGF
stimulation. PI(4,5)P2 was monitored using PLC-PH-GFP and confocal imaging. Recording was initiated upon addition of EGF and TMR-dextran in Na+-rich
buffer. During the initial 6 min, only green fluorescence was monitored. After 6 min excess TMR-dextran was washed and formed macropinosomes were
visualized in red channel. Insets show magnifications of indicated areas. (C) Quantification of PLC-PH-GFP localization to the plasma membrane during
EGF stimulation. Data are means ± SE of three separate experiments. (D) Detection of actin FBEs by imaging rhodamine-actin in cells before and after
treatment for 1 min with EGF in Na+-rich buffer. Images are representative of three experiments. Insets show regions at the edge of cells typically selected
for quantification. (E) Quantification of FBE cells before and after treatment with EGF in Na+-rich buffer or in pHc clamping medium. Data are means ± SE
of three separate experiments. The significance of the difference ± C. difficile toxin B (Tox B) was calculated using Student’s t test; ***, P < 0.001.
on March 18, 2013
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559Amiloride inhibits macropinocytosis by lowering pH • Koivusalo et al.
highly sensitive to pHc. Tiam1, Vav2, and Dock180 have
been implicated in epidermal growth factor receptor (EGFR)–
mediated activation of Rac1 and Cdc42 (Marcoux and Vuori,
2003; Tamás et al., 2003; Makino et al., 2006; Ray et al.,
2007). We attempted to determine the effect of pH on these
GEFs, but failed to observe consistent recruitment of either
Vav2 or Dock180 to the membrane of EGF-stimulated A431
cells. Tiam1, instead, was constitutively associated with the
membrane, as reported previously (Michiels et al., 1995). We
did not notice any significant changes in its distribution when
pHc was lowered from 7.8 to 6.8, and are therefore unable to
attribute the effects of pH to this GEF. We also considered the
possibility that acidification might affect the targeting or
retention of the GTPases in the membrane by altering the
surface charge. A polycationic stretch near the farnesylated
C terminus of Rac1 and Cdc42 is thought to contribute to their
targeting to the negatively charged plasmalemma (Heo et al.,
2006). To this end, cells were transfected with the constitu-
tively active Rac1-Q61L-GFP or with the charge-sensitive
probe R-Pre-mRFP, and their localization was visualized at
pHc 7.8 and 6.8 (Fig. S2). Lowering pHc to 6.8, however, had
no effect on the localization of these probes, suggesting that
altered membrane charge is not the likely explanation for the
reduced activation of the GTPases.
Other downstream steps or parallel pathways are also likely
to be impaired by cytosolic acidification during macropinocytosis.
One such target of pHc is cofilin, an actin-severing protein that
generates new FBEs (Condeelis, 2001; van Rheenen et al., 2007).
Frantz et al. (2008) showed that cofilin binding to PI(4,5)P2 is pH
sensitive, the affinity of the interaction weakening as the cytosol
becomes alkaline. The NHE-mediated alkalosis induced by
growth factors would be expected to release cofilin, contributing
to FBE formation and actin polymerization. The converse reac-
tion, i.e., the persistent attachment of cofilin to PI(4,5)P2 at more
acidic pH, could explain the inhibitory effect of amiloride on
macropinocytosis. Our experimental evidence, however, argues
against this mechanism and against a major role of cofilin in
EGF-induced actin polymerization in A431 cells. First, cofilin
phosphorylation, which is predicted to inactivate the protein,
increased upon EGF stimulation. Second, we found no evidence
for cofilin release from the membrane as a result of PI(4,5)P2
hydrolysis. Third, and most important, we failed to detect any
effect of the pH-dependent release of cofilin from PI(4,5)P2 on
FBE formation or actin polymerization. Mimicking the alkalini-
zation induced by EGF was insufficient to induce FBE or discern-
ible F-actin formation, whereas stimulation with the growth factor
under conditions where pH remained clamped at prestimulation
levels markedly activated FBE formation and actin polymer-
ization (Fig. 9). Moreover, the polymerization of actin and ac-
companying ruffling precede the alkalinization induced by EGF
(Fig. 2 A). Therefore, the sensitivity of cofilin to pH cannot ex-
plain the effects of amiloride on macropinocytosis.
Irrespective of the exact mechanism whereby decreased
cytosolic pH affects small GTPase activation and actin assem-
bly, our results indicate that amiloride and related compounds
are neither direct nor specific inhibitors of macropinocytosis.
Their inhibitory effects are the consequence of submembranous
at high concentrations amiloride directly inhibits autophosphory-
lation of the EGF receptor (Davis and Czech, 1985). Under the
conditions used in our experiments, however, the inhibitory ef-
fect of amiloride and its analogues on macropinocytosis appears
to be specific, caused by inhibition of NHE1. Indeed, inhibition of
exchange by substituting Na+ for NMG+ or K+ (in the absence
of nigericin) impaired macropinosome formation (Fig. 3), and
HOE-694 had no additional effect when added to Na+-free solu-
tions. These observations can be reconciled when considering the
changes in pHc induced by EGF. The growth factor stimulates
metabolic generation of H+ equivalents, but these are effec-
tively extruded by NHE1, which is activated concomitantly.
Indeed, in the presence of physiological [Na+] the stimulation
of the antiporter outstrips the rate of H+ generation, resulting in
a net alkalinization. The occurrence of a metabolic burst is
only unmasked when Na+/H+ exchange is prevented (Figs. 2
and 3). We therefore propose that macropinocytosis is not di-
rectly sensitive to amiloride or even to inhibition of NHE1, but
is instead impaired by the acidification that results when ex-
cess H+ production is uncompensated by the regulatory action
of the Na+/H+ antiporter.
If macropinocytosis is merely responding to the cytosolic
acidification, what makes it uniquely sensitive to amiloride and
its analogues? Other endocytic processes, including uptake of
transferrin through clathrin-coated pits, are also affected by
low pHc (Davoust et al., 1987; Sandvig et al., 1987; Cosson
et al., 1989). However, individual endocytic pathways display
differential sensitivity to changes is pHc: a modest acidification
(pHc ≈ 6.8) virtually eliminated macropinosome formation,
whereas inhibition of clathrin-mediated endocytosis requires a
more profound acidification (pHc ≤ 6.0; Davoust et al., 1987;
Sandvig et al., 1987; Fig. 4). Moreover, geometrical consider-
ations may accentuate the drop in pH experienced during
macropinocytosis. When Na+/H+ exchange is impaired, the H+
generated metabolically during signaling and actin polymer-
ization is likely to accumulate in the thin lamellipodia, where
diffusional exchange with the bulk cytosolic buffers is restricted.
Accordingly, our probes of submembranous pH revealed that
during macropinocytosis the acidification is more profound in
the immediate vicinity of the receptors than in the cytosol over-
all. Cell motility, another process dependent on extension of
lamellipodia, is similarly sensitive to the pHc and requires
NHE1 for optimal function (Lagana et al., 2000; Denker and
Barber, 2002; Stock et al., 2005).
The nature of the pH-sensitive step(s) in macropinocyto-
sis was analyzed by measuring individual events in the signal-
ing cascade while clamping pHc. Acidification caused only
modest changes in receptor phosphorylation (Fig. 5), which
in turn had negligible effects on adaptor binding (Fig. 6,
A and B) and on recruitment and activation of PI3K (Fig. 6, C–F),
a key reaction in macropinosome formation. In contrast, the
activation of Rac1/Cdc42 and their effectors was profoundly
inhibited (Figs. 7 and 8). This conclusion is consistent with
earlier observations of Frantz et al. (2007), who noted the pH
dependence of Cdc42 activation at the leading edge of migrat-
ing cells. We therefore conclude that the exchange factors that
activate Rac1/Cdc42 and/or the GTPases themselves are
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JCB • VOLUME 188 • NUMBER 4 • 2010 560
CFP variant optimized for FRET (Nguyen and Daugherty, 2005), and either
the CRIB domain from p21-activated kinase (PBD) published previously
(Machacek et al., 2009) or the Cdc42-binding CRIB domain from WASP
(CBD), amino acids 230–314, fused to the C terminus of YPet, a YFP variant
optimized for FRET (Nguyen and Daugherty, 2005). The EGFP coding re-
gion from the EGFP-C1 vector (Takara Bio, Inc.) was replaced with a PCR
product containing the CyPet or YPet coding regions flanked by an NcoI re-
striction site and a SGLASELGS linker containing a BamHI restriction site. The
PCR products of the Rac1, Cdc42, PBD, and CBD coding sequences were
inserted between the BamHI restriction site in the SGLASELGS linker and an
EcoRI restriction site in the downstream multiple cloning site of the vector.
The plasmids were transfected into A431 cells using Lipofectamine
LTX, Fugene6, or the Amaxa Nucleofection kit according to the manufac-
The effect of HOE-694 on NHE1 activity was measured as the rate of Na+-
induced recovery of pHc after an acid load. Dual-emission ratio fluores-
cence (640/590 nm) of SNARF-5F was used to measure pHc at 37°C.
Serum-starved cells grown on 18-mm coverslips were placed into Chamlid
imaging chambers (Live Cell Instrument, Inc.), loaded with 20 µM SNARF-5F
acetoxymethyl-ester for 20 min at 37°C and prepulsed with 30 mM NH4Cl
in Na+-rich buffer for 10 min. Cells were acidified by NH4Cl removal with
NMG+-rich buffer, and Na+/H+ exchange initiated by reintroduction of Na+-
rich buffer with or without HOE-694. pHc was calibrated using K+-rich buffer
containing 10 µg/ml nigericin (pHc-clamping buffer) as described previ-
ously (Thomas et al., 1979). pHsm was assessed by transfecting the cells
with membrane-targeted SEpHluorin/mCherry, measuring the SEpHluorin/
mCherry fluorescence emission ratio at the plasma membrane by spinning
disc confocal microscopy using conventional optics for green and red fluoro-
phores, and calibrating with K+/nigericin as above. pHc measured by
soluble SEpHluorin was assessed similarly, but the fluorescence was
captured by epifluorescence as detailed below.
Macropinocytosis and endocytosis assays
To measure macropinocytosis, serum-starved A431 cells grown on cover-
slips and placed in Chamlid chambers were incubated with 0.5 mg/ml
TMR-dextran and, where noted, stimulated with 100–200 ng/ml EGF in
the indicated buffer for 10 min at 37°C. Cells were washed and both DIC
and red fluorescence images of live cells were acquired. Where indicated,
the following inhibitors were used: 10 µM latrunculin B, 100 µM LY294002,
1 mM amiloride, or 10 µM HOE-694. In the case of latrunculin B and
LY294002 the cells were preincubated with the inhibitors at 37°C for 30 min
before EGF addition. Macropinocytosis was quantified as the number of
cells containing macropinosomes in the cells outlining each island.
Endocytosis was assessed by incubating the cells with 50 µg/ml
Alexa 546–conjugated transferrin in the indicated buffer for 15 min at
37°C, after which the cells were placed on ice and acid washed with 0.2 M
acetic acid in 150 mM NaCl and PBS to remove exofacial fluorescence.
The cells were then fixed and mounted on slides, and red fluorescence was
imaged and quantified.
Functional assays in pHc-clamped cells
To clamp pHc, the cells were preincubated at 37°C for 5 min in the presence
of 10 µg/ml of the K+/H+ ionophore nigericin in K+-rich buffer (K+/nigericin)
of predetermined pH before the addition of 100–200 ng/ml EGF.
Imaging of live cells was performed at 37°C in isotonic Na+-rich buffer
or in isotonic K+- or NMG+-rich buffer or pHc-clamping buffer as indi-
cated. pHc measurements were performed using a microscope (model
DM IRB; Leica) equipped with filter wheels (Sutter Instruments) to indepen-
dently alternate between 640- and 590-nm emissions for SNARF5-F
(Steinberg and Grinstein, 2007) or using conventional optics for green
and red for soluble SEpHluorin/mCherry. Light from an EXFO X-Cite 120
lamp (Exfo Life Sciences Group) was directed to the sample by using a di-
chroic mirror. A Plan-Apo 40x/NA 1.25 oil immersion objective was used.
Emitted light was captured by a CCD camera (Cascade II; Photometrics).
The filter wheel and camera were under the control of MetaMorph/Meta-
Fluor software (MDS Analytical Technologies). pHc measured with soluble
SEpHluorin was performed with the same system as above. The fluores-
cence of TMR-dextran taken up by macropinocytosis, transferrin–Alexa
546 taken up by endocytosis, rhodamine-phalloidin as well as rhodamine-
actin were imaged with a microscope (model DMIRE2; Leica) using a 63x
or 100x/NA 1.4 oil immersion objective under the illumination of an
acidification caused by metabolic H+ generation, unopposed
by the regulatory extrusion across the membrane. The unique
sensitivity of macropinocytosis, compared with other endocytic
processes, results from a complex convergence of circum-
stances: a large and sustained metabolic burst that occurs within
a diffusionally constrained compartment, the thin lamellipod.
These considerations must be taken into account when using
amiloride analogues as hallmarks of macropinocytosis because
not only are other processes likely to be inhibited by the pH
change, but macropinocytosis can overcome the inhibitory
effects of these compounds if means other than NHE1 are pro-
vided to regulate pHc.
Materials and methods
The acetoxymethyl-ester of SNARF-5, nigericin, EGF, TMR-dextran (Mw
10,000), transferrin-Alexa 546, and rhodamine-phalloidin were from
Invitrogen. HOE-694 was a gift from Dr. H.-J. Lang (Aventis Pharma,
Frankfurt am Main, Germany). Lipofectamine LTX was from Invitrogen,
Fugene6 from Roche, LY294002 from Enzo Life Sciences, Inc., latruncu-
lin B from EMD, G-Sepharose beads from GE Healthcare, and rabbit
skeletal muscle rhodamine-actin from Cytoskeleton, Inc. Mouse mono-
clonal anti-phosphotyrosine (4G10) and anti-GAPDH antibodies were
from Millipore, rabbit monoclonal anti-phospho-Akt (Ser 473) antibody
was from Cell Signaling Technology, mouse monoclonal anti-Rac1 and
anti-Cdc42 were from BD, and rabbit polyclonal anti-cofilin and anti–
phospho-cofilin were from Abcam. All other chemicals were from
Isotonic Na+-rich buffer contained 140 mM NaCl, 3 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 5 mM glucose ,and 20 mM Hepes, pH 7.4.
In NMG+-rich buffer NaCl and KCl were replaced by 143 mM NMG-
chloride, and in K+-rich buffer NaCl was replaced by 100 mM K-glutamate
and 43 mM KCl.
Cell culture and constructs
A431 cells were from American Type Culture Collection (Rockville, MD)
and were grown in DMEM with 10% fetal bovine serum at 37°C under
5% CO2. Before experiments the cells were serum starved for 2–16 h.
PBD-YFP was a gift from Dr. G. Bokoch (The Scripps Research Institute,
La Jolla, CA). Mouse p85-YFP was plasmid #1408 from Addgene, depos-
ited by Dr. L. Cantley (Harvard Medical School, Boston, MA). R-Pre-mRFP
and Rac1Q61L-GFP (Yeung et al., 2006) as well as PLC-PH-GFP (Várnai
and Balla, 2008) have been described previously. Membrane-targeted
SuperEcliptic (SE) pHluorin/mCherry was generated by first constructing
a chimeric construct consisting of the green, pH-sensitive fluorescent pro-
tein SEpHluorin (a gift of Dr. J. Rothman, Columbia University, New York,
NY; Miesenböck et al., 1998; Sankaranarayanan et al., 2000) fused
to the red, pH-insensitive mCherry (Shu et al., 2006). The EGFP coding
region from the EGFP-N1 vector (Takara Bio, Inc.) was replaced with a PCR
product containing the SEpHluorin coding region flanked by BamHI and
NotI restriction sites. The PCR product of the mCherry coding sequence
was inserted at the EcoRI and BamHI sites. The probe was targeted to the
plasma membrane by inclusion of the N-terminal motif of the Src family
kinase Lyn (Corbett-Nelson et al., 2006). The membrane targeting
sequence of Lyn was inserted between XhoI and EcoRI sites. The forward
and reverse oligonucleotide sequences for the Lyn membrane targeting se-
quence were 5-tcgagagaaatatgggatgtattaaatcaaaaaggaaagacgggAg-3
and 5-aattctcccgtctttcctttttgatttaatacatcccatatttctc-3, respectively.
The Rac1 FRET biosensor was reported previously (Kraynov et al.,
2000; Machacek et al., 2009), and here includes modifications to improve
FRET efficiency reported in Machacek et al. (2009). The Cdc42 biosensor
uses an intermolecular design as reported by several groups (Itoh et al.,
2002; Seth et al., 2003; Tzima et al., 2003; Hoppe and Swanson, 2004),
but here is further optimized by the use of different fluorescent proteins and
of a Cdc42-binding domain from WASP, a fragment shown to provide good
selectivity for activated Cdc42 in a previously developed biosensor with
a different design (Nalbant et al., 2004; Frantz et al., 2007; Machacek
et al., 2009). Both biosensors were generated by first constructing plas-
mids encoding either Rac1 or Cdc42 fused to the C terminus of CyPet, a
on March 18, 2013
Published February 15, 2010
561Amiloride inhibits macropinocytosis by lowering pH • Koivusalo et al.
In each experiment, all images were carefully inspected to verify that all
portions used to create the ratio image had a sufficiently high signal-
to-noise ratio. We targeted at least 300 gray level values (12-bit dynamic
range) above background in the lowest intensity regions within the cell
(S/n > 3). This was especially important in thin parts of the cell where
fluorescence was low.
Actin-free barbed end assay
Actin-free barbed ends were determined by a modification of previously
described methods (Chan et al., 1998; Frantz et al., 2008). In short,
serum-starved A431 cells on coverslips were incubated with or without
EGF in Na+-rich or pHc-clamping buffer for 1 or 3 min. To inhibit Rho
GTPases, cells were incubated in the presence of C. difficile toxin B
(50 ng/ml) for 3 h before EGF stimulation. To label FBEs the cells were per-
meabilized for 15 s in a buffer (20 mM Hepes, 140 mM NaCl, 3 mM
KCl, 2 mM MgCl2, 2 mM EGTA, 5 mM glucose, 1% BSA, and 0.5 mM
ATP, pH 7.5) containing 0.04% saponin and 0.02 µg/µl rhodamine-
labeled rabbit skeletal muscle actin. After 15 s the solution was diluted
with a 3x volume of permeabilization buffer without saponin and rhoda-
mine-actin, and incubation continued for 3 min followed by fixation. The
extent of FBE formation was calculated by measuring fluorescence intensity
of a band 0.3–0.5 µm wide at the edge of the cell (the edge of the protrud-
ing lamellipod in stimulated cells) and a band of the same width (0.5 µm)
inside the cell. The fluorescence intensity is reported as the ratio of the fluor-
escence at the edge to that in the cytosol, and comparison between experi-
ments was facilitated by normalizing to the cytosolic fluorescence.
Samples for Western blotting were scraped off the substratum in the pres-
ence of protease inhibitors (Sigma-Aldrich), 1 mM PMSF, 1 mM Na3VO4,
and 0.1 µM okadaic acid, subjected to SDS-PAGE, and transferred to nitro-
cellulose filters which were then blocked with 5% BSA or milk in TBS-Tween.
The primary antibody dilutions used were 1:10,000 for anti-P-Tyr, 1:5,000
for anti-P-Akt, 1:10,000 for anti-GAPDH, 1:1,000 for anti-Rac1 and
Cdc42, 1:1,000 for anti–phospho-cofilin, and 1:10,000 for anti-cofilin.
After incubating with horseradish peroxidase–conjugated secondary anti-
body the chemiluminescence of the immunoreactive bands was quantified
using the Fluorchem FC2 chemiluminescence system (Alpha Innotech). To
visualize actin, cells were fixed with 4% paraformaldehyde, permeabilized,
and stained with rhodamine-phalloidin.
Online supplemental material
Fig. S1 shows cofilin localization during macropinocytosis. Fig. S2 shows
the effect of cytosolic pH on the localization of surface charge probes.
Video 1 is a DIC illustration of the effect of pHc clamping on membrane pro-
trusions during EGF stimulation. Video 2 depicts Rac1 FRET ratio in pHc-
clamped, EGF-stimulated cells. Online supplemental material is available at
We thank Dr. Caterina DiCiano-Oliveira for help with PBD pull-downs and
Dr. Michael Glogauer for help with the FBE assay.
This work was supported by grant MOP4665 of the Canadian Institutes
of Health Research and a grant from the Ella and Georg Ehrnrooth Foundation
to M. Koivusalo. We gratefully acknowledge funding from National Institutes of
Health grants GM57464 and GM64346 (to K.M. Hahn), T32 GM008719,
and F30 F30HL094020-02 (to C. Welch). S. Grinstein holds the Pitblado
Chair in Cell Biology and is cross-appointed to the Department of Biochemistry
of the University of Toronto.
Submitted: 17 August 2009
Accepted: 25 January 2010
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EBQ100 lamp and using conventional optics for red fluorophores. Images
were captured by a CCD camera (model C4742-95-12ER; Hamamatsu
Photonics) under the control of Openlab software (PerkinElmer).
Imaging of transfected cells for construct localization analysis as
well as pHsm measurements were performed on a spinning disc confocal
microscopy system (Quorum Technologies, Inc.) based on an Axiovert
200M microscope (Carl Zeiss, Inc.), as detailed previously (Yeung et al.,
2008). Cells were maintained at 37°C using an environmental control sys-
tem (Live Cell Instrument). The samples were excited with diode-pumped
solid-state laser lines (Applied Research) using a 491-nm laser line and
520-nm emission filter for GFP, YFP, and SEpHluorin, and a 561-nm laser
line and 590-nm emission filter for mCherry and mRFP. A 63x/NA 1.4 oil
immersion objective was used. Images were acquired using a back-thinned
electron multiplier CCD camera (C9100-13 ImagEM; Hamamatsu Photonics)
driven by Volocity 4.1.1 software (PerkinElmer).
The software used for subsequent image analysis was MetaFluor for
pHc measurements and Volocity 4.1.1 for construct localization analysis
and all the other fluorescence intensity quantifications. All the quantifica-
tions were performed on background-subtracted images with 3–10 cells
analyzed in each experiment.
Rac1 and Cdc42 activity assays
The abundance of active (i.e., GTP-bound) small GTPases was quantified
by immunoblotting after a GST-PAK-PBD pull-down step, as described previ-
ously (Di Ciano et al., 2002).
Activation of both Rac1 and Cdc42 by FRET was measured in living
cells by monitoring the ratio of FRET (CyPet excitation and YPet emission
channels) to CyPet emission (CyPet excitation and emission channels), cor-
recting for emission bleed-through as described previously (Hodgson et al.,
2009) and briefly below. Cells were chosen for low expression levels
so that neither PBD-YPet nor CBD-YPet inhibited macropinocytosis. Time-
lapse sequences were acquired on an inverted epifluorescence microscope
(model IX81; Olympus), using a 40X UPlan FLN 1.3 N/A DIC lens (Olym-
pus), CCD camera (CoolSnapESII; Roper Industries), and MetaMorph soft-
ware. For emission ratio imaging, the following filter sets were used
(Chroma Technology Corp.): CyPet: D436/20, D470/40; FRET: D436/20,
HQ535/30; YPet: HQ500/20, HQ535/30. A dichroic mirror was
custom manufactured by Chroma Technology Corp. for compatibility with
all of these filters. Cells were illuminated with a 100-W Hg lamp through
an ND 1.0 neutral density filter. At each time point, three images were
recorded with the following exposure times: CyPet (1.2 s), FRET (1.2 s),
and YPet (0.4 s) with 2 × 2 binning. We routinely changed the order of ac-
quisition for all experiments to control for motion artifacts, using either the
order CyPet, FRET, YPet; or FRET, CyPet, YPet. We have used this approach
previously to show that the order of data acquisition did not affect the mea-
sured ratio (Nalbant et al., 2004; Pertz et al., 2006). In addition, measure-
ments of cells obtained in forward and reverse order were combined in the
final analysis. Time-lapse sequences were recorded at 10- or 20-s intervals
between frames for all biosensors, as described previously (Pertz et al.,
2006; Hodgson et al., 2009).
Ratio calculations to generate activity images were performed after
bleed-through and photobleaching correction methods described previ-
ously (Hodgson et al., 2009) because each component of the two chain bio-
sensor is not necessarily distributed equally throughout the cell. For
bleed-through correction, cells expressing CyPet or YPet alone were imaged
using the same imaging medium, exposure times, and intensities as those
listed above for the actual experiment. These measurements were used to
determine the amount of bleed-through of each fluorescent protein’s direct
emission into the FRET channel. Images from each channel were shade cor-
rected and background subtracted. Plotting FRET intensity as the depen-
dent variable versus CyPet or YPet intensity as the independent variable
yielded a linear relationship where the slope of the line defined the bleed-
through coefficient for that fluorescent protein and condition (0.25 for CyPet
and 0.15 for YPet using the conditions above). Using these parameters, the
ratio was derived using Eq. 1:
FRET Ratio FRET
− α⋅− β⋅
This equation corrects the raw FRET signal for bleedthrough from both CyPet
and YPet into the FRET channel, with subsequent calculation of the ratio.
For visual representations, a linear pseudocolor look-up table was
applied to all ratio images and the ratio values were normalized to the
lower scale value, which was chosen to exclude the bottom 5% of the
total histogram distribution, thereby avoiding spurious low intensity pixels.
on March 18, 2013
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on March 18, 2013
Published February 15, 2010