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INTRODUCTION |
Progressive acidification of vesicles in the endosomal pathway is
important for receptor and ligand sorting and vesicular fusion (1, 2).
Endosomal acidification is driven by a vacuolar-type H+
pump that is present in the endosomal-limiting membrane. To maintain electroneutrality, H+ entry into the endosomal aqueous
lumen must be accompanied by anion entry and/or cation exit. The
principal transportable intracellular anion is Cl
and
cation is K+. Studies of organellar pH in living cells and
isolated vesicles have provided evidence that Cl entry
may be the rate-limiting passive conductance in permitting active
H+ entry in endosomes (3-9). In isolated endocytic
vesicles from kidney proximal tubule (10) and liposomes reconstituted
with proteins from clathrin-coated vesicles (11), a protein kinase A-activated Cl conductance was characterized, and it was
proposed that activation of Cl channels might regulate
endosomal acidification by providing a shunt to dissipate the
interior-positive potential produce by the H+ pump. There
is also evidence that Na+/K+ pump activity in
early endosomes may alter the driving force for H+ entry
and thus regulate acidification (12-14). In Golgi, there is evidence
that both Cl and K+ conductances may
contribute to acidification (15-17), whereas acidification of
secretory granules in synaptic vesicles appears to require the
expression of a specific Cl channel (18). Although
measurements of endosomal pH have been reported utilizing ratioable
pH-sensitive fluorescent indicators (19-22), there have been no
measurements of ion concentrations in the endosomal lumen.
Physico-chemical considerations indicate that endosomal
[Cl] and pH should depend on the activity of the
vacuolar H+ pump, the magnitude of endosomal cation
(K+, Na+, and H+) and anion
(Cl and HCO3) conductances,
endosomal membrane potential, buffer capacity, Donnan potential, and
cytoplasmic pH and ion concentrations. Although attempts have been made
to model endosomal/organelle acidification mathematically (23, 24), the
paucity of information about key endosomal parameters precludes
meaningful predictions about endosomal regulatory processes. Taken in
reference to endosomal pH and cytoplasmic pH/[Cl],
endosomal [Cl] is a particularly important parameter
because of its implications for relative endosomal ion conductances and
membrane potential. If Cl conductance is the
rate-limiting ion conductance in endosomal acidification, then the
interior-positive endosomal electrical potential should produce marked
Cl accumulation in the endosomal aqueous lumen during acidification.
The purpose of this study is to develop and apply methodology to
measure endosomal [Cl] quantitatively in living cells.
For these measurements, we synthesized a ratioable long wavelength
fluorescent Cl indicator that is brightly fluorescent,
pH-insensitive, sensitive to [Cl] from 0 to >100
mM, biochemically stable, and membrane-impermeant. The
endosomal aqueous lumen in cultured cells was stained with Cl and pH indicators by fluid-phase endocytosis, and the
kinetics of endosomal [Cl] and pH were measured by
ratio image analysis. Pulse labeling, inhibitor addition, and ion
substitution maneuvers established quantitatively the role of
Cl conductance in endosomal acidification. An
unexpectedly low [Cl] early after endocytosis led us to
postulate that endosomal volume increases substantially during
acidification, which was supported experimentally using a novel ratio
imaging strategy to measure relative endosomal volume.
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MATERIALS AND METHODS |
Reagents
All chemicals for synthesis were purchased from Aldrich.
Aminodextran (Mr 40,000), 5-(and
6)-carboxyfluorescein (CF)1
succinimidylester, 5-(and 6)-carboxytetramethylrhodamine
succinimidylester (TMR-SE), CF-carboxytetramethyl-rhodamine
(TMR)-dextran (CF-TMR-dextran), calcein, sulforhodamine B,
6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ), and
2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethyl
ester (BCECF-AM) were obtained from Molecular Probes (Eugene, OR).
Nigericin, bafilomycin, valinomycin, monensin, and carbonyl cyanide
m-chlorophenylhydrazone (CCCP) were obtained from Sigma.
Cell Culture
J774.1 macrophages (ATCC no. TIB-67) were obtained from American
Type Cell Culture Collection (Manassas, VA) and grown in Dulbecco's
modified Eagles medium DME-H21 supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. CHO-K1 cells (ATCC no. CCL-61) were also obtained from the ATCC and
grown in Ham's F12K medium supplemented with 10% fetal bovine serum,
100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were
cultured on 18-mm diameter round glass coverslips at 37 °C in a 95%
air, 5% CO2 incubator and used just prior to confluence.
Synthesis of Ratioable Fluorescent Cl Indicator
Synthesis of 10,10'-Bis[3-carboxypropyl]-9,9'-biacridinium
Dinitrate (BAC) (Compound 3)--
To a stirred suspension of
10-[3-carboxypropyl]-9(10H)-acridone (Fig. 1A,
compound 1; prepared according to Ref. 25) (2.8 g, 10 mmol) in acetone (80 ml) was added zinc dust (13.3 g, 201 gram atom).
The mixture was stirred for 20 min at 30-40 °C. The flask was
cooled in ice-cold water, and 37% HCl (121 g, 1226 mmol) was added
dropwise over 4 h under inert atmosphere at 10 °C. The reaction
mixture was stirred overnight at room temperature, and 50 ml of
degassed water was added. A bright yellow precipitate was collected by
suction filtration, rinsed with water, and dissolved in 50 ml of a 5%
aqueous NaOH. The mixture was filtered, and the filtrate was
neutralized with acetic acid to give a bright yellow precipitate. The
precipitate was filtered, washed with water, dried, and recrystallized
from hot ethanol to yield 4.39 g (83%) of
10,10'-bis[3-carboxypropyl]-9,9'-biacridylidene (compound
2) as a yellow crystalline solid.
A suspension of compound 2 (5.3 g, 10 mmol) in 2 N nitric acid (250 ml) was heated for 1 h at 120 °C
until most of the brownish mass was dissolved. After cooling and
filtration, the precipitate was washed with dilute nitric acid, dried,
and recrystallized from dilute nitric acid to yield 4.81 g (74%)
of compound 3 as a yellow crystalline solid (absorption
maximum, 435 nm; molar extinction coefficient (
), 12,500 cm1 M1; quantum yield, 0.48).
1H NMR (400 MHz, Me2SO-d6): 
2.80-3.90 (m,
10H, alkyl CH2 and COOH), 5.58 (t, 4H, N-CH2,
J = 6.3 Hz), 7.64 (broad singlet, 4H, aromatic), 7.71 (t, 4H, aromatic, J = 6.0 Hz), 8.45 (t, 4H, aromatic, J = 8.8 Hz), and 9.04 (d, 4H, aromatic,
J = 9.2 Hz).
Synthesis of
10,10'-Bis[(3-N-succinimidyloxycarbonyl)propyl]-9,9'-biacridinium
Dinitrate (Compound 4)--
A solution of compound 3 (3.27 g, 5 mmol) in N,N-dimethylformamide (90 ml) was reacted with
N-hydroxysuccinimide (1.56 g, 13.6 mmol) and
N,N-dicyclohexylcarbodiimide (2.48 g, 12.05 mmol) at room
temperature for 24 h. The reaction mixture was diluted with
acetonitrile (15 ml) and stirred for 1 h, and the solid was removed by suction filtration. After washing in acetonitrile the filtrate was concentrated on a rotary evaporator, and the concentrate was added dropwise into a rapidly stirred mixture of ethyl
acetate/hexane (3:1, 500 ml). The yellow precipitate was collected by
suction filtration, and the filtrate was rinsed with ethyl
acetate/hexane (3:1, 50 ml). The product was further purified by
dissolving in N,N-dimethylformamide and precipitating in
ethyl acetate/hexane to yield 2.93 g (69%) of compound
4 as a yellow solid (melting point, 214-216 °C).
1H NMR (400 MHz, Me2SO-d6): 2.60-2.90 (m,
16H, alkyl CH2 and succinimidyl CH2), 5.62 (t,
4H, N-CH2, J = 6.3 Hz), 7.68 (broad singlet, 4H, aromatic), 7.76 (t, 4H, aromatic, J = 6.8 Hz), 8.49 (t, 4H, aromatic, J = 8.4 Hz), and 9.07 (d,
4H, aromatic, J = 9.7 Hz).
Synthesis of Biacridinium-tetramethylrhodamine Dextran
(BAC-TMR-Dextran, Compound 5)--
For synthesis of TMR-dextran,
5-(and 6-)-carboxytetramethyl rhodamine succinimidyl ester (26.4 mg,
0.05 mmol) was stirred with amino dextran (1.0 g, 0.025 mmol,
Mr 40,000) in aqueous NaHCO3 (25 ml,
0.1 M, pH 8.3-8.5) at room temperature for 2 h. The
dextran conjugate was purified by dialysis (25,000-Da cut-off) for
24 h against 0.1 M NaHCO3 and then against
water for 36 h at 4 °C. The TMR-dextran was lyophilized (molar
labeling ratio TMR/dextran, 1.08:1). For synthesis of BAC-TMR-dextran,
TMR-dextran (0.5 g, 0.0125 mmol) was reacted with compound 4 (0.25 g, 0.295 mmol) in aqueous NaHCO3 (50 ml, 0.1 M, pH 8.3-8.5) at room temperature for 3 h. The
reaction mixture was worked up as above to yield 0.46 g of
BAC-TMR-dextran (92% with respect to dextran); molar labeling ratio
biacridinium/TMR/dextran, 6.68:1.08:1.
Characterization of the Ratioable Fluorescent Cl
Indicator
Fluorescence spectra, molar extinction coefficient, and quantum
yield were measured by standard procedures using a Fluoromax-3 fluorimeter. Fluorescence quenching measurements were carried out at
peak excitation and emission wavelengths. Microliter aliquots of NaCl
(1 M stock) were added to 3 ml of compound (10 µM in 5 mM
Na2HPO4-NaH2PO4) at pH
7.4. Stern-Volmer constants (Ksv) were calculated from the slope of F0/F 
1 versus [Cl] plots (F0/F 1) = Ksv [Cl] where
F0 is BAC fluorescence in the absence of Cl
and F in the presence of Cl.
Endosome Labeling and Cell Perfusion
Endosomes were labeled by incubation of cells on coverslips with
BAC-TMR-dextran (18 mg/ml) or CF-TMR-dextran (3.5 mg/ml) for 2 min in
phosphate-buffered saline (PBS: 138 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM
Na2HPO4-NaH2PO4) at
37 °C. Coverslips were then washed five times in ice-cold PBS
containing 2% bovine serum albumin and mounted in a custom-built
perfusion chamber maintained at 37 °C in a PDMI-2 microincubator
(Harvard Apparatus, Holliston, MA). Cells were generally perfused with PBS. In some experiments the perfusate contained bafilomycin A1 (200 nM) or NH4Cl (5 or 10 mM). For
Cl-free experiments buffer Cl was replaced
by NO3.
Calibration Protocols
For in vivo Cl calibrations (BAC/TMR
fluorescence ratio versus [Cl]), perfusate
and endosomal [Cl] were equalized by incubation of
cells for up to 1 h at 37 °C in 120 mM
KCl/KNO3, 20 mM NaCl/NaNO3, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES pH 7.4, with [Cl] from
0-100 mM (NO3 replacing
Cl). Solutions contained the ionophores nigericin (10 µM), valinomycin (10 µM), CCCP (5 µM), monensin (10 µM), and the
H+ pump inhibitor bafilomycin (200 nM). For
in vivo pH calibrations (TMR/CF fluorescence ratio
versus pH) cells were incubated with 1 mM
CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM MES, 120 mM KCl, 20 mM NaCl, and the ionophore/bafilomycin mixture with pH
adjusted to 4.5-8.0 (in 0.5-pH unit intervals) (21).
Cytoplasmic pH was measured using BCECF by excitation ratio imaging
(440 and 490 nm) (26). Cytosolic [Cl] was measured
using SPQ as described previously (27).
Kinetics of Endosomal [Cl] and pH
After fluid-phase pulse labeling (2 min) with BAC-TMR-dextran or
CF-TMR-dextran and washing, the cells were perfused briefly with
ice-cold PBS in the perfusion chamber and then with PBS at 37 °C.
Sets of BAC and TMR images (for Cl) or CF and TMR images
(for pH) were acquired at specified times (generally 0, 5, 15, 30, and
45 min). In some experiments bafilomycin (200 nM) was added
to the perfusate from the beginning of the experiment or 45 min after
pulse labeling.
Endosomal Buffer Capacity
Buffer capacity (
) was determined from the rapid increase in
endosomal pH in response to addition of 5 mM
NH4Cl to the perfusate (5 mM NH4Cl,
130 mM NaCl, 2.7 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, and 10 mM HEPES pH 7.4). was computed from the equation:
= ([NH4Cl]/
pH)·10(pH(out) pH(final)),
where pH is the pH increase just after NH4Cl addition,
pHout is perfusate pH, and pHfinal is endosomal
pH after NH4Cl addition (28, 29).
Endosomal Volume Changes
Endosomes were pulse-labeled with the mixture of calcein (at
self-quenching concentration, 30 mM) and sulforhodamine 101 (at low concentration, 2 mM) in PBS (adjusted to 290 mosM)
for 1 min followed by washing with ice-cold PBS. The coverslip was
transferred to the perfusion chamber as described above. A series of
calcein (green) and sulforhodamine 101 (red) images were obtained
at specified times. Endosome expansion produced increased calcein
fluorescence because of volume dilution without change in
sulforhodamine 101 fluorescence. In some experiments bafilomycin (200 nM) was added to the perfusate. An in vitro
calibration of green-to-red fluorescence versus dilution
factor curve was obtained by collecting sets of images of thin films
(sandwiched between coverslips and glass slides) of PBS containing
calcein (30 mM) and sulforhodamine 101 (2 mM)
after specified dilutions with PBS.
Fluorescence Microscopy
Experiments were carried out using a Leitz upright fluorescence
microscope equipped with a coaxial-confocal attachment (Technical Instruments, San Francisco, CA) and a 14-bit cooled (30 °C) CCD camera (Photometrics) with a Tektronix back-thinned detector as described previously (19). Cell fluorescence was viewed with a ×100
oil immersion objective with a numerical aperture of 1.4 (Plan-apo,
working distance 0.17 mm, Nikon, Garden City, NY). The light source was
a stabilized Hg-Xe arc lamp (100 watt) lamp attenuated 5-50-fold using
neutral density filters. Cells were identified by transillumination
using dim red light (to avoid photobleaching), and focus was adjusted.
Images (500-ms acquisition time) were obtained using appropriate filter
sets for the BAC, TMR, CF, calcein, sulforhodamine 101, and BCECF
chromophores. Electronic shutters (Uniblitz, model D122, Vincent
Associates, Rochester, NJ) in the illumination and detection paths
minimized sample illumination. Custom filter sets (Chroma, Brattleboro, VT) were used for detection of BAC fluorescence (excitation, 470 ± 5 nm; dichroic, 505 nm; emission, 535 ± 20 nm) and TMR
fluorescence (excitation, 546 ± 5 nm; dichroic, 565 nm; emission,
590-nm cut-on). Serial image acquisitions indicated 1-3% BAC
photobleaching per image acquisition and <1% photobleaching for other
chromophores. In time course studies endosomes in different cells were
imaged for different time points.
Ratio Image Analysis
Custom software was written in Labview to compute
area-integrated background-subtracted pixel intensities. In each TMR
(red) image, four regions of cells containing well demarcated endosomes were identified by rectangular boxes, and for each region four nearby
regions outside of the endosome/cell were identified for background
computation. The same regions were automatically identified in the BAC
(green) image and displaced slightly if necessary because of chromatic
effects or slight sample movement. The average background was computed
for each selected endosome region from the per-pixel intensity of the
three lowest of the four selected background regions. After background
subtraction, red-to-green (R/G) intensity ratios were computed from
area-integrated intensities in each image. Three pairs of images were
analyzed for each time point. The same analysis routine was used with
CF-TMR images for pH measurements. Calcein/sulforhodamine images for
relative volume measurements were analyzed by measuring area-integrated
pixel intensities over individual endosomes in green (calcein) and red
(sulforhodamine) images. Background values were taken from three areas
without endosomes within the analyzed cell. Ratios were calculated for four endosomes in each image pair, and at least two image pairs were
analyzed per time point.
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RESULTS |
Characterization of a Ratioable Cl Indicator for
Measurement of Endosomal [Cl]--
The ratioable
fluorescent Cl indicator BAC-TMR-dextran (Fig.
1A, compound
5) was synthesized by covalently linking the
chloride-sensitive chromophore BAC (synthesized in five steps from
9(10H) acridon and 
-butyrolactone) and the
Cl-insensitive chromophore TMR to amino dextran (reaction
scheme shown in Fig. 1A). BAC fluorescence was sensitive to
Cl in the range 0 to >120 mM with a
Stern-Volmer quenching constant of 36 M1
(Fig. 1B, left). BAC fluorescence was insensitive
to pH in the range appropriate for cellular measurements (Fig.
1B, top right) and insensitive to cations,
non-halide anions (nitrate, phosphate, bicarbonate, and sulfate), and
albumin. The green-fluorescing BAC chromophore has fluorescence
excitation maxima at 365 and 434 nm and a broad emission spectrum with
peak at 505 nm (Fig. 1B, bottom right). The
red-fluorescing TMR chromophore is not sensitive to pH or ion
concentrations so that the ratio of red TMR fluorescence to green BAC
fluorescence gives [Cl].
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Fig. 1.
Synthesis and fluorescence properties of
BAC-TMR-dextran. A, reaction scheme (see "Materials
and Methods" for details). B, left,
Stern-Volmer plot for fluorescence quenching of BAC-dextran by
Cl. F0 is BAC fluorescence in the absence of
Cl and F in the presence of Cl (fitted
Stern-Volmer constant, 36 M1). Top
right, pH dependence of BAC fluorescence. Bottom
right, excitation and emission spectra of BAC-dextran in 10 mM sodium phosphate (pH 7.4).
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Cl Accumulation in Endosomes--
Fig.
2A shows fluorescence emission
spectra of BAC-TMR-dextran, indicating the Cl-sensitive
green BAC fluorescence and the Cl-insensitive red TMR
fluorescence. For cell studies, two cell lines with different
properties were used (J774 macrophages and CHO cells). Fig.
2B (left) shows a calibration of BAC-TMR-dextran red-to-green fluorescence ratio (R/G) versus
[Cl] in aqueous solution (filled circles)
and J774 cells (open circles). Cl was replaced
by nitrate in the solution measurement. For cell measurements,
endosomes were labeled by fluid-phase endocytosis with a 2-min
incubation with BAC-TMR dextran in saline. Representative cell images
are shown in Fig. 2B (right). As found in
previous studies (20-32), endosomes were seen as distinct bright spots on a dark background. The fluorescent spots became larger with increasing chase time, corresponding to progression from early endosomes to multivesicular bodies to lysosomes. The red TMR
fluorescence seen in Fig. 2B (right) colocalized
well with the green BAC fluorescence. Endosome labeling was not seen
when cells were incubated for 2 min with BAC-TMR-dextran at 4 °C
instead of 37 °C (images not shown).
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Fig. 2.
Calibration of BAC-TMR-dextran fluorescence
ratio versus [Cl]. A,
fluorescence emission spectra of BAC-TMR-dextran (excitation: 434 nm
BAC, 540 nm TMR) in 10 mM sodium phosphate (pH 7.4)
containing indicated [Cl]. B,
left, calibration of BAC-TMR-dextran fluorescence
versus [Cl] in solution (filled
circles) and living cells (open circles). Indicator
red-to-green fluorescence ratios (R/G) were measured by fluorescence
microscopy as described under "Materials and Methods." For solution
measurements Cl was replaced by
NO3. For cell measurements J774 cells were
pulse-labeled with BAC-TMR-dextran for 2 min and incubated for 1 h
with buffers containing specified [Cl] and ionophores
(see "Materials and Methods" for details). Data are mean ± S.E. (smaller than symbols) for measurements on three separate sets of
experiments. Right, representative images of TMR red
fluorescence and BAC green fluorescence in J774 cells in calibration
study with 60 mM [Cl].
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Calibration of R/G versus [Cl] in endosomes
was done by incubating BAC-TMR-dextran-loaded cells with a high
K+ buffer containing an ionophore/bafilomycin mixture to
equalize solution and endosomal [Cl] as described in
"Materials and Methods." R/G was measured in endosomes from 8-10
different cells in three separate cell cultures. Fig. 2B
(left) shows that R/G versus [Cl]
in endosomes is not significantly different from that in solution, indicating that BAC-TMR-dextran fluorescence is
Cl-selective and thus insensitive to other components
present in the endosome lumen.
The R/G versus [Cl] calibration was used to
determine endosomal [Cl] under physiological
conditions. Fig. 3A shows TMR
and BAC images of J774 cells at 0, 15, and 45 min after pulse labeling
together with a pseudocolored ratio image with a [Cl]
scale. Endosomal [Cl] was fairly uniform in each image
and increased with chase time. Fig. 3B summarizes the
kinetics of endosomal [Cl] in J774 and CHO cells as a
function of chase time at 37 °C. Endosomal [Cl]
progressively increased and attained steady-state values after an
~45-min chase time. Interpretation of these data in terms of driving
forces requires knowledge of endosomal pH as well as cytoplasmic pH and
[Cl] (see below). Fig. 3C shows that
Cl accumulation in endosomes was reversed by the addition
of the vacuolar H+ pump inhibitor bafilomycin
A1. Here the bafilomycin was added after the 45-min chase
period. These data suggest that H+ pump activity drives
Cl accumulation in endosomes.
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Fig. 3.
Time course of endosomal [Cl]
after pulse labeling. A, fluorescence micrographs of
J774 cells at 0, 15, and 45 min after pulse labeling showing red TMR
fluorescence (top), green BAC fluorescence
(middle), and pseudocolored ratio image with
[Cl] scale (bottom). B, time
course endosomal [Cl] after 2-min pulse labeling with
BAC-TMR-dextran computed from measured R/G (right hand
axis). Data are shown as mean ± S.E. (n = three sets of cell cultures, 8-10 cells from each culture at each time
point). C, time course of endosomal [Cl]
following bafilomycin (200 nM) addition to the perfusate at
45 min after labeling (n = 3).
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Relationship between Endosomal Acidification and Cl
Accumulation--
Endosomal pH was measured under the same conditions
used in endosomal [Cl] measurements. The ratioable pH
indicator CF-TMR-dextran was used in which CF green fluorescence is
pH-sensitive and TMR red fluorescence is pH-insensitive (21). Fig.
4A shows a calibration plot of
TMR-to-CF red-to-green fluorescence ratio (R/G) versus pH in
endosomes. The perfusate contained high [K+] and
ionophores/bafilomycin to equalize external and endosomal pH. R/G was
sensitive to pH in the range appropriate for endosomal measurements.
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Fig. 4.
Endosomal acidification. A,
in situ calibration of CF-TMR-dextran red-to-green
fluorescence ratio (R/G) as a function of pH. J774 cells were
pulse-labeled with CF-TMR-dextran for 2 min and incubated for 1 h
with buffers containing ionophores and bafilomycin at specified pH (see
"Materials and Methods" for details). Data are shown as mean ± S.E. of measurements for three sets of experiments. B,
time course of endosomal acidification after 2-min pulse labeling with
CF-TMR-dextran (n = three sets of experiments). Where
indicated bafilomycin (200 nM) was added to the perfusate.
C, representative measurement of endosomal buffer capacity
showing prompt increase in endosomal pH after addition of 5 mM NH4Cl. The average endosomal buffer capacity
was computed from pH (S.E., n = 4)
(inset).
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Fig. 4B summarizes the time course of endosomal pH in J774
and CHO cells as a function of chase time. Endosomes were labeled with
CF-TMR-dextran for 2 min as done for the [Cl]
measurements using BAC-TMR-dextran. As reported in other cell types
(19-21) endosomes progressively acidified. Bafilomycin addition to the
perfusate alkalinized the endosomal lumen as expected (33).
To relate changes in [Cl] and pH quantitatively,
endosome buffer capacity was measured. Buffer capacity was determined
from the prompt pH increase following addition of NH4Cl (5 or 10 mM) to the perfusate (Fig. 4C) (34). Rapid
entry of NH3 produces luminal alkalinization, which depends
on the amount of NH3 entry and endosomal buffer capacity.
The endosomal buffer capacities of 36 ± 1 mM/pH unit
for J774 cells and 50 ± 3 mM/pH unit for CHO cells
(Fig. 4C, inset) are similar to that of 43 mM/pH unit measured in opossum kidney cells (29).
Unlike the bafilomycin-induced alkalinization, NH3-induced
alkalinization occurs without net movement of charge across the endosomal-limiting membrane. If Cl exit from endosomes
accompanying bafilomycin-induced alkalinization is the result of charge
coupling to maintain electroneutrality, then it is predicted that
NH3-induced alkalinization should not be accompanied by
Cl exit. Fig. 5A
shows that after a 45-min chase following BAC-TMR-dextran pulse
labeling, bafilomycin addition resulted in Cl exit,
whereas comparable alkalinization produced by NH4Cl
addition did not affect endosomal [Cl]. This finding in
cells also supports the conclusion in solution studies (Fig.
1B, top right) that BAC-TMR-dextran fluorescence is pH-insensitive.
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Fig. 5.
Mechanistic analysis of Cl
accumulation in endosomes. A, J774 cells were
pulse-labeled with BAC-TMR-dextran, chased at 37 °C for 45 min, and
perfused with PBS containing 200 nM bafilomycin
(filled circles) or 5 mM NH4Cl
(open circles) (S.E., n = three sets of
experiments). [Cl] was determined from R/G (right
hand axis). B, J774 cells were pulse-labeled as in
A except that bafilomycin (200 nM) was present
in the perfusate (filled circles), or PBS was replaced by
Cl-free PBS (nitrate-replacing Cl) during
the 2-min pulse labeling. C, cytoplasmic pH was measured
using 440 nm/490 nm excitation ratio imaging of the pH indicator BCECF.
Calibration of excitation ratio versus pH shown together
with measurements on J774 and CHO cells (S.E., n = 3)
(errors smaller than circle size). Intracellular pH was manipulated
using ionophores in high [K+] solutions as described
under "Materials and Methods." D, cytoplasmic
[Cl] measured using the Cl-sensitive
fluorescent indicator SPQ. Top, representative experiments
showing the time course of cytoplasmic SPQ fluorescence in response to
perfusion of J774 cells with PBS followed by calibration solutions
containing high [K+] and ionophores with indicated
[Cl]. Bottom, average cytoplasmic
[Cl] for J774 and CHO cells (S.E., n = 3).
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The time courses of endosomal acidification (Fig. 4B) and
[Cl] increase (Fig. 3B) suggest that the
entry of positive charge due to H+ during endosomal
acidification can be accounted for by the entry negative charge caused
by Cl. For example, a decrease in pH from 6.5 to 6.0 (where buffer capacity measurements are accurate) in J774 cells
produces an H+ influx of 18 mM (pH = 36 mM/pH unit × 0.5 pH unit), similar to the measured
increase in [Cl] of 15 mM. As a further
test of the conclusion that Cl conductance is the major
endosomal conductance, the time course of endosomal
[Cl] was measured in the continuous presence of
bafilomycin in the perfusate after pulse labeling (Fig. 5B,
closed circles). Endosomal [Cl] was < 10 mM initially, so that there was a substantial
[Cl] gradient driving Cl entry
(cytoplasmic [Cl] ~45 mM, see below). A
significant counterion conductance to K+ or other ions
would have produced an increase in [Cl]. Fig.
5B also shows the time course of endosomal
[Cl] after 2 min of pulse labeling with BAC-TMR-dextran
in a zero Cl solution (nitrate replacing
Cl) (open circles). Endosomal
[Cl] increases as in Fig. 3B, except that
[Cl] are lower than those obtained when BAC-TMR-dextran
is internalized in the Cl-containing PBS.
Cytoplasmic pH and [Cl]--
Cytoplasmic pH was
measured by excitation ratio imaging (440 nm/490 nm) using the
fluorescent pH indicator BCECF. Fig. 5C shows that
cytoplasmic pH was 7.31 ± 0.04 in J774 cells and 7.35 ± 0.04 in CHO cells. Cytoplasmic [Cl] was measured using
the quinolinium-type Cl indicator SPQ. Cells were labeled
with SPQ by overnight incubation, and SPQ fluorescence was measured
continuously. Cells were initially perfused with the physiological
buffer used for the endosomal [Cl] and pH measurements
and then with a series of calibration solutions containing high
K+, ionophores, and specified [Cl] (Fig.
5D). Cytoplasmic [Cl] was 44 ± 2 mM in J774 cells and 47 ± 3 mM in CHO cells.
Endosomes Swell during Acidification and Cl
Accumulation--
The low [Cl] just after
BAC-TMR-dextran internalization was an unanticipated observation
because endosomes presumably sample the perfusate [Cl]
of >130 mM. Because it seems unlikely that a mechanism
could or should exist for the rapid pumping of Cl out of
endosomes just after internalization, we postulated that quasi-static
physico-chemical mechanisms are responsible for the low
[Cl]. This may occur, for example, if endosomes are in
a relatively low volume, collapsed state as they form, so that the
Donnan effects of membrane proteins might create an interior-negative
potential that excludes Cl during the budding process.
Subsequent H+ and Cl entry would result in
endosome swelling and decreased density of fixed negative charges.
Indeed, there is morphometric evidence that endosome volume increases
soon after internalization (35, 36).
To test the hypothesis that endosomes swell after internalization, we
adapted a ratio imaging approach developed recently to measure
osmolality in microcompartments (37). Endosomes were pulse-labeled with
a mixture of calcein at self-quenching concentration (30 mM) and sulforhodamine 101 at low (non-self-quenching)
concentration (2 mM). The ratio of calcein green
fluorescence to sulforhodamine 101 red fluorescence (G/R) provides a
semi-quantitative measure of relative endosome volume. Fig.
6A shows the dependence of G/R on dilution of the internalization solution. Dilution produces an
increase in G/R because of decreased calcein self-quenching.
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Fig. 6.
Time course of relative endosomal
volume. A, in vitro calibration of
green-to-red fluorescence ratios (G/R) of PBS containing calcein (30 mM) and sulforhodamine 101 (2 mM) diluted by
indicated factors with PBS (S.E., n = 3). B,
fluorescence micrographs showing green calcein and red sulforhodamine
101 fluorescence at 0 and 15 min after 2-min pulse labeling with 30 mM calcein and 2 mM sulforhodamine 101. C, time course of relative endosomal volume
(V/Vo) deduced from G/R (right hand axis) in
J774 macrophages pulse-labeled as in B (S.E.,
n = 3). Where indicated, bafilomycin (200 nM) was present in the perfusate.
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Fig. 6B shows fluorescence micrographs at 0 and
15 min after endosome pulse labeling with the
calcein-sulforhodamine 101 mixture. The green calcein fluorescence
(relative to red sulforhodamine 101 fluorescence) was greater at 15 min, indicating less self-quenching and hence increased endosome
volume. Quantitative analysis of G/R ratios in Fig. 6C
showed an ~2.5-fold increase in relative endosomal volume
(V/V0) over 15 min, after pulse labeling. The increase in
endosome volume was largely blocked by inhibition of acidification by bafilomycin.
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DISCUSSION |
The purpose of this study was to measure endosomal
Cl concentration to determine whether endosomal
acidification is accompanied by Cl accumulation. As
described in the Introduction, Cl is the major
intracellular anion, and its transport across organellar membranes has
been proposed to regulate endosomal acidification. It is not possible
to predict endosomal [Cl] a priori because
of its many determinants including endosomal H+ pump
activity, ion permeabilities, buffer capacity, membrane and Donnan
potentials, and fusion/budding dynamics. For example, endosomal
[Cl] can be driven above its electrochemical
equilibrium concentration if Cl is the principal
transported ion that accompanies active H+ influx, provided
that initial endosomal [Cl] is fairly high and/or
buffer capacity is low. To measure endosomal [Cl] we
synthesized and validated a fluorescent fluid-phase marker of
endocytosis suitable for quantitative ratio image analysis. The new
indicator was applied to measure the kinetics of endosomal [Cl] in two cell lines, and the results were evaluated
mechanistically by analysis of bafilomycin effects, ion substitution,
endosomal volume changes, and cytoplasmic [Cl] and pH.
BAC-TMR-Dextran as a Ratioable Indicator for Measurement of
Endosomal [Cl]--
There were a number of
requirements for the ratioable Cl indicator. Bright (high
molar extinction and quantum yield) long wavelength fluorescence was
needed to obtain adequate fluorescence signals from small endosomes on
an autofluorescent cellular background. Quinolinium-type
Cl indicators, which have been used extensively to
measure cytoplasmic [Cl] (38), were not suitable for
measurement of endosomal [Cl] because of the dim blue
fluorescence, the need for UV excitation, and the imperfect
Cl specificity. Strict insensitivity of the indicator to
pH and good sensitivity to [Cl] in the range 0-100
mM was needed for measurement of endosomal [Cl] as well as chemical stability in the endosomal
environment. Yellow fluorescent protein-based Cl
indicators were not suitable because of the strong pH sensitivity and
relatively low Cl sensitivity (39). The
biacridinium-dextran (BAC-TMR-dextran) conjugate synthesized here had
the required bright long wavelength fluorescence, pH insensitivity,
Cl sensitivity, and chemical stability. The main
limitation was the susceptibility of the biacridinium chromophore to
photobleaching, which was several times greater than that for
fluorescein. However, adequate biacridinium images could be obtained
with <3% photobleaching using relatively high concentrations during
pulse labeling and using brief low intensity illumination.
Our approach for measuring endosomal [Cl] involved
quantitative ratio imaging of fluorescently labeled vesicles in
adherent cells. An important component of the measurement process was
the sensitive imaging hardware, consisting of an objective with
efficient light collection (numerical aperture, 1.4) and low
autofluorescence, electronic shutters to minimize light exposure, and a
high quantum efficiency-cooled CCD camera detector. Further,
custom-written software for determination of background-subtracted
endosome fluorescence made possible the quantitative determination of
endosomal [Cl], pH, and relative volume by ratio imaging.
Endosomal Cl Accumulation Parallels
Acidification--
Endosomal acidification in J774 and CHO cells was
accompanied by accumulation of Cl. Cl entry
was blocked by bafilomycin, even though a substantial
cytoplasmic-to-endosomal Cl concentration gradient was
present. The presence of a significant parallel conductance
(e.g. K+, H+) would have
permitted Cl entry. After endosomal acidification and
Cl accumulation, alkalinization by bafilomycin but not
NH4Cl resulted in Cl exit, supporting the
conclusion that charge coupling is responsible for parallel
Cl and H+ transport. The molar quantity of
Cl entry was comparable with that of H+ entry
as determined from measurements of endosomal acidification and buffer
capacity. Similar results were obtained when internalization was done
with Cl-free extracellular solution; although absolute
endosomal [Cl] was lower than that when internalization
was done in Cl-containing solutions, the increases in
[Cl] were comparable. Together, these findings indicate
that Cl is the major conductance in endosomes of J774 and
CHO cells and that Cl transport closely parallels
H+ transport. However, our data do not exclude a small
contribution from K+ conductance or H+ leak,
the latter occurring during endosomal alkalinization following bafilomycin addition (Fig. 4B). In addition, these results
provide the first data on endosomal [Cl]. If
Cl is the principal endosomal conductance, the slightly
greater endosomal versus cytoplasmic [Cl] at
45 min after endocytosis suggests a mildly interior-positive endosomal
membrane potential (10-20 millivolts) in the steady-state. Measurements of endosomal membrane potential and luminal
[K+] are needed to define experimentally the remaining
driving forces to complete a first-order biophysical description of
endosomal H+/ion transport mechanisms.
Endosomal Acidification Is Accompanied by Increased Luminal
Volume--
The low [Cl] of 17 mM (J774)
and 28 mM (CHO) just after endocytosis was an unexpected
observation, because extracellular fluid contains >130 mM
Cl. As shown schematically in Fig.
7, we reasoned that endosomal [Cl] should be low at the time of endosome budding from
the plasma membrane, probably because of the high interior-negative
Donnan potential of a partially collapsed nascent endosome. Subsequent acidification accompanied by Cl entry is predicted to be
accompanied by a volume increase. It is not possible to predict a
priori the magnitude of the volume increase because of the many
factors involved (including changing activity/osmotic coefficients, the
quantity of impermeant osmolytes, membrane fusion events, etc.).
Morphometric studies of labeled endosomes by electron microscopy
support the possibility that endosomal volume increases early after
internalization (35, 36), although from static morphometry measurements
it is difficult to deduce quantitative time course information. We
developed an experimental approach to estimate changes in relative
endosomal volume based on the self-quenching of the fluid-phase
fluorescent indicator calcein (40). It was found that endosome volume
increased ~2.5-fold over 15 min after internalization and that the
volume increase was blocked by inhibition of endosomal acidification by
bafilomycin. Endosomal acidification is thus accompanied by active
Cl accumulation and endosome swelling.
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Fig. 7.
Schematic showing increasing endosomal
[Cl] and volume during progressive acidification.
See "Discussion" for explanations.
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In summary, our results establish for the first time a quantitative
ratio imaging method to measure endosomal chloride concentration. The
measurement of endosomal [Cl] should have numerous
biological applications in the functional analysis of putative
intracellular Cl channels such as ClC3 (18, 41), ClC5
(42, 43), and CFTR (causing cystic fibrosis, Refs. 8 and 44). The
"proton sponge" hypothesis (45) for efficient gene delivery by
non-viral vectors (proposed to increase endosome buffer capacity and
hence Cl accumulation and swelling) should be amenable to
direct experimental validation. With suitable chemical modification, it
should also be possible to measure [Cl] in selected
endosomal subcompartments (e.g. internalization of labeled transferrin in recycling endosomes) and in organelles of the
secretory pathway. Fluorophore labeling approaches have been developed
to target fluorescent probes to organellar sites, including retrograde
transport of labeled toxins (46), expressed single-chain antibody
trapping of labeled hapten complexes (47), TGN38-based retrieval of
labeled antibodies (48), expressed avidin trapping of labeled biotin
(17), and trapping engineered probes by expressed cysteine-containing
helices (49).
Indicator
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Cardiovascular
Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman@itsa.ucsf.edu.
