1 Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109-0620, USA
2 The Program in Cellular and Molecular Biology, University of Michigan Medical
School, Ann Arbor, MI 48109-0620, USA
* Author for correspondence (e-mail: kenchris{at}umich.edu)
Accepted 22 October 2001
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Summary |
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Key words: Lysosome, Endocytosis, Bafilomycin A1
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Introduction |
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Unlike calcium, pH in vacuolar compartments has been well characterized.
Lysosomes maintain a pH of 4.0-5.0, and the intermediate compartments,
comprised of pinosomes, phagosomes, early endosomes and late endosomes, are
less acidic (Mellman et al.,
1986). Macropinosomes, which are formed from cell surface ruffles
that close into endocytic vesicles containing extracellular fluid (pH 7.2),
acidify within 10 minutes to pH 5.5 (Tsang
et al., 2000
). Rapid acidification is also characteristic of
phagosomes and endosomes (Fuchs et al.,
1989
; Hackam et al.,
1999
; McNeil et al.,
1983
). Experimental alkalinization of vacuolar compartments has
indicated requirements for acidic pH in a number of cellular processes,
including antigen presentation (Watts,
1997
), delivery of toxins and viral capsids across endosomal
membranes (Draper and Simon,
1980
; Lord et al.,
1999
; Marsh and Helenius,
1980
) and bacterial escape from phagosomes
(Beauregard et al., 1997
).
Hence, it is commonly assumed that pH is the chief controlling variable in
vacuolar regulation mechanisms. Other ions found in vacuolar compartments,
such as calcium, could be of comparable importance. However, these ions have
been more difficult to measure and manipulate in acidic compartments. The
elucidation of their relative importance and roles has awaited development of
appropriate analytical strategies.
The calcium-binding affinities of the many fluorescent probes for measuring
calcium are sensitive to pH, ionic strength and temperature
(Grynkiewicz et al., 1985;
Lattanzio and Bartschat, 1991
;
Tsien, 1980
). Thus, accurate
fluorometric measurement of vacuolar [Ca2+] at constant temperature
and ionic strength requires knowledge of compartment pH, as well as the
Kd of the fluorescent probe for calcium at that pH,
temperature and ionic strength. The reported measurements of calcium in
endosomes and phagosomes have identified dramatic decreases in
[Ca2+] in those compartments relative to extracellular calcium
([Ca2+]ext)
(Gerasimenko et al., 1998
;
Lundqvist et al., 2000
).
However, these studies probably underestimated calcium concentrations, because
experiments designed to measure the effects of pH on probe calcium-binding
affinity were carried out using saturating levels of calcium. To measure
calcium accurately in acidic environments, both probe calcium-binding
affinities and pH must be known precisely.
The present studies characterized calcium dynamics in macrophage vacuolar compartments using four experimental stages. First, the calcium-binding affinities of fluorescent probes were measured over the range of pH found in these compartments. Second, both pH and free calcium were measured in individual organelles using ratiometric fluorescence microscopy and the calibrated fluorescent probes. These measurements were used to obtain pH-corrected values of [Ca2+]lys. Third, as a confirmation of the ratiometric measurements, lysosomal calcium concentrations were measured by fluorescence lifetime imaging microscopy. Finally, pharmacological manipulations were applied to examine the relationships between vacuolar calcium, vacuolar pH, and endocytosis. We report that lysosomes contain high concentrations of calcium, and that lysosomal calcium content is influenced by both endocytosis and lysosomal pH. This newfound relationship between pH and vacuolar calcium in macrophages has important implications for organelle trafficking and cellular phenomena presently thought to be regulated by vacuolar pH.
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Materials and Methods |
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Cell culture
Mouse bone-marrow-derived macrophages were obtained from the femurs of
female C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME) and were cultured
in vitro as previously described (Swanson,
1989). Five to eight days after starting the culture, cells were
plated onto 25 mm circular coverslips in 6-well dishes at a density of
2x105 cells/cover slip and incubated overnight in DME, with
10% heat-inactivated FBS and 100 U/ml pen-strep (DME-10F; Gibco BRL,
Gaithersburg, MD). Some experiments used macrophages that were activated by
overnight incubation in DME-10F with 100 ng/ml LPS (List Biological, Campbell,
CA) and 100 U/ml IFN-
(R&D Systems).
Determination of fluorescent probe equilibrium dissociation
constants
Calcium-binding affinities of fluorescent probes were measured in
calibration buffer (CB; 130 mM KCl, 1 mM MgCl2, 15 mM Hepes, 15 mM
MES, pH 4-7.2) at 20-22°C. Solutions in which calcium concentrations were
40 µM were prepared using EGTA (neutral pH) or BAPTA (lower pH) calcium
buffers. Briefly, a 100 mM stock solution of CaEGTA was prepared using the
`pH-metric' method described previously
(Tsien and Pozzan, 1989
). A
100 mM stock of K2EGTA was also prepared. Similar stock solutions
were prepared using BAPTA (100 mM CaBAPTA and 100 mM K2BAPTA). By
changing the molar ratio of CaEGTA and K2EGTA, calcium buffers were
produced with ionized calcium levels that ranged from 17 nM to 38 µM under
neutral pH conditions. Other calcium solutions (
40 µM ionized calcium)
were prepared from a 1 M stock solution of anhydrous CaCO3 that was
first boiled to drive off CO2 then pH-adjusted to 7.2 with 5 M
HCl.
Calibration of the ratiometric calcium probe fura dextran (furaDx) was
performed by measuring the steady state fluorescence excitation spectra of
dyes in solutions of known pH and calcium concentration. Excitation
wavelengths were varied from 300-420 nm (5 nm band pass) and fluorescence
recorded at 510 nm (10 nm band pass) using a spectrofluorometer (PTI, Trenton,
NJ). At each pH and calcium concentration, fluorescence spectra were recorded,
then analyzed by plotting log [Ca2+]free vs log 340-380
ratio for furaDx and determining the x-intercept. Microsoft Excel Visual Basic
programs were used for spectral acquisition and data analysis. Data analysis
used methods previously described
(Grynkiewicz et al.,
1985).
Analogous calibrations were obtained for the fluorescence lifetime probe Oregon Green BAPTA-1 dextran (OGBDx). Fluorescence lifetime decays were recorded on a modified spectrofluorometer (PTI, Trenton, NJ). A 520 nm long pass filter selected the fluorescence emission wavelength illuminating a fast photomultiplier (R6780; Hamamatsu, Japan) connected to time-correlated single photon counting electronics and control software (TimeHarp; Picoquant A/G, Germany). Pulsed excitation was provided from a picosecond modelocked Ti:Sapphire laser that was pumped by a frequency-doubled Nd:YVO4 solid-state laser (Spectra Physics, Santa Clara, CA). The near infrared output from the Ti:Sapphire laser was pulse-picked to 8 MHz repetition and frequency doubled to 490 nm. The laser was coupled to the spectrofluorometer via a graded-index multi-mode optical fiber. Fluorescence decays were acquired as a function of solution calcium concentration and data was processed using FluoFit software (PicoQuant A/G, Germany) by fitting to a double exponential decay model and determining the fraction of [Ca2+]bound and [Ca2+]free probe. The Kd was obtained by calculating [Ca2+]free where [Ca2+]bound/[Ca2+]free=1.
FuraDx and OGBDx remained soluble at the pH values used in these studies.
The solubility of BAPTA-based probes is somewhat reduced below pH 6.0, but at
low concentrations it remains soluble at pH3
(Tsien, 1980
). However, in our
studies the Ca2+-binding moiety was coupled to dextran, a highly
soluble sugar, which increased solubility over a wide pH range. To maintain
solubility, all probe solutions were diluted to
1 mg/ml, well below the
manufacturer's specified solubility limit. To test whether all probe was
dissolved under the conditions of the experiments, we examined solutions of
dissolved probe by light scattering. Since insoluble probe would increase
scattering, we compared the 90° scattering from solutions of furaDx at pH
4-7.2. No significant increase in light scattering was observed, indicating
that solubility issues did not complicate our analysis.
Fluorescent labeling of lysosomes
Macrophage lysosomes were labeled by endocytosis of both pH and calcium
probes. Cells on coverslips were washed three times with Ringer's buffer (RB;
155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM
NaH2PO4, 10 mM Hepes and 10 mM glucose, pH 7.2-7.4) and
placed in a Leiden chamber (Harvard Apparatus, Cambridge, MA). Macrophages
were then pulsed for 15 minutes at 37°C with 0.3-1.0 mg/ml fluorescein
dextran (FDx), 0.3-1.0 mg/ml Oregon Green dextran (OGDx), and 1 mg/ml fura
dextran (furaDx); all dextrans had an average molecular weight of 10,000. All
lysosomal fluorescent probes were dissolved in RB or Ca2+-free RB
(pH 7.2-7.4) at concentrations 1 mg/ml. Loading solutions also contained
10 ng/ml rM-CSF, to stimulate pinocytosis. The cells were washed five times
with RB, chased for
120 minutes in RB at 37°C, and observed using the
ratiometric fluorescence microscope. For experiments in which cells were
treated with <2 mM calcium, the cells were pulsed as described above, then
chased for
120 minutes in calcium-free RB supplemented with calcium from a
400 mM CaCO3 stock solution.
For fluorescence lifetime measurements, macrophage lysosomes were loaded
with Oregon Green BAPTA-1 dextran (OGBDx; 1 mg/ml, average molecular weight
10,000) by pulse-labeling for 15 minutes as described above. These cells were
washed five times in RB, chased for 120 minutes in RB at 37°C and
observed using the fluorescence lifetime imaging microscope.
Measurement of lysosomal pH and [Ca2+]lys using
ratiometric fluorescence microscopy
Fluorescence images of labeled cells (em=510 nm) were
collected using 340 nm, 380 nm, 440 nm and 485 nm excitation. Images collected
at these four excitation wavelengths allowed calculation of both pH and
Ca2+ levels; 340 nm corresponds to the excitation maximum of
calcium-bound furaDx, while 380 nm is the excitation maximum of calcium-free
furaDx. The intensity of the FDx and OGDx fluorescence at 440 nm excitation is
pH-independent, while fluorescence at 485 nm excitation changes as a function
of pH. Consequently, pH was determined from the FDx and OGDx fluorescence
(440/485 ratios), and calcium was measured from the furaDx fluorescence
(340/380 ratios) using appropriate probe Kd for the
measured pH of the organelle.
All images were processed using Metamorph software. Prior to or following each experiment, a background image was collected while blocking the excitation source and leaving all other parts of the light path unchanged. This background image was subtracted from all other images. In addition, fluorescent background signals were determined by histogram analysis of cell-free regions of each image; these were also subtracted from other images.
To obtain an organelle pH ratio, the background-subtracted 485 nm image (I485bs) was divided by the background-subtracted 440 nm image (I440bs) and multiplied by 1000 to obtain a ratio image (R485/440=1000xI485bs/I440bs). Next, a binary image was generated from the two primary fluorescence images (Binary485*440=I485bsxI440bs), adjusting the intensity threshold to include only the labeled organelles. By overlaying that binary image onto the ratio image, measurements of ratios could be restricted to the organelles of interest, and then exported to Excel for further data processing.
Organelle pH was calibrated by equilibrating fluorescently labeled
macrophages for at least 10 minutes in CB (pH 3.5-7.2) containing 10 µM of
the H+ ionophore nigericin and 10 µM of the K+
ionophore valinomycin. Fluorescence images were acquired at various
extracellular pH levels using excitation at 440 nm and 485 nm
(Beauregard et al., 1997).
These images were processed as described above to obtain a pH standard curve
for calibrating the experimental pH ratio values. In organelles loaded with
both FDx and OGDx, plots of average intensity ratios (R485nm/440nm)
vs pH gave a nearly linear response between pH 7.2 and 3.5, as has been shown
previously (Downey et al.,
1999
). These points were fit using a linear least squares
algorithm and used to convert intensity ratios to pH values.
To measure calcium ratios, background-subtracted 340 nm and 380 nm images
were combined into ratio images
(R340/380=1000xI340bs/I380bs), and the
fluorescence ratios of individual organelles were collected and exported to
Excel spreadsheets, using binary masks as described above for pH measurements.
Calcium probes were calibrated by first incubating labeled macrophages with
nigericin and valinomycin in CB (pH 7.2) for 10-15 minutes, then adding
ionomycin (final concentration 10 µM) and K2EGTA (final
concentration 10 mM) and incubating for an additional 3-5 minutes.
Fluorescence images were recorded using appropriate excitation wavelengths
(340 nm and 380 nm) and the resulting ratio was used to estimate the value in
the absence of calcium (i.e. Rmin). This buffer was then replaced
with 10 µM ionomycin with excess (10 mM) Ca2+ in CB (pH 7.2) and
incubated for 3-5 minutes; images were recorded at the appropriate excitation
wavelengths to yield the ratio for bound probe (i.e. Rmax). All
images were background-subtracted, as described above. Values for
Rmin and Rmax and Q (the ratio of the fluorescence of
the unbound probe at high and low calcium at 380 nm excitation) were used to
calculate [Ca2+]lys according to the method described
previously (Grynkiewicz et al.,
1985). The Kd used for calcium estimation was
calculated according to the measured pH of each individual organelle using the
constants for BAPTA from Tsien (Tsien,
1980
) and the methods for correcting the Kd
for temperature, ionic strength and pH of Bers et al.
(Bers et al., 1994
).
Measurement of [Ca2+]lys using fluorescence
lifetime imaging microscopy
Macrophage lysosomes were loaded with OGBDx by endocytosis as described
above. [Ca2+]lys was subsequently obtained with the
fluorescence lifetime imaging microscope, using the ratio of two defined delay
times after the laser pulse: 1.0 nanosecond (T1) and 3.0
nanoseconds (T2). The ratio of the amplitude of the free
(T1) and calcium-bound (T2) probes, obtained during
nanosecond measurement windows was used to obtain the overall free calcium
concentration. These results were calibrated using a similar approach to the
ratiometric measurements described above except that Rmin and
Rmax were determined from the ratio of T1/T2,
and 8.74x10-4 M (pH 4.0) was used for the probe
Kd.
Measurement of [Ca2+]cyt
FFP-18AM, a fura-2-like probe that labels cell membranes, was dissolved in
DMSO and loaded into macrophages as an acetoxymethyl ester. Cells were
incubated for 30 minutes at 37° in RB containing 1 µM FFP-18 AM and 1%
Pluronic F-127 (Calbiochem, La Jolla, CA), washed with RB prior to
measurement. Fluorescence images were acquired using 340 and 380 nm excitation
(em=510 nm). After appropriate background subtraction, as
described for measurement of organelle pH and [Ca2+]lys
using ratiometric fluorescence microscopy, a binary image was generated by
adjusting the intensity threshold to include only the labeled cells. By
overlaying the binary image onto the two images, ratio measurements of labeled
cells were collected for export to Excel and further data processing. Ratios
were calibrated using the Ca2+ ionophore ionomycin, as described
above.
Manipulation of pH, calcium and magnesium
Lysosomal pH was increased using bafilomycin A1 (final
concentration 500 nM from a 100 µM stock in DMSO) or ammonium chloride
(final concentration 10 mM), added to cells in RB.
All experiments were performed in the presence of millimolar
Mg2+. Since the affinity of BAPTA for Mg2+ is several
orders of magnitude lower than its affinity for calcium, interferences from
Mg2+ were not expected. However, as a control experiment,
macrophage lysosomes were loaded with the pH and calcium probes using our
standard protocols, then chased for 120 minutes in Mg2+-free RB
prior to making the measurements. Additionally, pH and calcium calibrations
were performed in Mg2+-free CB.
Microscopy
Ratiometric imaging
Ratiometric images were acquired using an inverted research microscope
(TE300; Nikon, Japan) equipped with phase-contrast transmitted light and
mercury arc lamp excitation with epifluorescence optics. Several dichroic
mirror sets (Omega Optical, Brattleboro, VT) were used; a double excitation
set for both fura and fluorescein dyes (XF79: 340HT15, 380HT15, 440DF20,
485DF15 excitation filters in wheel; 505DRLPXR dichroic mirror and 535DF35
emission filter in cube) was used for the majority of experiments, while a
single generic blue dichroic (XF12: 340HT15 and 380HT15 excitation filters in
wheel; 420DCLP dichroic mirror and 435ALP emission filter in cube) was used
where necessary to increase microscope sensitivity for the fura-based calcium
probes. An excitation filter wheel (Lambda 10-2, Sutter Instruments, Novato,
CA), containing band pass filters, was used to select the excitation
wavelength. Both the transmitted light path and the fluorescence excitation
path contained shutters (Uniblitz, Rochester, NY) to control illumination of
the cells. A temperature-controlled imaging chamber (Harvard Apparatus,
Cambridge, MA) maintained sample temperature at 37°C. A cooled scientific
CCD camera (Quantix; Photometrics, Tucson, AZ) recorded fluorescence and
transmitted light images. For some experiments a lens-coupled GEN IV
intensifier (VSH-1845; Videoscope Intl., Dulles, VA) was inserted in front of
the CCD camera. Metamorph software (Universal Imaging, West Chester, PA)
controlled the camera, shutters, and filter wheels during all experiments.
Fluorescence lifetime imaging
The fluorescence lifetime imaging microscope was an inverted research grade
microscope (TE300; Nikon, Japan) equipped with both phase-contrast and
epi-fluorescence optics and shutters (Uniblitz, Rochester, NY). Fluorescence
excitation was provided via a graded index multi-mode fiber optic, coupled to
a mode-locked Ti:Sapphire laser (Tsunami, 1 picosecond pulses, 81 MHz,
835-1005 nm; Spectra Physics, Mountain View, CA), which was pulse picked to 8
MHz and frequency doubled (415-500 nm), and pumped by a solid-state
frequency-doubled Nd:YVO4 laser (532 nm, Millennia V; Spectra
Physics, Mountain View, CA). The fiber was mechanically agitated to scramble
the coherence of the laser. A dichroic mirror set (XF115:475AF40 excitation
filter, 505DRLP dichroic mirror, and 510ALP emission filter in the cube, Omega
Optical, Brattleboro, VT) reflected the excitation light onto the sample and
selected for the green fluorescence prior to the picosecond gated intensified
CCD camera (PicoStar HR; La Vision A/G, Germany). A DEL-150 computer board
(Becker & Hickl A/G, Germany) produced electronic time delays relative to
the laser pulse. DaVis software (La Vision A/G, Germany) controlled the
camera, laser shutter, and delay board during image acquisition. For these
experiments, images were collected in 1000 picosecond windows, with delay
times of 1.0 and 3.0 nanoseconds after the pulse. Images of fluorescence at
the two delay times were then analyzed ratiometrically to infer changes in
fluorescence lifetimes of the fluorophores.
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Results |
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Because chelator affinity for calcium is altered by pH, fluorescent calcium
probes are typically used within a limited pH range. The calcium probes used
in this study are structural variants of the calcium chelator BAPTA, whose
calcium affinity remains relatively constant between pH 6.0 and pH 7.5
(Tsien and Pozzan, 1989), but
changes by several orders of magnitude between pH 6.0 and 4.0. Calcium levels
are typically calculated from the probe's fluorescent response assuming that
the reported Kd, measured at neutral pH, applies over the
pH range of the experiments. Such assumptions are useful above pH 6.0, but are
invalid at lower pH ranges where the probe Kd is more
sensitive to solution pH. Hence, to use BAPTA-based probes at low pH, the
probe's Kd for calcium at that pH must be accurately known
and applied to the calculation of [Ca2+]. To determine the
relationship between probe Kd and pH, the calcium-binding
affinities of the calcium probes were measured between pH 4.0 and 7.0. The
affinities of furaDx and OGBDx for calcium were similar to those previously
described for BAPTA (Fig. 1)
(Tsien, 1980
;
Bers et al., 1994
). This result
was not surprising, considering the structural similarities between the
calcium-binding moieties of furaDx, OGBDx and BAPTA. Further, this result
indicated that published relationships between solution pH and BAPTA affinity
for calcium (Tsien, 1980
;
Bers et al., 1994
) could be
used to calibrate the measurements with furaDx and OGBDx.
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This relationship between probe affinity and solution pH allowed us to measure [Ca2+] over a large pH range (4.0-7.2). As for any equilibrium probe, the highest probe sensitivity is obtained when measured [Ca2+] is within a log unit of the Kd. Thus, optimal measurement is a function of both probe Kd, which varies with pH, and solution [Ca2+]. At pH 4.0, furaDx and OGBDx reliably measured calcium between 10-2 and 10-4 M; at higher pH, probe Kd was lower, and the range of measurable [Ca2+] was also lower (e.g. 10-4 to 10-6 M at pH 5.0). Although nearly all measurements were within this measurement window, we discarded data in which the [Ca2+] fell outside one log unit of the probe Kd.
Measurement of [Ca2+]lys
To measure calcium in macrophage lysosomes, a fluorescent probe cocktail
containing FDx, OGDx and furaDx was loaded into lysosomes by endocytosis. Upon
visualization in the microscope, the dyes were compartmentalized in tubular
and vesicular structures typical of lysosomes and late endosomes
(Swanson et al., 1987;
Swanson, 1999
), indicating
that the probes were effectively trafficked to lysosomal compartments within
the cell.
The spectral responses of FDx and OGDx were used to determine pH of individual lysosomes, which was then used to calibrate the spectral response of furaDx in those same organelles. Lysosomal pH was 4.0±0.1 and measured lysosomal furaDx ratios nearly all fell between Rmin and Rmax. Using the calculated relationship between pH and probe affinity (Fig. 1), [Ca2+]lys was determined to be 6.0±0.9x10-4 M (n=24 cells; Fig. 2).
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To corroborate the ratiometric fluorescence imaging results, [Ca2+]lys was also measured by fluorescence lifetime imaging, using the fluorescence lifetime probe OGBDx. Lifetime measurements did not allow simultaneous measurement of both pH and calcium in individual organelles, so we calibrated OGBDx using its Kd at pH 4.0, as determined by the ratiometric measurements. Fluorescence lifetime microscopic measurement of OGBDx reported a [Ca2+]lys of 4.0±0.7x10-4 M (n=18 cells; Fig. 2), similar to that obtained using ratiometric methods. Thus, [Ca2+]lys was found by two different methods to be lower than extracellular calcium ([Ca2+]ext=2 mM) and substantially higher than the cytosolic calcium concentrations ([Ca2+]cyt=50-150 nM).
Measurements of [Ca2+]lys in the absence of
magnesium
As all experiments were performed in the presence of millimolar
Mg2+, it was possible that probe response reflected
[Mg2+] instead of, or in addition to, [Ca2+]. Since the
affinity of BAPTA for Mg2+ is several orders of magnitude lower
than its affinity for calcium, interferences from Mg2+ were not
expected. However, in light of previous biological roles proposed for
Mg2+, interpretation of our Ca2+ measurements required
that we examine the effects of [Mg2+] on our measurements.
[Ca2+]lys was measured in RB without added
Mg2+, calibration of the probe's fluorescence response was
performed in the absence of Mg2+ (Mg2+-free CB), and
ratiometric fluorescence measurements in Mg2+-free and
Mg2+-containing buffers were compared. As predicted from in vitro
measurements, there were no significant differences between the ratiometric
fluorescence measurements of pH and [Ca2+]lys in the
presence or absence of Mg2+ (data not shown), indicating that
Mg2+ has no measurable effect on the fluorescence response of the
calcium probes.
Changes in [Ca2+]ext and
[Ca2+]cyt affect [Ca2+]lys
Lysosomal calcium levels may result from calcium influx into the lysosomes
via endocytosis. To ascertain the role of endocytosis in determining measured
lysosomal calcium levels, we labeled the lysosomes with furaDx and the pH
probe cocktail, then incubated cells in a range of extracellular calcium
concentrations. [Ca2+]lys was similar in cells incubated
in RB containing 2 mM calcium or 500 µM calcium, but was slightly reduced
in cells incubated with RB containing 100 µM calcium
(Fig. 2). These results suggest
that influx of calcium via endocytosis contributes to the high
[Ca2+]lys. [Ca2+]lys was also
measured in cells that had been incubated in the absence of Ca2+.
In cells chased in 10 mM EGTA, [Ca2+]lys dropped to
10-5 M. [Ca2+]lys lower than
10-5 M could not be detected with furaDx because of the low pH.
Since the Kd of CaEGTA at pH 4 is nearly 1 M, the EGTA
inside the lysosomes was probably not buffering
[Ca2+]lys to low levels. Instead, extracellular EGTA
should have had two different effects that would lower
[Ca2+]lys. First, by reducing
[Ca2+]ext, it reduced the amount of calcium entering
lysosomes by endocytosis. Second, because EGTA can reduce
[Ca2+]cyt to low levels
(Larsen et al., 2000
), it may
have depleted lysosomes of calcium through its effect on
[Ca2+]cyt (see below). Thus,
[Ca2+]lys may be affected by both
[Ca2+]ext and [Ca2+]cyt. In cells
that were chased in different calcium concentrations or in EGTA, lysosomal pH
remained constant (Fig. 2),
indicating that the altered lysosomal Ca2+ levels do not affect
lysosomal pH.
Increasing lysosomal pH reduces [Ca2+]lys
Experimental manipulation of pH levels have allowed identification of
important roles for vacuolar acidification in physiology and pathogenesis. It
is possible that lysosomal pH could also affect lysosomal Ca2+
levels. To determine the relationship between lysosomal pH and
[Ca2+]lys, [Ca2+]lys was measured
after manipulation of lysosomal pH. Macrophages were treated with bafilomycin
A1, an inhibitor of the H+-ATPase that increases pH of
acidic compartments. After 45 minutes in bafilomycin A1, lysosomal
pH increased from 4 to 7 and [Ca2+]lys decreased from
0.6 mM to 285 nM (Fig. 2). We
next examined the time-course of this effect
(Fig. 3). Cells incubated for
1-3 hours in RB without bafilomycin A1 maintained constant low pH
and high [Ca2+]lys (data not shown). However, after
addition of bafilomycin A1, pH increased from 4 to 7 over 45
minutes, and [Ca2+]lys decreased correspondingly. These
results demonstrate a profound relationship between lysosomal pH and
[Ca2+]lys.
|
It was possible that this dramatic result reflected a process unique to
bafilomycin A1. Hence, the pH-dependence of
[Ca2+]lys was also examined using the weak base ammonia,
which rapidly increases the pH of acidic compartments
(Poole and Ohkuma, 1981).
Within 60 seconds of adding 10 mM ammonium chloride, pH increased and
[Ca2+]lys decreased in magnitude similar to that
observed using bafilomycin A1
(Fig. 4). Returning cells to RB
without ammonium chloride produced a rapid reacidification and recovery of
high [Ca2+]lys. Together, these studies indicated that
although [Ca2+]lys did not affect lysosomal pH
(Fig. 2), lysosomal pH had a
profound effect on [Ca2+]lys.
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Elevation of lysosomal pH released calcium into the cytoplasm
Two mechanisms could account for the pH-dependence of
[Ca2+]lys. First, pH-dependent calcium-binding molecules
present in the organelle could selectively bind calcium at increased pH,
leading to lysosomal sequestration of calcium and reduced
[Ca2+]lys. Second, pH-dependent calcium channels or
transporters could release calcium from the lysosome into the cytosol at
increased pH. These possibilities can be distinguished by measuring
[Ca2+]cyt as lysosomal pH is increased, since calcium
released from lysosomes during alkalinization should increase
[Ca2+]cyt. [Ca2+]cyt was measured
in macrophages labeled with FFP-18 (Fig.
5A). When lysosomal pH was increased in non-activated macrophages,
small increases in cytosolic calcium levels could be observed (data not
shown). Significantly larger effects of lysosomal pH on cytosolic calcium were
observed in activated macrophages (Fig.
5A), where lower resting cytosolic calcium and larger lysosomal
compartments presumably produced greater relative effects on total cytosolic
calcium (Cohn, 1978;
Lowry et al., 1999
).
Complementary time-lapse measurements of the rapid drop in
[Ca2+]lys associated with elevation of lysosomal pH are
shown in Fig. 5B
(Fig. 5C shows control).
Additionally, similar measurable increases in cytosolic calcium were observed
in non-activated macrophages when thapsigargin was used to inhibit calcium
uptake by the ER (data not shown). Although these experiments cannot rule out
calcium efflux into the cytosol from other calcium storage organelles, the
effects of ammonium chloride would be greatest upon the most acidic organelles
(i.e. lysosomes). Therefore, these results indicated that calcium was being
released from lysosomes into cytosol upon alkalinization. The reduced
[Ca2+]lys observed at increased pH may reflect the
presence of pH-dependent calcium channels or transporters in the lysosomal
membrane.
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Discussion |
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The contribution of endocytosis to
[Ca2+]lys
Vacuolar accumulation of fluid-phase probes by pinocytosis is directly
proportional to extracellular concentrations of those probes
(Swanson and Silverstein,
1988; Swanson,
1999
). If calcium were to behave analogously, its concentration in
lysosomes would be proportional to [Ca2+]ext. Instead,
lowering [Ca2+]ext from 2 mM to 500 µM did not
appreciably alter [Ca2+]lys, indicating that at or near
physiological [Ca2+]ext, [Ca2+]lys
is regulated independent of endocytosis. However, incubation of cells in 100
µM [Ca2+]ext or in EGTA-containing buffers decreased
[Ca2+]lys. These observations could be explained by a
mechanism in which endocytosis provides a source of calcium for the vacuolar
compartment, but other factors prevent [Ca2+]lys from
exceeding 600 µM.
The relationship between lysosomal pH and
[Ca2+]lys
Alterations of [Ca2+]ext or
[Ca2+]lys did not alter lysosomal pH. A previous study
of the pH-dependence of [Ca2+] in endocytic compartments, carried
out on fibroblast endosomes, showed that increases in extracellular calcium
led to reduced acidification (Gerasimenko
et al., 1998). The differing results of the two studies suggests
that the pH-dependence of [Ca2+]lys may not apply to all
endocytic organelles.
Although changing [Ca2+]lys did not measurably affect lysosomal pH, increases in lysosomal pH dramatically lowered [Ca2+]lys. Slow alkalinization with bafilomycin A1 produced an equally slow lowering of [Ca2+]lys. Rapid alkalinization with ammonium chloride produced a rapid decrease in [Ca2+]lys by several orders of magnitude. Because increases in [Ca2+]cyt could be detected when lysosomal pH was increased with ammonium chloride, we infer that vacuolar calcium was moving into cytoplasm, possibly via pH-dependent calcium channels or pumps.
High [Ca2+]lys was rapidly restored by removal of
ammonium chloride, indicating that calcium can be delivered into lysosomes
from cytoplasm. Accordingly, the sensitivity of
[Ca2+]lys to pH may reflect an equilibrium relationship
between pH and calcium across the lysosomal membrane. We propose a mechanism
similar to that described for calcium accumulation in yeast vacuoles
(Dunn et al., 1994;
Ohsumi and Anraku, 1983
).
First, the proton ATPase in the lysosomal membrane maintains an acidic lumenal
pH and a gradient of protons across that membrane. Second, the proton gradient
drives the accumulation of calcium via a calcium/proton exchange protein in
the lysosomal membrane. Conditions that elevate lysosomal pH reduce the proton
gradient and consequently reduce the calcium concentration gradient that can
be maintained across that membrane.
Implications for cell biology and pathogenesis
The most striking finding of the present studies is that experimental
treatments that increase the pH of vacuolar compartments in macrophages lower
vacuolar calcium levels proportionally. This implies that cellular processes
previously attributed to vacuolar acidification may be equally attributable to
vacuolar decalcification. These processes could include receptor-ligand
dissociation in endosomes, penetration of cellular membranes by bacterial
toxins and viral capsids, and the processing and loading of antigen onto MHC
class II molecules (Mellman et al.,
1986). We have observed reductions in
[Ca2+]pino by two orders of magnitude as pH decreases
from 7.2 to 6.2 in newly formed pinosomes, followed by significant increases
in [Ca2+]pino as the pinosome matures (K.A.C.,
unpublished), implying that low calcium concentration is a distinct
physiological feature of early endosomes. In light of these observations and
those described in this study, it may be appropriate to re-examine any
processes in which a primary role for pH has not been supported by independent
experimental approaches.
This relationship between vacuolar pH and vacuolar calcium could also
affect membrane fusion between late endosomes, lysosomes and other organelles.
Various studies have demonstrated that raising lysosomal pH increases
lysosomal secretion (Tapper and Sundler,
1990), that increasing [Ca2+]cyt leads to
lysosomal secretion (Andrews,
1995
), and that endocytosed calcium may provide a source of
calcium that facilitates vesicle fusion
(Peters and Mayer, 1998
).
Perhaps transient or experimentally induced alkalinization of lysosomes
releases calcium that allows membrane fusion with plasma membrane or with
other organelles.
Although plasma membrane and ER are the principal regulated sources of cytosolic calcium, the vacuolar compartment could serve as an additional source of [Ca2+]cyt. The magnitude of the changes in [Ca2+]cyt observed here in response to alkalinization were small relative to total cytosolic calcium, but it may be that, under some circumstances, transient alkalinization of late endosomes or lysosomes releases sufficient calcium to produce an intracellular signal.
A number of bacterial and fungal pathogens survive within macrophage
vacuolar compartments, and their mechanisms for survival may require
manipulation or monitoring of vacuolar [Ca2+]. For example,
survival of Histoplasma capsulatum inside macrophage vacuoles
requires that organism to secrete a calcium-binding protein
(Kugler et al., 2000;
Sebghati et al., 2000
). This
protein could be required to maintain [Ca2+] sufficiently high to
allow growth in the relatively alkaline vacuole
(Eissenberg et al., 1988
). For
Salmonella typhimurium, regulation of gene expression in phagosomes
has been linked with both pH and divalent cations
(Alpuche-Arande et al., 1992
;
Vescovi et al., 1996
). A full
explanation of this regulatory system will require the development and
application of methods for distinguishing the contributions of vacuolar pH and
calcium.
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