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Department of Biological Sciences, Wayne State University, Detroit, MI 48202
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Abstract |
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600,000 times faster than those of video frames, we have
characterized Ca2+ waves within cells in exquisite detail
to reveal Ca2+ signaling routes. Polarized neutrophils
exhibited a counterclockwise rotating ryanodine-sensitive juxtamembrane
Ca2+ wave during temporal calcium spikes. During
stimulation with fMLP, a chemotactic factor, two Ca2+ waves
traveling in opposite directions around the perimeter of the cell
emanated from sites of stimulation (the clockwise wave is verapamil
sensitive). Phagocytosed targets exhibit counterclockwise
Ca2+ waves traveling about their periphery originating from
the plasma membrane. This study: 1) outlines the technology to observe
Ca2+ signaling circuitry within small living cells; 2)
shows that extracellular spatial information in the form of a
chemotactic factor gradient is transduced into intracellular chemical
patterns, which provides fresh insights in signaling; 3) suggests that
a line of communication exits between the cell surface and phagosomes;
and 4) suggests that spatiotemporal Ca2+ patterns
contribute to drug actions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study spatiotemporal Ca2+ waves in small
cells, such as neutrophils, mast cells, and lymphocytes, a technique
with high spatial and temporal resolution is required. High resolution
perpendicular to the optical axis a microscope (lateral or
x-y) is provided by conventional microscopy, which provides
a resolution of 200 nm according to Rayleigh’s equation. The
temporal resolution required depends on the dynamics of the events
under study. Assuming that a wave travels at an unmyelinated axon
velocity (10 m/s), a 50-ns exposure time leads to an acceptable wave
displacement of 500 nm while the shutter is open. Some blurring is
anticipated, but the displacement is a fraction of the cell size.
Previous studies in other cell types have reported
Ca2+ wave velocities of 25 µm/s
(16, 17, 18). In this case, a 1-ms exposure yields a
displacement of 25 nm, which is much smaller than the Rayleigh
distance. Thus, depending on the underlying mechanism(s), biological
wave phenomena may require shutter speeds of 50 ns to 1
ms.
Several high speed microscopy techniques have been developed to explore
this time regimen. To shorten exposure times, Zoghbi et al.
(20) excited intracellular fluorescence using a
single-shot 7-ns laser pulse. Although these short pulses eliminated
blurring during exposure, one could not collect a consecutive image
series of Ca2+ signals. Recent developments in
high speed imaging have relied primarily on charge-coupled device
(CCD)3 technology.
This approach has yielded capture rates of 100 to 1000 frames/s
(19, 20, 21, 22) wherein the exposure time was equal to the frame
readout time. We have recently developed a high speed microscopic
imaging technique that captures individual images with 50 ns to
ms exposure times at up to 1000 frames/s (23, 24, 25, 26),
which allows us to collect stop-action movies of extremely rapid cell
signaling events.
In the present study, we extend our high speed microscopy studies to intracellular Ca2+ signaling. Our work suggests novel elements of Ca2+ signaling processes in human neutrophils including Ca2+ pattern reorientation during chemotactic stimulation and plasma membrane-to-phagosome signaling after phagocytosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Indo-1-acetoxymethyl ester (indo-1-AM) and BAPTA-AM were purchased from Molecular Probes (Eugene, OR).
Cells
Human peripheral blood neutrophils were purified using Ficoll-Hypaque step density gradient centrifugation. The cells were labeled with indo-1-AM at 5 µg/ml for 20 min. at 37°C, as described (27).
Phagocytosis of erythrocytes
SRBCs (Alsevers; Rockland Scientific, Gilbertsville, PA) were opsonized with rabbit anti-sheep E Ab (ICN Pharmaceuticals, Costa Mesa, CA) as described (28). SRBCs were added to neutrophils adherent to quartz coverslips at E:T 20:1 for 30 min at 37°C. Nonadherent SRBCs were removed by gentle washing.
Microscopy
Cells were observed microscopically at 37°C. Cells were suspended in HBSS containing glucose but not phenol red. Experiments were performed with quartz or Swiss glass coverslips; some glass contains chromium inclusions, which absorb and emit light in the same region as indo-1. Each microscope slide was scanned to identify morphologically polarized cells with well-defined uropods.
Microfluorometry and excitation spectrophotometry
Quantitative microfluorometry and excitation spectroscopy were performed on single cells using a Peltier-cooled PMT D104 system (Photon Technology, Lawrenceville, NJ) attached to a Zeiss Axiovert 35 (Carl Zeiss, New York, NY) fluorescence microscope. A monochromator and a fiber-optically coupled xenon lamp were controlled by FeliX software (Photon Technology). During microfluorometry, the excitation and emission wavelengths were set using excitation at 350 nm (10 nm band-pass), and emission was detected using a 400LP dichroic mirror and a 405DF43 emission filter. The PMT output was plotted as a function of time. For excitation spectroscopy, a 400-nm long-pass dichroic mirror and either a 405DF43 emission filter or a 490DF20 emission filter were used. Excitation spectra were processed by FeliX software. The excitation spectra shown are an average of 10 spectra each accumulated with a 0.2-s integration time and a 1-nm step size.
High speed imaging and emission spectroscopy
High speed imaging was performed using an Axiovert 135
fluorescence microscope with a quartz condenser, quartz objective, and
an AttoArc mercury lamp (Carl Zeiss). In the high wavelength region, a
365WB50 exciter, a 400LP dichroic mirror, and a 490DF20 emission filter
were used. To image the low wavelength indo-1 region, a 365WB50
exciter, 400LP dichroic mirror and a 418LP emission filter or, for
improved contrast, a 355HT15 exciter, a 390LP dichroic reflector, and a
405DF43 emission filter were used. To increase light collection
efficiency, the bottom port of the microscope was used. This port was
fiber-optically coupled to the input of an Acton-150 (Acton
Instruments, Acton, MA) imaging spectrophotometer. The fiber optic
coupling results in a substantial gain in light collection efficiency
(29). In comparison with a similar Zeiss inverted scope
using optical elements to relay the light to a PMT, the throughput of
this modified microscope was improved greatly. The exit side was
connected to a liquid N2-cooled intensifier
attached to a Peltier-cooled I-MAX-512 camera (approximately -20°C)
(Princeton Instruments, Trenton, NJ) (27, 28, 29). A Gen-II
tube was used to provide maximal efficiency in the violet-blue region
of the spectrum (30). The camera was controlled by a high
speed Princeton ST-133 interface and a Stanford Research Systems
(Sunnyvale, CA) DG-535 delay gate generator (26). To
improve computer acquisition times, the size of the pixel array was
adjusted. A Dell Precision 410 workstation with an 800-MHz processor,
1.0-Gb RAM, 16-Mb onboard cache, and a high speed Lava Dual PCI
enhanced port (Lava Computer, Toronto, Canada) was used. Winspec
(Princeton Instruments) software was used. Winspec CPU calls were given
system priority to enhance the instrument’s duty cycle. Data were
acquired without reporting to the monitor to further improve system
speed. Data capture used a software-allocated RAM disk. For emission
spectroscopy, the mirror in the Acton unit was replaced with a ruled
grating (300 grooves/mm) (23). A schematic diagram of the
apparatus is shown in Fig. 1
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, calcium spikes lasting for 200 ms occur
once every 20 s. Because only a few seconds of high speed
acquisition were possible (depending on the duty cycle of the
instrument), it was necessary to note the arrival time of calcium
spikes in the live spectrum-analysis mode of the Winspec software and
then manually trigger high speed acquisition before the arrival of the
calcium spike. Thus, in many cases "clipping" at the beginning or
end of the movies occurred whereas in other cases the spike was missed
altogether. The instability of the Windows operating environment also
contributed to data loss. The number of independent experiments is
listed below as n. The number of successful high speed movies is given
as m.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fluorescent calcium probe indo-1 was chosen because it can be
used in nonratiometric experiments (31), a
necessity at high data acquisition speeds. Fig. 2a shows four indo-1 emission
spectra collected at different Ca2+
concentrations using microspectrophotometry. Emission intensity is a
function of Ca2+ concentration and wavelength;
the lower wavelength peak at 415 nm increases with
Ca2+ concentration, whereas the higher wavelength
peak decreases in intensity. According to Fig. 2a, cells
imaged at 415 nm should brighten as Ca2+ levels
increase, whereas cells imaged at 490 nm should darken. This is
confirmed in Fig. 2, b and c, wherein the local
Ca2+ signal becomes brighter in Fig. 2b at 415 nm but dimmer in a separate experiment (Fig. 2c) at 490 nm. We have also confirmed the presence of
Ca2+ spikes in polarized neutrophils
(15) using quantitative microfluorometry at 415 nm
(Fig. 2d) and 490 nm (Fig. 2e). In both
experiments the interspike interval is 20 s. This
interval decreased to 10 s. after exposure to neutrophil
activating stimuli such as FMLP, as previously described (32, 33). Spikes were not observed for resting cells or unlabeled
polarized cells (data not shown). These data show that the spike
duration is 210 ms, although it does not provide spatial details.
Because the dynamic range is greater near 415 nm, this spectral region
was used in subsequent experiments.
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a shows that the emission intensities differ when
monitored at 415 and 490 nm, we sought to confirm the similarity in the
excitation properties of indo-1 at these emission wavelengths in this
system. We therefore performed excitation spectroscopy on adherent
indo-1-labeled neutrophils. Fig. 3 shows
representative excitation spectra of an indo-1-labeled neutrophil as
measured at the emission wavelengths of 415 and 490 nm. As these
spectra show, the wavelength dependence of indo-1 excitation is very
similar for the two emission wavelengths. Thus, the excitation
properties of indo-1 do not depend on the emission wavelength.
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Inasmuch as fluorescent Ca2+
indicators may act as intracellular buffers, we next confirmed that the
indo-1 labeling protocol did not lead to significant buffering of
Ca2+ signals. To accomplish this goal,
quantitative microfluorometry experiments were conducted on
indo-1-labeled neutrophils, as described above, except that different
concentrations of indo-1 were used in the labeling procedure. After
labeling, the cells were washed extensively then resuspended in HBSS
(without Ca2+, Mg2+, or
phenol red). Fig. 4 shows quantitative
microfluorometry of polarized neutrophils labeled with different
concentrations of indo-1 for 20 min. Although the
Ca2+ spike interval may vary slightly from cell
to cell, the amplitude of the spike is clearly dependent on the
concentration of indo-1 used in the labeling protocol. Importantly, at
an indo-1 concentration of 35 µg/ml, the amplitude of the
Ca2+ spikes decreases. This reduction in
amplitude at 35 µg/ml is likely due to the ability of indo-1 to
buffer Ca2+ signals. Thus, we find no evidence
for indo-1-mediated buffering at the indo-1 concentration used in these
studies (5 µg/ml). We next independently confirmed that indo-1 in
neutrophils was responding to Ca2+. Previous
studies have shown that neutrophils are able to polarize during
conditions of Ca2+ buffering (34).
We therefore studied polarized neutrophils labeled with indo-1 in HBSS
in the absence of external divalent cations. Cells were studied in a
microscope chamber that allowed the addition of external solutions. The
intracellular Ca2+ buffer BAPTA-AM was chosen
because other molecules, such as
2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[carboxymethyl]aminoquinolone,
would interfere with the fluorescence studies. BAPTA-AM was added to
cells at a final concentration of 30 µM during observations at
37°C. As Fig. 5a shows, 2
min after addition of BAPTA-AM to polarized neutrophils, the intensity
of the Ca2+ spikes begin to diminish
progressively. BAPTA-AM diffuses into the cell where it is cleaved to
form BAPTA. We suggest that the reduction in indo-1 intensity is due to
the buffering capacity of BAPTA; intracellular
Ca2+ buffering capacity increases as BAPTA-AM
enters the cell, thereby progressively decreasing spike amplitude.
Furthermore, the constancy of the Ca2+ spike
amplitudes in Figs. 2d, 4, and 5b (and data not
shown) indicate that significant photobleaching is not occurring under
the conditions used. Therefore, the gradual reduction in
Ca2+ spike intensity in Fig. 5a cannot
be explained by photobleaching. However, one might argue that the
Ca2+ signaling simply stopped in a manner
unrelated to intracellular Ca2+ buffering by
BAPTA; e.g., the cell may have simply returned to a resting morphology.
In our experience with leukocytes and tumor cells,
Ca2+ spikes do not significantly diminish in
intensity during our observations. Fig. 5b shows a
representative example of an indo-1-labeled neutrophil as the cell
polarity was relaxed. This illustrates the fact that
Ca2+ spikes end abruptly. Thus, our experiments
were performed at an indo-1 concentration that 1) was substantially
below the level of indo-1-mediated buffering and 2) responded to
Ca2+.
Although data presented above suggest that photobleaching is not
significant during these experimental conditions, we sought to
rigorously exclude this possibility. We therefore performed experiments
over extended periods of time to ascertain the level of photobleaching.
Ca2+ spikes were studied in polarized neutrophils
during continuous illumination with a mercury lamp. Fig. 6 shows a representative long duration
experiment covering 10 min of observation. In this experiment, the
Ca2+ spike intensity decreased by 0.2%/min.
Similar results were obtained in other experiments. Therefore,
photobleaching is not a significant problem in these studies. This
result is not surprising because previous workers have noted that
photobleaching is not a significant problem for indo-1-labeled adherent
cells for up to 30 min of observation (35).
Temporal characterization of Ca2+ signal detection
Indo-1-labeled neutrophils were observed while adherent to quartz
coverslips. The cells were observed from the basal to apical surfaces,
as illustrated in Fig. 7A. To
test the effect of shutter speed on images, the CCD chip was
electronically gated for various times. Polarized neutrophils display
repetitive Ca2+ spikes (15, 33).
Fig. 8 shows six experiments of
indo-1-labeled polarized cells at increasingly shorter shutter speeds
(2 s to 50 ns). At relatively long exposure periods, the cell may
appear somewhat brighter at the center such as in the second frame of
Fig. 8a. This is due to the randomization of the bright
indo-1- Ca2+ complex while the electronic shutter
is open. Because neutrophils and most other cells are thicker near the
center, they might appear brighter at the center at slow shutter speeds
when there is no Ca2+ spike occurring (e.g., a
spike is present in Fig. 2a, frame 3, but not in
frame 2). No clear spatiotemporal patterns emerge
until a shutter speed of 200 µs is reached. The image is improved by
the shorter exposure time which 1) reduces the distance
Ca2+ waves can travel, 2) reduces the ability of
indo-1:Ca2+ complexes to move away from regions
of high Ca2+ concentration, and 3) reduces the
contribution of background fluorescence within the cell by a factor of
104 (from 2 s to 200 µs). Thus, gating
times of 
200 µs can detect Ca2+ signaling
routes in this particular experiment.
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Fig. 9 shows one cell with each
Ca2+ spike divided into multiple frames using a
50-ns exposure time and a 20-ms delay time between frames.
Ca2+ does not rise uniformly during a spike: it
is highly asymmetrical and time dependent (Fig. 9A). The
Ca2+ signal follows a pathway near the cell
surface in a counterclockwise direction, as viewed from the basal to
apical surfaces (Fig. 7A). The direction of
Ca2+ wave travel, in this case counterclockwise,
is not meant in an absolute sense, but rather in the laboratory frame
of reference (Fig. 7A). Thus, the terms counterclockwise and
clockwise are used only for illustrative purposes to differentiate
between the types of high speed waves (Fig. 7B). The
Ca2+ signal begins near the center of the
lamellipodium and then travels unidirectionally in all cells observed
(Fig. 9A). The calcium wave traveled with a velocity of
180 ± 16 µm/s. Furthermore, the Ca2+ wave
terminated when it returned to the ignition site. Thus, for polarized
neutrophils, the ignition site, termination site, and velocity did not
vary significantly. Moreover, intracellular Ca2+
signals possess inherent asymmetries (i.e., directionality), like the
unidirectional propagation of action potentials along an axon.
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B, an FMLP (50
nM)-charged micropipet was within several micrometers of the cell. FMLP
was thereby delivered to a specific region of the plasma membrane, as
confirmed using a fluorescent FMLP analog (data not shown). As shown in
Fig. 9A, Ca2+ signaling begins near
the center of the lamellipodium and then propagates around the
perimeter of the cell in a counterclockwise direction. When this
Ca2+ wave reaches the FMLP binding site, a second
Ca2+ wave moving in a clockwise direction
emanates from this point (Fig. 9B) which eventually becomes
a new lamellipodium. The two waves propagate around the perimeter of
the cytoplasm, cross at the opposite side of the cell and then continue
until they return to the FMLP binding site. The counterclockwise and
clockwise waves travel with the same velocity of 180 µm/s. These
spatial and temporal findings are generally true, but the total
distance traveled depends on the location of FMLP binding. For example,
when FMLP is applied at the uropod (data not shown), the first wave
does not travel as far before splitting into two waves. The next
Ca2+ spike is a single Ca2+
wave that begins at the FMLP binding site and propagates in a
counterclockwise direction (Fig. 9C). These events were
restricted to productive engagement of the formyl peptide receptor
because the antagonist Boc-Phe-Leu-Phe-Leu-Phe did not affect the
Ca2+ signaling pattern (data not shown).
To characterize these signals, pharmacological probes were used. When
neutrophils were exposed to saturating doses of ryanodine, which blocks
endoplasmic reticulum (ER) Ca2+ release, the
cells did not polarize normally. We therefore titered ryanodine to find
that 10 µg/ml did not significantly affect cell shape. When the cells
were observed with high speed imaging, a Ca2+
wave was initiated at the lamellipodium but disappeared during
propagation (Fig. 10A).
Thus, the counterclockwise Ca2+ release is
ryanodine sensitive, suggesting the involvement of ryanodine receptors
in wave propagation. In contrast, the plasma membrane channel blocker
verapamil had no effect on the counterclockwise wave in polarized cells
(data not shown). However, verapamil (100 µM) did block the
clockwise-propagating wave in neutrophils exposed to FMLP during
imaging experiments (Fig. 10B). This verapamil concentration
is consistent with interference with K+ channels.
The clockwise wave was also blocked by chelation of extracellular
Ca2+ using EDTA. In this experiment
indo-1-labeled adherent cells were washed three times with
Ca2+-free PBS then suspended in PBS containing 1
mM EDTA. When polarized cells were stimulated with FMLP, only one
counterclockwise Ca2+ wave was observed (Fig. 10C). Therefore, it seems likely that plasma membrane
channels participate in propagation of the clockwise
Ca2+ wave. In addition, the actions of these
Ca2+ inhibitors reconfirm that these waves (or
spikes) are due to Ca2+. Because the clockwise
wave is not initiated in the presence of ryanodine, the role of
ryanodine receptors in this pattern element cannot be discerned. These
waves correlate with cell polarity and receptor binding and represent a
very early intracellular event in direction finding. Thus,
extracellular spatial information is transduced into intracellular
spatial information.
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We next tested the potential breadth of Ca2+
signaling waves during a distinct physiological process. Phagocytosis
has been associated with Ca2+ spikes
(15). Indo-1-labeled neutrophils were incubated with
IgG-opsonized SRBCs on the 37°C stage of the microscope.
Ca2+ signaling was imaged at high speed (50 ns
exposure time and 20 ms delay time). As Fig. 11 shows, the
Ca2+ signal begins at the lamellipodium and then
propagates in a counterclockwise direction about the periphery of the
cell. As this first Ca2+ wave passes the target,
another Ca2+ wave appears to split off from the
first, followed by its propagation in a counterclockwise manner about
the perimeter of the target. As the differential interference contrast
image illustrates (Fig. 11, frame 1), the dark circular
regions in the fluorescence micrographs correspond to the two
IgG-opsonized targets. By adjusting the focus of the microscope,
the targets were found to be internalized, although the phagosome
nearer the lamellipodium appeared to have completed phagocytosis just
before Ca2+ imaging was initiated. In this
example, the Ca2+ wave appears to travel
intracellularly from one phagosome to a neighboring phagosome (Fig. 11, frame 15). In other examples of multiple target uptake where
the targets are separated by several micrometers (data not shown), the
Ca2+ wave travels from the region of the cell
surface to the phagosomes. Thus, there appear to be at least two routes
of Ca2+ signaling in the vicinity of phagosomes.
When cells were treated with ryanodine as described above,
Ca2+ wave propagation about the phagosomes was
not observed (data not shown). This is consistent with the reported
intracellular origin of Ca2+ signals mediating
phagolysosome formation. Because phagocytosis takes place at very low
Ca2+ levels (37), we suggest that
these Ca2+ waves promote the formation of
phagolysosomes, which does require Ca2+ signaling
(38, 39). Although these Ca2+ waves
resemble those associated with chemotactic stimulation, they differ in
their location. Thus, a diversity of Ca2+
signaling routes can be observed during physiological events.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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High speed microscopy has revealed that Ca2+ is not homogeneously distributed within a polarized cell during a spike; instead, Ca2+ waves follow apparently specific and highly reproducible signaling routes. In other words, the Ca2+ spike of a neutrophil is a rapidly traveling Ca2+ wave when resolved spatiotemporally. These findings provide exciting new insights in cell signaling. For example, we speculate that the Ca2+ signaling routes defined above reflect the subcellular control mechanisms participating in cell orientation and phagolysosome formation. Morphologically polarized cells exhibit one ryanodine-sensitive Ca2+ wave propagating in a counterclockwise direction around a cell. During chemotactic stimulation, two Ca2+ waves traveling in clockwise and counterclockwise directions, likely associated with both external and internal Ca2+ stores, are observed. The clockwise Ca2+ wave does not occur immediately after binding; ligated receptors must wait until the counterclockwise-rotating wave reaches the FMLP binding site. When the single wave reaches this site, Ca2+ signals are propagated in both directions. The counterclockwise wave and local receptor activation may act synergistically to reach a signaling threshold that allows ignition of a clockwise wave. This wait state, or phase delay, was anticipated by our temporal studies of neutrophil activation (47). These two Ca2+ waves orient in the direction of an extracellular ligand, like an intracellular compass.
Multiple Ca2+ waves are also seen after phagocytosis of IgG-opsonized SRBCs. However, in this case, the waves were found well within the cytoplasm of the neutrophil where they traveled in a counterclockwise direction around the target. We speculate that Ca2+ waves traveling about the perimeter of the phagosome participate in phagolysosome formation. However, these phagosome-associated Ca2+ waves differ from prevailing models of phagolysosome formation in that they were observed only in the presence of a pre-existing Ca2+ wave traveling near the plasma membrane or a neighboring phagosome. Thus, in addition to the physiological information encoded within the frequencies and amplitudes of Ca2+ spikes (2, 3, 4, 5, 6, 7, 8), high speed microscopy has revealed a Ca2+ spike may contain multiple types of spatiotemporal patterns, which we believe are also rich in information.
Our studies have shown that a direct line of communication exists between the Ca2+ signaling apparatus of the plasma membrane and that of the phagosome. Thus, the phagosome remains in communication with the plasma membrane despite having pinched off from the plasma membrane. Several recent studies have suggested a role for the ER in phagocytosis. 1) The microorganisms Legionella and Brucella can reside within an ER-like region of phagosomes (48). 2) A proteomic analysis has identified the presence of ER components within phagosomes (49). 3) Mutants deficient in the ER proteins calreticulin and calnexin are deficient in phagocytosis. Moreover, green-fluorescent protein-labeled calreticulin and calnexin demonstrated a link between the ER and phagocytic cup (50). Thus, we suggest that a strand of the ER mediates the line of communication indicated by the present study. Although we have not studied the mechanism of Ca2+ signal migration from cell surfaces to intracellular membranes, a direct coupling between the L-type Ca2+ channels of the plasma membrane and ryanodine receptors on internal ER membranes is a possibility (51, 52), given that we have preliminary evidence suggesting the presence of L-type channels on neutrophils (our unpublished observations). The characteristics of the assembly and regulation of this signaling conduit in living cells will likely contribute to a molecular understanding of why certain receptors do not promote phagolysosome fusion and certain microorganisms escape phagolysosomal destruction.
In addition to its location, a wave is also described by a velocity.
The velocity of the perimembrane Ca2+ wave is 180
µm/s. Although this velocity has been observed in all of the
experiments described here, a spherically expanding
Ca2+ wave with a velocity of 30 µm/s has
been observed during neutrophil adherence (our unpublished
observations), which presumably represents a distinct propagation
mechanism. The velocity of 180 µm/s is substantially slower than
the velocity of membrane depolarization seen in unmyelinated axons, yet
faster than that seen for other Ca2+ waves
(16, 17, 18, 19) or expected based on the measured diffusion
coefficient of Ca2+ (53). A
mechanism involving both membrane potential and short range diffusion
might account for these observations. One likely participant is
inositol 1,4,5-triphosphate, which is generated during FMLP stimulation
of neutrophils (54). Inositol 1,4,5-triphosphate promotes
Ca2+ release from the ER, which in turn triggers
store-operated Ca2+ influx across the plasma
membrane (55). Recently, phosphoinositol 3-kinase-
knockout mice have been shown to be defective in orientation in the
presence of a chemotactic factor (56); this defect may be
associated with the second clockwise Ca2+
wave.
The literature reports a Ca2+ concentration of
400 nM during neutrophil activation (27). This
concentration is spatially and temporally averaged over an entire cell
or a population of cells. However, our data show that the
Ca2+ concentration is not temporally or spatially
uniform. The Ca2+ concentration near a plasma
membrane may be far greater than the average concentration. For
example, a concentration of 25 µM Ca2+ is
required to mediate neutrophil degranulation in permeabilized cells
(57), yet concentrations of only 400 nM are observed in
stimulated neutrophils. Inspection of Figs. 8 and 10 show that local
Ca2+ levels near plasma membranes and phagosomes
are far higher than its bulk concentration, thus potentially accounting
for the apparent discrepancy. Moreover, enzymes and cytoskeletal
structures presumed to be insensitive to
Ca2+ due to micromolar
Kd may actually be sensitive when the
spatiotemporal dynamics of the signal are considered.
The high speed microscopic imaging techniques outlined in this and previous papers (23, 24, 25, 26) were designed to permit analyses of chemical wave propagation events within immune cells and should be generally applicable. For example, interactions been leukocytes and targets such as microbes and tumor cells is one potential area of interest. Preliminary studies in this laboratory suggest Ca2+ signaling coherence between neutrophils and endothelial cells during their interactions. We speculate that intracellular signaling may be understood in terms of spatiotemporal variables. Because enzymes can respond to Ca2+, ATP, NADPH, pH, cAMP, and other intracellular conditions and because Ca2+, NAD(P)H, and pH exhibit various signaling patterns (23, 24, 25, 26), a variety of superimposed spatiotemporal patterns could be generated. Hence, a few chemicals, each with a set of characteristic patterns, could generate many spatiotemporal enzyme activity patterns. Using gene knockout mice, it should also be possible to identify proteins participating in various pattern elements. Lastly, we speculate that differences in emergent chemical patterns may explain certain difficulties encountered during rational drug development.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Howard R. Petty, Department of Biological Sciences, Wayne State University, Detroit, MI 48202. E-mail address: hpetty{at}biology.biosci.wayne.edu
3 Abbreviations used in this paper: CCD, charge-coupled device; indo-1-AM, indo-1-acetoxymethyl ester; ER, endoplasmic reticulum.
Received for publication June 24, 2002. Accepted for publication October 23, 2002.
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 S. Dewitt, W. Tian, and M. B. Hallett Localised PtdIns(3,4,5)P3 or PtdIns(3,4)P2 at the phagocytic cup is required for both phagosome closure and Ca2+ signalling in HL60 neutrophils J. Cell Sci., February 1, 2006; 119(3): 443 - 451. [Abstract] [Full Text] [PDF]
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 S. G. Elner, H. R. Petty, V. M. Elner, A. Yoshida, Z.-M. Bian, D. Yang, and A. L. Kindzelskii TLR4 Mediates Human Retinal Pigment Epithelial Endotoxin Binding and Cytokine Expression Invest. Ophthalmol. Vis. Sci., December 1, 2005; 46(12): 4627 - 4633. [Abstract] [Full Text] [PDF]
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I. Harfi, F. Corazza, S. D'Hondt, and E. Sariban Differential Calcium Regulation of Proinflammatory Activities in Human Neutrophils Exposed to the Neuropeptide Pituitary Adenylate Cyclase-Activating Protein J. Immunol., September 15, 2005; 175(6): 4091 - 4102. [Abstract] [Full Text] [PDF]
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 H. Han, A. Stessin, J. Roberts, K. Hess, N. Gautam, M. Kamenetsky, O. Lou, E. Hyde, N. Nathan, W. A. Muller, et al. Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils J. Exp. Med., August 1, 2005; 202(3): 353 - 361. [Abstract] [Full Text] [PDF]
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S. Fache, J. Dalous, M. Engelund, C. Hansen, F. Chamaraux, B. Fourcade, M. Satre, P. Devreotes, and F. Bruckert Calcium mobilization stimulates Dictyostelium discoideum shear-flow-induced cell motility J. Cell Sci., August 1, 2005; 118(15): 3445 - 3458. [Abstract] [Full Text] [PDF]
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 A. J. Clark and H. R. Petty Differential Intracellular Distributions of Inositol Trisphosphate and Ryanodine Receptors Within and Among Hematopoietic Cells J. Histochem. Cytochem., July 1, 2005; 53(7): 913 - 916. [Abstract] [Full Text] [PDF]
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C. Witt, S. Raychaudhuri, and A. K. Chakraborty Movies, measurement, and modeling: the three Ms of mechanistic immunology J. Exp. Med., February 22, 2005; 201(4): 501 - 504. [Abstract] [Full Text] [PDF]
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 A. L. Kindzelskii, V. M. Elner, S. G. Elner, D. Yang, B. A. Hughes, and H. R. Petty Toll-Like Receptor 4 (TLR4) of Retinal Pigment Epithelial Cells Participates in Transmembrane Signaling in Response to Photoreceptor Outer Segments J. Gen. Physiol., July 26, 2004; 124(2): 139 - 149. [Abstract] [Full Text] [PDF]
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A. L. Kindzelskii, R. G. Sitrin, and H. R. Petty Cutting Edge: Optical Microspectrophotometry Supports the Existence of Gel Phase Lipid Rafts at the Lamellipodium of Neutrophils: Apparent Role in Calcium Signaling J. Immunol., April 15, 2004; 172(8): 4681 - 4685. [Abstract] [Full Text] [PDF]
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J.-B. Huang, A. L. Kindzelskii, A. J. Clark, and H. R. Petty Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells: Their Apparent Roles in Cell Motility and Invasion Cancer Res., April 1, 2004; 64(7): 2482 - 2489. [Abstract] [Full Text] [PDF]
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 R. G. Worth, M.-K. Kim, A. L. Kindzelskii, H. R. Petty, and A. D. Schreiber Signal sequence within Fcgamma RIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion PNAS, April 15, 2003; 100(8): 4533 - 4538. [Abstract] [Full Text] [PDF]
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 C. J. Weijer Visualizing Signals Moving in Cells Science, April 4, 2003; 300(5616): 96 - 100. [Abstract] [Full Text] [PDF]
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