Using high sensitivity fluorescence imaging with shutter speeds ∼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.

Transmembrane signaling is conventionally viewed as the appearance of a second messenger such as Ca2+, cAMP, or phosphoproteins within the cytoplasm of a cell (1). However, recent studies have shown that signaling can be mediated by temporal changes in second messenger concentrations. For example, intracellular Ca2+ concentration changes can lead to differential gene expression (2). Intracellular Ca2+ levels oscillate as well, with frequency-encoded information (3, 4, 5, 6, 7, 8). In addition to temporal Ca2+ changes, its concentration may also vary spatially within a cell. Many studies have reported static Ca2+ gradients (9, 10), which presumably reflect Ca2+ source and sink terms some distance apart. However, such images contain little temporal information. Experiments to derive spatiotemporal information generally represent long time-scale variations in “static” patterns, not inherent dynamics. Examples of slowly changing Ca2+ patterns include hepatocytes, endothelial cells, neutrophils, and exocrine cells (11, 12, 13, 14, 15). Spatiotemporal Ca2+ waves have been observed in large cells including oocytes and myocytes and in cell layers (16, 17, 18, 19). However, it has been impossible to observe such waves in small cells due to rapid wave motion, intermixing of cytoplasmic labels during image acquisition, and comparatively high background levels at typical exposure times.

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.

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).

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).

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.

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.

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 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.

FIGURE 1.

Schematic diagram of the apparatus for high speed imaging and spectroscopy. The diagram shows a Zeiss IM135 scope, as described in Materials and Methods, interfaced to the detection and data-handling electronics.

FIGURE 1.

Schematic diagram of the apparatus for high speed imaging and spectroscopy. The diagram shows a Zeiss IM135 scope, as described in Materials and Methods, interfaced to the detection and data-handling electronics.

Close modal

As illustrated in Figs. 4–6, 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.

FIGURE 4.

Representative examples of Ca2+ spikes within polarized neutrophils using different loading concentrations of indo-1-AM. Quantitative microfluorometry studies showing Ca2+ spikes within separate polarized neutrophils after loading with various extracellular concentrations of indo-1-AM from 1 to 35 μg/ml. The intensity of the Ca2+ spikes increases as the loading concentration of indo-1-AM is increased. However, the intensity decreases at 35 μg/ml, which is due to the buffering capacity of indo-1. For this reason, a concentration of 5 μg/ml, which gives a sufficient intensity but does not promote buffering, was generally used in these studies. To conserve space, data are plotted as relative (not absolute) intensity vs time. (The baseline fluorescence intensity was greatly increased in cells labeled at an indo-1-AM concentration of 35 μg/ml.) Bar, 2000 counts (n = 3).

FIGURE 4.

Representative examples of Ca2+ spikes within polarized neutrophils using different loading concentrations of indo-1-AM. Quantitative microfluorometry studies showing Ca2+ spikes within separate polarized neutrophils after loading with various extracellular concentrations of indo-1-AM from 1 to 35 μg/ml. The intensity of the Ca2+ spikes increases as the loading concentration of indo-1-AM is increased. However, the intensity decreases at 35 μg/ml, which is due to the buffering capacity of indo-1. For this reason, a concentration of 5 μg/ml, which gives a sufficient intensity but does not promote buffering, was generally used in these studies. To conserve space, data are plotted as relative (not absolute) intensity vs time. (The baseline fluorescence intensity was greatly increased in cells labeled at an indo-1-AM concentration of 35 μg/ml.) Bar, 2000 counts (n = 3).

Close modal
FIGURE 5.

Effect of Ca2+-sequestering agent BAPTA on Ca2+ spikes in neutrophils. Indo-1-labeled neutrophils were allowed to polarize on coverslips. Ca2+ spikes were followed over time using a PMT. When BAPTA-AM was added to the chamber to a final concentration of 30 μM, the fluorescence intensity of indo-1 gradually diminished (a), in contrast to the effect of BAPTA, when neutrophils become spherical the Ca2+ spikes abruptly end without gradual diminution (b). Bar, 2000 counts (n = 3).

FIGURE 5.

Effect of Ca2+-sequestering agent BAPTA on Ca2+ spikes in neutrophils. Indo-1-labeled neutrophils were allowed to polarize on coverslips. Ca2+ spikes were followed over time using a PMT. When BAPTA-AM was added to the chamber to a final concentration of 30 μM, the fluorescence intensity of indo-1 gradually diminished (a), in contrast to the effect of BAPTA, when neutrophils become spherical the Ca2+ spikes abruptly end without gradual diminution (b). Bar, 2000 counts (n = 3).

Close modal
FIGURE 6.

Effect of prolonged illumination on Ca2+ spike intensity. Indo-1-labeled neutrophils were allowed to polarize on coverslips. Ca2+ spikes were followed over extended periods of time using a PMT. A small loss in Ca2+ spike intensity was observed. In this representative example, a 3% reduction in spike intensity was found after 12 min. Thus, photobleaching of the fluorescent indo-1:Ca2+ complexes is not likely to seriously affect the spatial analyses of Ca2+ spikes shown below. Bar, 2000 counts (n = 3).

FIGURE 6.

Effect of prolonged illumination on Ca2+ spike intensity. Indo-1-labeled neutrophils were allowed to polarize on coverslips. Ca2+ spikes were followed over extended periods of time using a PMT. A small loss in Ca2+ spike intensity was observed. In this representative example, a 3% reduction in spike intensity was found after 12 min. Thus, photobleaching of the fluorescent indo-1:Ca2+ complexes is not likely to seriously affect the spatial analyses of Ca2+ spikes shown below. Bar, 2000 counts (n = 3).

Close modal

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. 2,a 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. 2,a, 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. 2,b at 415 nm but dimmer in a separate experiment (Fig. 2,c) at 490 nm. We have also confirmed the presence of Ca2+ spikes in polarized neutrophils (15) using quantitative microfluorometry at ∼415 nm (Fig. 2,d) and 490 nm (Fig. 2 e). 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.

FIGURE 2.

Emission spectroscopy, imaging, and microfluorometry of indo-1. a, Indo-1 and various Ca2+ concentrations were encased in a thin layer of gelatin and then studied by emission microspectrophotometry. The emission peaks are sensitive to Ca2+ concentration (28 ). Bars b and c approximate the spectral regions used in panels b and c. b and c, Ca2+ signaling was studied by high speed microscopy using a 100-ns shutter speed. In b, an emission filter in the region of 415 nm was used and showed a bright region high in Ca2+ rotating about the periphery of the cell (arrow). In c, an emission filter at 490 nm was used. At 490 nm, the emission intensity of indo-1 decreases at a higher calcium concentration, thus leading to a dark region high in Ca2+ rotating about the cell periphery (arrow). ×1260; n = 4, m = 22). d and e, Ca2+ spikes within polarized neutrophils. The spectral regions used in d and e were 415 and 490 nm, respectively. Bar, 2000 counts (n = 4).

FIGURE 2.

Emission spectroscopy, imaging, and microfluorometry of indo-1. a, Indo-1 and various Ca2+ concentrations were encased in a thin layer of gelatin and then studied by emission microspectrophotometry. The emission peaks are sensitive to Ca2+ concentration (28 ). Bars b and c approximate the spectral regions used in panels b and c. b and c, Ca2+ signaling was studied by high speed microscopy using a 100-ns shutter speed. In b, an emission filter in the region of 415 nm was used and showed a bright region high in Ca2+ rotating about the periphery of the cell (arrow). In c, an emission filter at 490 nm was used. At 490 nm, the emission intensity of indo-1 decreases at a higher calcium concentration, thus leading to a dark region high in Ca2+ rotating about the cell periphery (arrow). ×1260; n = 4, m = 22). d and e, Ca2+ spikes within polarized neutrophils. The spectral regions used in d and e were 415 and 490 nm, respectively. Bar, 2000 counts (n = 4).

Close modal

Several additional experiments were performed to characterize the physical and chemical properties of indo-1 in neutrophils. Although Fig. 2,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.

FIGURE 3.

Excitation spectroscopy of indo-1. Indo-1-labeled neutrophils were examined by excitation microspectrophotometry. Excitation wavelengths were scanned using a xenon lamp and monochrometer. Emission intensities were detected at 490 nm (trace a) and 405 nm (trace b) using emission filters and a PMT assembly (Photon Technologies). Note the similarities in the excitation spectra (n = 3).

FIGURE 3.

Excitation spectroscopy of indo-1. Indo-1-labeled neutrophils were examined by excitation microspectrophotometry. Excitation wavelengths were scanned using a xenon lamp and monochrometer. Emission intensities were detected at 490 nm (trace a) and 405 nm (trace b) using emission filters and a PMT assembly (Photon Technologies). Note the similarities in the excitation spectra (n = 3).

Close modal

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. 5,a 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. 2,d, 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. 5,a 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. 5 b 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).

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. 7,A. 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. 8,a. 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. 2 a, 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.

FIGURE 7.

Summary of the observational geometry and wave directions observed. A, Cells were observed on an inverted fluorescence microscope at 37°C from the basal toward the apical surfaces. B, With this observational geometry, polarized neutrophils were found to exhibit Ca2+ waves traveling in either counterclockwise (cc) or clockwise (c) directions. C, Ca2+ waves originate from several cellular sites including the lamellipodium, ligand binding sites, and the plasma membrane near sites of phagocytosis.

FIGURE 7.

Summary of the observational geometry and wave directions observed. A, Cells were observed on an inverted fluorescence microscope at 37°C from the basal toward the apical surfaces. B, With this observational geometry, polarized neutrophils were found to exhibit Ca2+ waves traveling in either counterclockwise (cc) or clockwise (c) directions. C, Ca2+ waves originate from several cellular sites including the lamellipodium, ligand binding sites, and the plasma membrane near sites of phagocytosis.

Close modal
FIGURE 8.

Effect of exposure time on the appearance of indo-1 labeled polarized neutrophils. Images with exposure times of 2 s to 50 ns were obtained with a 30-ms duty cycle. Clear spatiotemporal Ca2+ patterns can be recognized at 200 μs and below (n = 3). ×960.

FIGURE 8.

Effect of exposure time on the appearance of indo-1 labeled polarized neutrophils. Images with exposure times of 2 s to 50 ns were obtained with a 30-ms duty cycle. Clear spatiotemporal Ca2+ patterns can be recognized at 200 μs and below (n = 3). ×960.

Close modal

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. 9,A). The Ca2+ signal follows a pathway near the cell surface in a counterclockwise direction, as viewed from the basal to apical surfaces (Fig. 7,A). 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. 7,A). Thus, the terms counterclockwise and clockwise are used only for illustrative purposes to differentiate between the types of high speed waves (Fig. 7,B). The Ca2+ signal begins near the center of the lamellipodium and then travels unidirectionally in all cells observed (Fig. 9 A). 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.

FIGURE 9.

High speed imaging reveals that Ca2+ spikes are Ca2+ waves propagating in the perimembrane region of polarized neutrophils. A, Micrographs of indo-1-labeled cells were acquired using a 100-ns shutter speed and a 30-ms duty cycle. A–C, Micrograph sequences shown in represent consecutive spikes observed for a single polarized neutrophil. A Ca2+ wave begins at the lamellipodium and propagates in a counterclockwise direction. B, At a time between the sequence shown in A and B, a micropipet charged with 50 nM FMLP was discharged within a few micrometers of the cell surface to stimulate formyl peptide receptor on one side of the cell (at the top of the micrograph). In this sequence, the Ca2+ signal begins at the old lamellipodium and then propagates about the periphery of the cell until it reaches the FMLP binding site. At this point, the counterclockwise wave becomes two Ca2+ waves traveling in opposite directions. Both Ca2+ waves continue propagating around the cell until they return to the FMLP binding site. C, The next Ca2+ spike begins at the site of FMLP binding (arrow) and then propagates about the periphery of the cell as a single unidirectional wave. Thus, environmental directional cues are transduced into oriented spatial patterns (n = 4, m = 34). ×840.

FIGURE 9.

High speed imaging reveals that Ca2+ spikes are Ca2+ waves propagating in the perimembrane region of polarized neutrophils. A, Micrographs of indo-1-labeled cells were acquired using a 100-ns shutter speed and a 30-ms duty cycle. A–C, Micrograph sequences shown in represent consecutive spikes observed for a single polarized neutrophil. A Ca2+ wave begins at the lamellipodium and propagates in a counterclockwise direction. B, At a time between the sequence shown in A and B, a micropipet charged with 50 nM FMLP was discharged within a few micrometers of the cell surface to stimulate formyl peptide receptor on one side of the cell (at the top of the micrograph). In this sequence, the Ca2+ signal begins at the old lamellipodium and then propagates about the periphery of the cell until it reaches the FMLP binding site. At this point, the counterclockwise wave becomes two Ca2+ waves traveling in opposite directions. Both Ca2+ waves continue propagating around the cell until they return to the FMLP binding site. C, The next Ca2+ spike begins at the site of FMLP binding (arrow) and then propagates about the periphery of the cell as a single unidirectional wave. Thus, environmental directional cues are transduced into oriented spatial patterns (n = 4, m = 34). ×840.

Close modal

Because chemotactic agents cause Ca2+ release from internal stores and an influx of extracellular Ca2+ (36), we tested the ability of FMLP to affect Ca2+ patterns. A physiologically relevant dose of 50 nM FMLP triggered two Ca2+ waves traveling in opposite directions (clockwise and counterclockwise). In the experiment of Fig. 9,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. 9,A, 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. 9,B) 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. 9 C). 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. 10,A). 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. 10,B). 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. 10 C). 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.

FIGURE 10.

Pharmacological agents suggest that clockwise and counterclockwise Ca2+ waves originate from extracellular and intracellular sources. A, Suboptimal doses of ryanodine interfere with the propagation of counterclockwise waves. B, Verapamil blocks the appearance of clockwise waves during FMLP addition. C, The appearance of the clockwise waves was also blocked in a Ca2+-free buffer containing EDTA (n = 5, m = 28). ×920.

FIGURE 10.

Pharmacological agents suggest that clockwise and counterclockwise Ca2+ waves originate from extracellular and intracellular sources. A, Suboptimal doses of ryanodine interfere with the propagation of counterclockwise waves. B, Verapamil blocks the appearance of clockwise waves during FMLP addition. C, The appearance of the clockwise waves was also blocked in a Ca2+-free buffer containing EDTA (n = 5, m = 28). ×920.

Close modal

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.

FIGURE 11.

Phagosomes exhibit counterclockwise traveling Ca2+ waves. High speed fluorescence microscopy of indo-1-labeled neutrophils was performed using the 355HT15/390LP/405DF43 filter set. Cells were allowed to bind and phagocytose IgG-opsonized SRBCs. The erythrocytes were not labeled with indo-1 and therefore appear dark in the micrographs. A polarized neutrophil with associated erythrocytes are shown in the differential interference contrast image of frame 1. In addition to the counterclockwise wave traveling near the cell surface (Fig. 3, trace a), an additional counterclockwise wave appears to split off the perimembrane wave (frames 12 and 13) and travel about the upper target (frames 13–20). A third Ca2+ wave appears to split off from the first target (frame 15) and then travel about the perimeter of second target (frames 15–25), while the original Ca2+ wave (frames 240) continues its journey about the cell periphery (n = 3, m = 15). ×1340.

FIGURE 11.

Phagosomes exhibit counterclockwise traveling Ca2+ waves. High speed fluorescence microscopy of indo-1-labeled neutrophils was performed using the 355HT15/390LP/405DF43 filter set. Cells were allowed to bind and phagocytose IgG-opsonized SRBCs. The erythrocytes were not labeled with indo-1 and therefore appear dark in the micrographs. A polarized neutrophil with associated erythrocytes are shown in the differential interference contrast image of frame 1. In addition to the counterclockwise wave traveling near the cell surface (Fig. 3, trace a), an additional counterclockwise wave appears to split off the perimembrane wave (frames 12 and 13) and travel about the upper target (frames 13–20). A third Ca2+ wave appears to split off from the first target (frame 15) and then travel about the perimeter of second target (frames 15–25), while the original Ca2+ wave (frames 240) continues its journey about the cell periphery (n = 3, m = 15). ×1340.

Close modal

Intracellular Ca2+ plays a pivotal role in many cell functions. It has been linked with neutrophil functions such as production of reactive oxygen species, chemotaxis, phagolysosome formation, degranulation, adherence, and integrin recycling (9, 10, 15, 34, 40, 41, 42, 43, 44, 45). At the cellular level, Ca2+ changes are often seen as temporally short and intense bursts, also known as spikes. Ca2+ spikes occur during neutrophil adherence, phagocytosis, and migration (15, 41). However, the spatiotemporal dynamics of Ca2+ signaling in neutrophils and many other cell types are essentially unknown. To map dynamic signaling events, we have used modern electro-optic technology and an efficient optical microscope (23, 24, 25) that allows both high repetition rates and short exposure times. When indo-1-labeled cells are imaged for a relatively long period of time, the indo-1:Ca2+ emission resembles that obtained using conventional video microscopy. However, when the CCD chip is gated for short periods of time, punctate indo-1:Ca2+ emission is observed traveling near the plasma membrane. A theoretical study by Simon and Llinas (46) predicted intense submembrane Ca2+ profiles at submicrosecond time scales. Our experimental findings are consistent with these calculations. Moreover, the repetition rates available with our apparatus make it is possible to follow the trafficking of Ca2+ signals from place to place within a living cell.

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-γ 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.

1

This work was supported by National Institutes of Health Grant CA74120 (to H.R.P.).

3

Abbreviations used in this paper: CCD, charge-coupled device; indo-1-AM, indo-1-acetoxymethyl ester; ER, endoplasmic reticulum.

1
Petty, H. R..
1993
.
Molecular Biology of Membranes
Plenum Press, New York.
2
Dolmetsch, R. E., R. S. Lewis, C. C. Goodnow, J. I. Healy.
1997
. Differential activation of transcription factors induced by Ca2+ response amplitude and duration.
Nature
386
:
855
3
Berridge, M. J..
1997
. The AM and FM of calcium signaling.
Nature
386
:
759
4
Gu, X., N. C. Spitzer.
1995
. Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients.
Nature
375
:
784
5
Putney, J. W..
1998
. Calcium signaling: up, down, up, down what’s the point?.
Science
279
:
191
6
De Koninck, P., H. Schulman.
1998
. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.
Science
279
:
227
7
Goldbeter, A..
1996
.
Biochemical Oscillations and Cellular Rhythms
Cambridge University Press, Cambridge.
8
Tsien, R. W., R. Y. Tsien.
1990
. Calcium channels, stores, and oscillations.
Annu. Rev. Cell Biol.
6
:
715
9
Sawyer, D. W., J. A. Sullivan, G. L. Mandell.
1985
. Intracellular free calcium localization in neutrophils during phagocytosis.
Science
223
:
663
10
Brundage, R. A., K. E. Fogarty, R. A. Tuft, F. S. Fay.
1993
. Chemotaxis of newt eosinophils: calcium regulation of chemotactic response.
Am. J. Physiol.
34
:
1527
11
Thorn, P., A. M. Lawrie, P. M. Smith, D. V. Gallacher, O. H. Peterson.
1993
. Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate.
Cell
74
:
661
12
Kasai, H., Y. X. Li, Y. Miyashita.
1993
. Subcellular distribution of Ca2+ release channels underlying Ca2+ waves and oscillations in exocrine pancreas.
Cell
74
:
669
13
Thomas, A. P., D. C. Renard, T. A. Rooney.
1991
. Spatial and temporal organization of calcium signalling in hepatocytes.
Cell Calcium
12
:
111
14
Jacob, R..
1990
. Imaging cytoplasmic free calcium in histamine stimulated endothelial cells and in fMet-Leu-Phe stimulated neutrophils.
Cell Calcium
11
:
241
15
Marks, P. W., F. R. Maxfield.
1990
. Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils.
J. Cell Biol.
110
:
43
16
Lechleiter, J. D., D. E. Clapham.
1992
. Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes.
Cell
69
:
283
17
Wussling, M. H., H. Salz.
1996
. Nonlinear propagation of spherical calcium waves in rat cardiac myocytes.
Biophys. J.
70
:
1144
18
Newman, E., K. R. Zahs.
1997
. Calcium waves in retinal glial cells.
Science
275
:
844
19
Takashima, I., M. Ichikawa, T. Iijima.
1999
. High-speed CCD imaging system for monitoring neural activity in vivo and in vitro, using a voltage-sensitive dye.
J. Neurosci. Methods
91
:
147
20
Zoghbi, M. E., P. Bolanos, C. Villalba-Galea, A. Marcano, E. Hernandez, M. Fill, A. L. Escobar.
2000
. Spatial Ca2+ distribution in contracting skeletal and cardiac muscle cells.
Biophys. J.
78
:
164
21
Takemura, H., S. Yamashina, A. Segawa.
1999
. Millisecond analyses of Ca2+ initiation sites evoked by muscarinic receptor stimulation in exocrine acinar cells.
Biochem. Biophys. Res. Commun.
259
:
656
22
Paemeleire, K., P. E. Martin, S. L. Coleman, K. E. Fogarty, W. A. Carrington, L. Leybaert, R. A. Tuft, W. H. Evans, M. J. Sanderson.
2000
. Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26.
Mol. Biol. Cell
11
:
1815
23
Petty, H. R., R. G. Worth, A. L. Kindzelskii.
2000
. Imaging sustained dissipative patterns in the metabolism of individual living cells.
Phys. Rev. Lett.
84
:
2754
24
Petty, H. R., A. L. Kindzelskii.
2000
. High-speed imaging of sustained metabolic target patterns in living neutrophils during adherence.
J. Phys. Chem. B
104
:
10952
25
Petty, H. R., A. L. Kindzelskii.
2001
. Dissipative metabolic patterns respond during neutrophil transmembrane signaling.
Proc. Natl. Acad. Sci. USA
98
:
3145
26
Kindzelskii, A. L., H. R. Petty.
2002
. Apparent role of traveling metabolic waves in periodic oxidant release by living cells.
Proc. Natl. Acad. Sci. USA
99
:
9207
27
Sehgal, G., K. Zhang, R. F. Todd, L. A. Boxer, H. R. Petty.
1993
. Lectin-like inhibition of immune complex receptor-mediated stimulation of neutrophils: effects on cytosolic calcium release and superoxide production.
J. Immunol.
150
:
4571
28
Worth, R. G., L. Mayo-Bond, J. G. J. van de Winkel, R. F. Todd, III, H. R. Petty, A. D. Schreiber.
2001
. The cytoplasmic domain of FcγRIIA (CD32) participates in phagolysosome formation.
Blood
98
:
3429
29
Roper Scientific Technical Note No. 6. www.roperscientific.com.
30
Roper Scientific Technical Note No. 11. www.roperscientific.com.
31
Haugland, R. P..
1996
.
Handbook of Fluorescent Probes and Research Chemicals
Molecular Probes, Eugene.
32
Kindzelskii, A. L., M. M. Eszes, R. F. Todd, III, H. R. Petty.
1997
. Proximity oscillations of complement type 4 (αX, β2) and urokinase receptors on migrating neutrophils.
Biophys. J.
73
:
1777
33
Petty, H. R..
2001
. Neutrophil oscillations: temporal and spatiotemporal aspects of cell behavior.
Immunol. Res.
23
:
125
34
Pierini, L. M., M. A. Lauson, R. J. Eddy, B. Henedey, F. R. Maxfield.
2000
. Oriented endocytic recycling of α5β1 in motile neutrophils.
Blood
95
:
2471
35
Wahl, M., M. J. Lucherini, E. Gruenstein.
1990
. Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations.
Cell Calcium
11
:
487
36
Merritt, J. E., R. Jacob, T. J. Hallam.
1989
. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils.
J. Biol. Chem.
264
:
1522
37
Di Virgilio, F., B. C. Meyer, S. Greenberg, S. C. Silverstein.
1988
. Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels.
J. Cell Biol.
106
:
657
38
Majeed, M., N. Perskvist, J. D. Ernst, K. Orselius, O. Stendahl.
1998
. Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosis in human neutrophils.
Microb. Pathog.
24
:
309
39
Jaconi, M. E., P. D. Lew, J. L. Carpentier, K. E. Magnusson, M. Sjögren, O. Stendahl.
1990
. Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils.
J. Cell Biol.
110
:
1555
40
Hallett, M. B., E. V. Davies, A. K. Campbell.
1990
. Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal.
Cell Calcium
11
:
655
41
Maxfield, F. R., B. A. Kruskal.
1987
. Cytosolic free calcium increases before and oscillates during frustrated phagocytosis in macrophages.
J. Cell Biol.
105
:
2685
42
Jaconi, M. E., J. M. Theler, W. Schlegel, R. D. Appel, S. D. Wright, P. D. Lew.
1991
. Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family.
J. Cell Biol.
112
:
1249
43
Lew, P. D., C. B. Wollheim, F. A. Waldvogel, T. Pozzan.
1984
. Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils.
J. Cell Biol.
99
:
1221
44
Lawson, M. A., F. R. Maxfield.
1995
. Ca2+- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils.
Nature
377
:
75
45
Demaurex, N., A. Monod, D. P. Lew, K. Krause.
1994
. Characterization of receptor-mediated and store-regulated Ca2+ influx in human neutrophils.
Biochem. J.
297
:
595
46
Simon, S. M., R. R. Llinas.
1985
. Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release.
Biophys. J.
48
:
485
47
Albrecht, E., H. R. Petty.
1998
. Cellular memory: neutrophil orientation reverses during temporally decreasing chemoattractant concentrations.
Proc. Natl. Acad. Sci. USA
95
:
5039
48
Mesresse, S., O. Steele-Mortimer, E. Moreno, M. Desjardins, B. B. Finlay, J. P. Gorvel.
1999
. Controlling the maturation of pathogen-containing vacuoles: a matter of life and death.
Nat. Cell Biol.
1
:
E183
49
Garin, J., R. Diez, S. Kieffer, J.-F. Dermine, S. Duclos, E. Gagnon, R. Dadoul, R. Rondeau, M. Desjardins.
2001
. The phagosome proteome: insight into phagosome functions.
J. Cell Biol.
152
:
165
50
Muller-Taubenberger, A., A. N. Lupas, H. Li, M. Ecke, E. Simmeth, G. Gerisch.
2001
. Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis.
EMBO J.
20
:
6772
51
Lu, X., L. Xu, G. Meissner.
1994
. Activation of the skeletal muscle calcium release channel by a cytoplasmic loop of the dihydropyridine receptor.
J. Biol. Chem.
269
:
6511
52
Marty, I., M. Robert, M. Villaz, K. De Jonh, Y. Lai, W. A. Catterall, and M. Ronjat, M. 1994. Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle. Proc. Natl. Acad. Sci. USA 91:2270.
53
Nasi, E., D. Tillotson.
1985
. The rate of diffusion of Ca2+ and Ba2+ in a nerve cell body.
Biophys. J.
47
:
735
54
Synderman, R., C. D. Smith, M. W. Verghese.
1986
. Model for leukocyte regulation by chemoattractant receptors: roles of a guanine nucleotide regulatory protien and polyphosphoinositide metabolism.
J. Leukocyte Biol.
40
:
785
55
Itagaki, K., K. B. Kannan, D. H. Livingston, E. A. Deitch, Z. Fekete, C. J. Hauser.
2002
. Store-operated calcium entry in human neutrophils reflects multiple contributions from independently regulated pathways.
J. Immunol.
168
:
4063
56
Hannigan, M., L. Zhan, Z. Li, Y. Ai, D. Wu, C.-K. Huang.
2002
. Neutrophils lacking phosphoinositide 3-kinase γ show loss of directionality during N-formyl-Met-Leu-Phe-induced chemotaxis.
Proc. Natl. Acad. Sci. USA
99
:
3603
57
Smolen, J. E., S. J. Stoehr, L. A. Boxer.
1986
. Human neutrophils permeabilized with digitonin respond with lysosomal enzyme release when exposed to micromolar levels of free calcium.
Biochim. Biophys. Acta
886
:
1