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Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils

During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]i increased rapidly from < or = 100 nM to > or = 400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of approximately 200 nM. The initial Ca2+ spike was insensitive acutely to removal of extracellular Ca2+ but was abolished by treatments expected to deplete internal Ca2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lamellipod activity and suppressed the Ca2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca2+ spike on stimulation. Although cell flattening and lamellipod activity were induced by chemoattractant when the normal Ca2+ response was blocked, cells failed to polarize and crawl, indicating that Ca2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.

The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca 2+ signal may trigger the cytoskeletal reorganiTation requlred for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca 2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca 2+ signals. To identify these messengers, Ca 2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]t increased rapidly from ~100 nM to >.~400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of '~ 200 nM.
The initial Ca 2+ spike was insensitive acutely to removal of extracellular Ca 2+ but was abolished by treatments expected to deplete internal Ca 2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lametlipod activity and suppressed the Ca 2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca 2+ spike on stimulation. Although cell flattening and larneUipod activity were induced by chemoattractant when the normal Ca 2+ response was blocked, cells failed to polarize and crawl, indicating that Ca 2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca 2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca 2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus. D RECTIONAL persistence during locomotion in a stimulus gradient and rapid reorientation when the direction of the stimulus gradient changes are virtually certain to involve a complex web of highly interactive intracellular processes that stabilize directionality in a way that can be immediately and effectively overridden. Although components of the signal transduction pathways that control cell orientation have been identified in many types of cells, little is known about the mechanisms involved in translating the spatio-temporal patterns of the stimulus into appropriate changes in intracellular organization. The ubiquity of pathways in which Ca 2÷ serves as a second messenger and the Ca2+-sensitivity of a large number of cytoskeletal proteins suggests that Ca 2+ could play an important role. Thus considerable effort has been devoted to characterizing changes in the spatial distribution of [Ca2+]i in response to agonists and understanding their physiological significance (e.g., Albritton and Meyer, 1993;Girard and Clapham, 1993;Thorn et al., 1993).
Large free-living amebae often exhibit a gradient of [Ca2+]i, with the highest concentration in the rear of the cell (except during phagocytosis, when [Ca2+]i rises in the region of the presumptive phagosome; Taylor et al., 1980). A similar Ca 2 gradient in newt eosinophils reverses direction when the stimulus gradient is reversed during chemotaxis (Brundage et al., 1991). Many proteins that affect the organization of the actin cytoskeleton are sensitive to [Ca 2+] (e.g., Yin and Stossel, 1979), and [Ca 2+] has opposite effects on the ATPase activities of myosin II and some isoforms of myosin I (Collins and Matsudaira, 1991; see Pollard and Korn, 1973). The two types of myosin are distributed differently in locomotory cells, myosin I being confined mostly to the front of the cell and myosin II to the rear (Fukui et al., 1989). An in vitro contraction model consisting of a gel of myosin II and actin mixed with filamin and gelsolin contracts away from the high concentration end of a [Ca2+]i gradient (Janson et al., 1991). The spatial distribution of the Ca 2+calmodulin complex, which likely reflects the distribution of its activated target proteins, including those that activate myosin II, is similar to that of [Ca2÷]i in migrating fibroblasts, as demonstrated with a fluorescent analog of calmodulin (Hahn et al., 1992). Ca 2+ also seems to play a role, via integrins, in regulating the attachment of human neutrophils to the substratum (Marks et al., 1991). Collectively these observations suggest that a gradient of [Ca2÷]~ could stabilize the organization of cytoskeletal components responsible for cell polarity.
They also raise the question of how the spatial pattern of [Ca2+]~ itself arises. In secretory epithelia (see Thorn et al., 1993), the [Ca2+]i gradient results from spatial variations in the density of InsP3-sensitive internal stores. In newt eosinophils during chemotaxis, however, the direction of the [Ca2+]i gradient changes rapidly in response to relocating the stimulus source (within tens of seconds; Brundage et al., 1991) and well before the cell reorientation which enables it to follow the stimulus. Thus [Ca2+]i must be controlled at least partly by messenger pathways that allow spatial characteristics of the stimulus to be translated acutely into a new spatial distribution of Ca 2+. Brundage et al. (1993) showed that fluctuations in [Ca2+]~ appear to correlate with changes in cell shape, speed, and orientation. The next step toward understanding the origin and the physiological significance of the [Ca2+]~ gradient is to identify chemoattractant-sensitive messengers that affect Ca 2+ homeostasis and to determine how they might produce particular spatial patterns of [Ca2÷]i. These experiments were designed to determine whether chemoattractant-induced changes in [Ca2+]~ result from influx across the plasma membrane or discharge of internal stores or both, to identify which if any of the most common second messengers affect [Ca2+]~ and to investigate the relation between changes in [Ca2+]i and cell shape, polarity, and locomotion when [Ca2+]~ was perturbed by experimental intervention. Newt eosinophils injected with fura-2 were activated with chemoattractant applied uniformly in the medium (to evoke chemokinesis) and stimulus-sensitive second-messenger pathways identified by quantifying changes in [Ca2+]~ in response to treatments expected to alter these pathways. Cell morphology and locomotion were monitored simultaneously. The results show that chemoattractantinduced increases in [Ca2+]~ can originate exclusively from internal stores, at least some of which appear to be sensitive to inositol-l,3,4-biphosphate (InsP3) ~. The data also suggest that diacyl glycerol (DAG) and protein kinase C (PKC) activity suppress [Ca2+]i. Ca 2+ homeostasis appears to be required for polarization and locomotion but not for cell ac-1. Abbreviations used in this paper: ACM, amphibian culture medium; DAG, diacyl glycerol; DOG, dioctanoyl glycerol; InsP3, inositol-l,3,4biphosphate; PKC, protein kinase C; SPS, sulfated polysaccharides. tivation and flattening. Both Ca 2÷ and PKC activity seem to affect cell shape. A scheme is proposed for messenger-mediated changes in the spatial distributions of [Ca2+]i and PKC activity in response to changes in the external chemoattractant gradient during chemotaxis, by means of which these two agents could serve as initiators of cell reorientation, perhaps overriding spatial patterns arising from variations in the density of Ca 2÷ stores.

Cell Preparation and Experimental Protocols
Newt eosinophils were prepared by a modification of the technique of Koonce et al. (1984). Newts were bled by clipping the tip of the tail and 0.2-0.3 ml of blood collected in 1 ml heparinized amphibian culture medium (ACM,pH 7.2,and containing,mM: 64 NaC1,4.3 KC1,1.4 CaCI2,1.3 MgSO4,0.7 NaH2PO4,19.2 Hepes and 4.4 glucose). 0.1 ml of the mixture was spread on a 25-mm circular coverslip. Cells settled and attached in "o30 min. The coverslip was washed in ACM to remove erythrocytes and clamped into a frame to form a cell chamber, which was filled with 0.4-0.6 ml ACM and mounted on the stage of an inverted microscope. During the first 5-10 rain on the microscope, most cells were polarized and active and became quiescent over the next 10-15 min, first withdrawing lamellipods, and then becoming round and loosening attachments to the substrate. Cells were injected as they were undergoing this process, using a Picospritzer II (General Valve Corp., E. Hanover, NJ). The injectate contained 0.5-1.0 mM fura-2 (potassium salt) dissolved in 20 mM Pipes or 10 mM glutamic acid/potassium glutamate (pH 7.2). When necessary, the cells were allowed to recover until lCa2+]i was <100 nM (10-20 min). Cell volume was ,°4 pl, injection volume <8% cell volume and [fura-2]i <40 #M unless otherwise indicated. Methods used to determine these values are described below.
Phase-contrast images were acquired at 0.5 s -I and fura-2 fluorescence image pairs at 0.1 s -~ . The basic experimental protocol was to record images as ACM was replaced with 10% newt serum in ACM (the only substance known to be chemotactic for these cells; see Brundage et al., 1993), with various agents added either to the injectate or to the bathing medium. At least one cell from each blood sample was injected with fura-2 alone and treated only with 10% newt serum and the data used to assess other "control" treatments, such as injection with nominally inactive peptides and other agents and exposure to the solvents of drugs introduced into the medium.

Imaging and Data Analysis
Phase-contrast and fluorescence in,ages at excitation wavelengths of 340 and 380 nm were acquired using the Digital Imaging Microscope described by Brundage et al. 0991) and a Nikon 40× UVF objective (NA 1.4). The modified Zeiss IM35 inverted microscope was equipped with two cameras. A video camera (Dage MTI CCD72, Michigan City, IN, from which the infra-red filter was removed) passed to a video disk recorder (Panasonib TQ-2028F) phase-contrast images acquired using transmitted illumination through a 680-rim high-pass filter. A low noise, slow-readout, thermoelectrically cooled CCD camera (model CH220, TI 4849 detector, Photometrics, Tucson, AZ) acquired fura-2 fluorescence image pairs that were tramferred directly to the disk of a PDP-11/73 microcomputer (Digital Equipment Corporation, Maynard, MA). A mask in the optical path to the cooled CCD camera allowed only half the chip to be exposed. The image acquired at the first excitation wavelength (filter 340/DFI0 nm; Omega Optical, Brattleboro, VT) was shifted electronically to the masked half of the chip as the second excitation filter (380/DFI0 run) was being positioned. The duration of each exposure was 0.5 s and the interval between the two images of a pair 0.1 s. The size of each pixel in the fluorescence image was 0.526 #In and the size of the field read from the CCD controller onto computer disk 100 The J'oumal of Cell Biology, Volume 127, 1994 X 150 ~m (one-quarter of the chip) for the image at each excitation wavelength. The PDP-11/73 controlled the cooled CCD camera shutter, image transfer from the CCD chip to disk, the excitation filter wheel and shutter (MVI, Avon, MA) and phase-contrast image acquisition by the video disk recorder. All analysis was performed on Silicon Graphics Personal Iris workstations (Mountain View, CA), connected to the PDP via Ethernet, using software developed in the laboratory. Phase-contrast images stored on video disk were digitized by the image acquisition interface of a Silicon Graphics GTX workstation. Images were stored on erasable optical laser disks (Introl Corp., Minneapolis, MN). Composite [Ca2+]i and phasecontrast images were generated with Silicon Graphics UNIX utilities software and 35-mm photographic film exposed by a Matrix Color Graphic Recorder (Agfa, Matrix Division, Orangeburg, NY) interfaced to a Personal Iris.
Computation of [CaZ+]~. Image pairs of fura-2 fluorescence at ~x3,~0 and ~x3s0 were stored by the camera controller on hard disk as an alternating time-lapse series. Background fluorescence was determined by averaging intensities of a 4400-pixel region of the field never entered by the cell during recording, and that value was subtracted from each pixel of each image in the sequence. The concentration of Ca 2+ at each pixel was then calculated from the relation developed by Tsien and coworkers (Grynkiewicz et al., 1985; see Moore et al., 1990). The spatially averaged [Ca2+]i values shown in the graphs were calculated by averaging [Ca2+]i values of all pixels within the perimeter of the cell, which was marked by an automated tracing program based on a threshold/flood-fill algorithm, operating on the bleach-corrected, Ca2+-insensitive fura-2 images calculated as described below.

Determination of lntracellular Concentrations of lnjectate Constituents
A procedure was developed for quantifying the intracellular concentrations of fura-2 and other injected compounds, based on the fact that many cells were spherical until exposed to chemoattractant. Thus cell volume could be calculated by measuring cell diameter in phase-contrast or fluorescence images. The optical system was calibrated by injecting a cell-sized microdroplet of fura-2 into silicone oil in the cell chamber (Moore et al., 1990). Phase-contrast images of the spherical droplet were recorded by time-lapse video as the pipette was lowered to the bottom glass surface of the chamber. The volume of the microdroplet was calculated from sphere diameter measured from the video images. When the droplet contacted the glass surface, it adhered and flattened to form a disk whose total intensity was recorded with the cooled CCD camera, at the same exposure and camera gain used in cell experiments. The area of the field from which fluorescence intensity was recorded was 200 × 150 Itm and so included the entire region surrounding the fluorescent disk. Background fluorescence at both wavelengths was recorded from an empty field and subtracted before further data processing. The calibration factor (CF) for fura-2 is given by [fura-2] (mol/liter x sphere volume (1) CF (mol/count) = (1) microdroplet intensity (counts).
Intensities in both equations are the Ca2+-insensitive values calculated from the 340 and 380 image pairs (to avoid recording a third image at the isosbestic wavelength of fura-2), given by where B is the ratio of the dynamic range of the 340 to the 380 fluorescence intensity determined by calibrating fura-2 in solutions of <10 -7 and >10 -3 M [Ca 2÷] on the microscope (Becker and Fay, 1987). Intracellular concentrations of other components of the injectate were calculated from their ratio to fura-2 concentration in the injectate, assuming this ratio to be unaffected by experimental protocols.

Introduction of Drugs into the Medium
Extracellular agents were introduced by replacing the bathing medium. In the case of acute presentation of 5 mM EGTA to reduce extracellular [Ca2+], it was essential to determine the time required for [Ca 2+] inunedi-ately adjacent to the cell to become less than resting intracellular [Ca2+]. Normal medium containing fura-2 was added to the chamber with activated uninjeeted cells and the kinetics of the decrease in [Ca 2+] next to a cell recorded as this medium was replaced with new medium containing the same concentration of fura-2 and 5 mM EGTA. The field diaphragm in the epifluorescence excitation path was focused on the coverslip and constricted to illuminate a region ,~30/~m in diameter, producing the quasi-confocal optical conditions required to record fluorescence from a layer of solution ~<20-ttm deep at the surface of the coverslip (see Hiraoka et al., 1990). The thickness of this layer was verified independently as follows. The cell was positioned so that part of its anterior granule-containing region occupied ",,30% of the illuminated field. Upon changing the medium, external [Ca2+]i fell from >1 mM to <1/zM within I0 s and to <50 aM within 20 s. The actual depth of the in-focus solution layer that contributed significantly to the fura-2 fluorescence images was assessed by monitoring the decrease in fluorescence intensity in focal planes above the coverslip as the objective was moved upward and the image of the field diaphragm became blurred. Simultaneous phase-contrast images of the cell recorded by the video camera showed that when the top (medium-facing) surface of the cell was optimally focused (~10 #m above the coverslip), fluorescence intensity had decreased by >50%. Thus, fura-2 fluorescence >110 tzm above the coverslip did not contribute significantly to the fluorescence used to calculate [Ca 2+] just outside the cell, which was much less than cytoplasmic [Ca 2+] when Ca 2+ spikes occurred in cells injected with fura-2 and recorded as they were exposed to medium containing 5 mM EGTA.

Chemoattractant Causes [Ca2÷], to Increase and Cells to Flatten, Polarize, and Crawl
When resting eosinophils were exposed to chemoattractant, the first response was a sudden transient increase in [Ca2+]j by several hundred nM, followed by flattening, polarization (i.e., expansion of a single advancing lameUipod at one end, constriction of the cell cortex and location of the nucleus at the other) and locomotion. These events are illustrated in the first two figures, showing a series of phase-contrast and [Ca2÷]i images of a typical cell (Fig. 1) and a graph of spatially averaged [Ca2÷]i from the same cell (Fig. 2). Lamellipods are apparent in the phase-contrast image acquired at the peak of the initial Ca 2÷ transient ( Fig. 1 Both the kinetics and the spatial characteristics of the responses were consistent from cell to cell, except as noted. In 46 cells, resting [Ca2÷]~ (averaged for 1-2 min before stimulation) was 85 5:5 nM (mean + standard error). Peak [Ca2÷]i in response to chemoattractant was 417 5:16 nM (defined as the maximal value within 2 min of stimulation) and the time to peak 34 5:3 s. The range of activation peaks was broad (214-708 nM), and in many cells, [Ca2+]~ rose from the resting level to the peak within one 10-s interval, suggesting that sampling frequency was too low for the true peak to be recorded. Other cells, like that shown in Fig. 1, produced several spikes within 2 min of stimulation, and occasionally the initial spike was not the largest. The postactivation level of [Ca~÷]~ was 201 5:12 nM (29 cells), averaged for 1 min between the Ca 2÷ spikes that occurred in most cells during chemokinesis. For example, [Ca2+]i val- ues for the cell in Figs. 1 and 2 were averaged between 6 and 7 rain after activation to determine the postactivation level.
Included in these mean values are data from seven cells injected with the inactive analog of the peptide inhibitor of PKC, two cells injected with the peptide inhibitor of cAMPdependent protein kinase, one cell exposed to 0.1% DMSO (the solvent for several agents added to the medium), four cells exposed to 1,3-dioctanoyl glycerol (DOG) (a diacyl glycerol not found in live cells), and 2 cells injected with de-N-sulfated beparin, a control compound for the sulfatedpolysaccharide InsP3 receptor blockers. None of these cells was different from the other 30 cells, which were injected with fura-2 alone and exposed only to ACM and 10% newt serum.
Some cells did not flatten and move across the substrate in response to chemoattractant but instead elongated verti-cally into the medium with only the nuclear end attached. Although these cells were actively changing shape, horizontal velocity in the plane of the substrate was near zero, and, except for the initial spike, [Ca2+]i was well below active levels measured in crawling cells. Some of these cells eventually flattened and crawled, and this change was accompanied by an increase in [Ca2+],, suggesting that Ca 2+ changes and locomotion are tightly coupled. The only [Ca2+]i values from these "swimming" cells included in the mean values given above are resting and peak values, which were within the range of the other control cells. None of the agents used to perturb [Ca2+]~ in the experiments to be described affected the frequency with which this type of response occurred. It was observed less often when the coverslips were washed in hot 5 N HC1.
[Ca2÷]~ measured within 1 rain of injection with fura-2 was often higher than values recorded 10-20 rain later, probably because of Ca 2÷ influx produced by injection. Cells with postinjection values of 0.5-1 #M usually flattened, polarized, and crawled for a few minutes. As [Ca2÷]~ declined, some of these cells resumed their original spherical shape, while others were tightly stuck to the coverslip and remained flattened but inactive, as indicated by the absence of lamelli- Figure 7. Images showing effects of Ca 2+ depletion from internal stores and injection of exogenous Ca 2+ buffer on responses to stimulation. The cell whose [Ca2+]i image is shown in A is the same as that in Figs. 3 C and 4 C. The color bar in A has two different scales, from 50 to 90 nM for the image on the left, 140 s after EGTA application, and 10--40 nM for the image on the fight, 470 s after EGTA. The cell in B was exposed to 5 mM EGTA for 20 rain, which reduced [Ca2+]i to <50 nM (Fig. 8. A) and blocked the stimulus-induced Ca 2+ spike. The cell in C was injected with fura-2 to a final intracellular concentration of 55 ~,M, calculated as described in Materials and Methods. Pseudocolor images of [Ca2+]~ in C are for the cell shown in Fig. 8   pods, shape changes, and locomotion. While the initial Ca 2÷ spike produced by activation of spherical cells appeared to be spatially uniform, in flattened cells it was spatially graded, with the highest [Ca2÷]~ in the perinuclear region.

[Ca2+]~ Spikes Are Independent of Ca ~ Influx from the External Medium
The increase in [Ca2+]i elicited by chemoattractant could come from influx across the plasma membrane, from discharge of internal stores, or both. Relative contributions of these two sources were assessed by measuring [Ca'+]~ in the presence of extracellular EGTA during random migration in spatially uniform chemoattractant (chemokinesis) and in response to stimulation. In 4 of 9 cells treated with 5 mM EGTA during chemokinesis, Ca ~+ spikes and cell polarity and locomotion persisted for at least 10 min (the duration of the treatment after the recording period). The time course of changes in average [Ca2+]~ in one of these cells is shown in Fig. 3 A and phase-contrast micrographs in Fig. 4 Fig. 4, asterisks in C, the time points of Ca 2+ images shown in Fig. 7 A. detection with fura-2 is that Ca 2+ storage sites are most dense in the perinuclear region.
Another group of cells was exposed to 5 mM EGTA before activation with chemoattractant. In general, treatment with EGTA for a few minutes produced no effect on the initial Ca 2+ response to chemoattractant sti ulafion, although longer treatments depleted internal stores, as described in the following section. The graph in Fig. 5 shows the Ca 2+ response of a cell incubated in EGTA for 10 min, and then activated by chemoattractant. The cell produced a robust initial Ca 2+ spike followed by several smaller ones before [Ca2+]i ultimately declined to <50 nM. Thus the Ca 2÷ spike produced by stimulation does not require Ca 2÷ influx across the plasma membrane. The phase-contrast micrographs of this cell in Fig. 6 show that although its initial morphological response to stimulation was normal, it failed to polarize, inset, in that its nucleus assumed a position in the center of the cell and lamellipods formed simultaneously at several locations and extended in many directions. Also the cell never crawled. These responses were typical of three other ceils exposed for 2-10 min to EGTA before stimulation.

Ca 2+ Homeostasis Is Required for Polarization and Locomotion but Not for Flattening and LameUipod Extension
To learn which chemoattractant-induced morphological changes were sensitive to [Ca2+]i, some ceils were treated with EGTA for/>15 min and others injected with fura-2 to high intracellular concentrations (55-400 IzM) to buffer changes in free [Ca2+], The effects of EGTA are shown by the phase-contrast images in Fig. 7 B. The cell was slightly shown). On stimulation, the cell flattened further and extended lamellipods in several directions away from the cytoplasmic extension containing the nucleus. There was no detectable change in [Ca2+]i during the 10-min recording period after stimulation with chemoattractant ( Fig. 8 A). The cell in Fig. 7 C was injected with 1 mM fura-2 to a final intracellular concentration of 55 #M. As shown by the graph in Fig. 8 B, [Ca2+]i rose slowly to reach a peak of 400 nM 2.5 min after stimulation. The distribution of [Ca2+]i at 540 s is typical of the pattern observed throughout the stimulus post-recording period, with [Ca2+]~ highest in the perinuclear region. This cell also failed to polarize, in that the nucleus became centrally located, and although the cell elongated, lameUipods formed at both ends of the long axis.

Sulfated Polysaccharide Blockers of InsP3 Receptors Obliterate Both the Ca t÷ Spike and Morphological Responses to Chemoattractant
The fact that discharge of internal stores is sufficient to produce increases in [Ca2+]~ in response to chemoattractant suggests that the effect could be mediated by InsP3. Cells were therefore injected with one of several sulfated polysaccharides (SPS), which block binding of InsP3 to its receptor (ECs0 6.9 #g/ml for pentosan polysulfate on InsP3 receptors from liver microsomes; Tones et al., 1989; see also Ghosh et al., 1988;Hill et al., 1987). Phase-contrast and [CaZ+]i images of two cells injected with fura-2 and either heparan sulfate or an inactive heparin analog, de-Nsulfated heparin, are shown in Fig. 9. In contrast to the inactive compound (Figs. 9 B and 10 B), heparan sulfate completely abolished the Ca 2÷ response to chemoattractant (Fig. 10 A). Identical results were obtained with heparin sulfate and pentosan polysulfate. The phase-contrast images in Fig. 9 A show that the heparan-injected cell also exhibited no morphological response to chemoattractant. This result was surprising in view of the persistence of chemoattractant-induced shape changes under conditions in which Ca 2÷ responses to chemoattractant were altered by injection of high concentrations of fura-2 or prolonged exposure to EGTA (Fig. 7). Thus the sulfated polysaccharides blocked not only the Ca 2+ spike but also Ca2+-independent flattening and lamellipod extension. Several investigators have reported that sulfated polysaccharides inhibit PKC (K~ for pentosan polysulfate in vitro 0.32 #g/ml; Herbert and Maffrand, 1991). The ubiquity of the coproduction of diacyl glycerol and InsP3 by receptor-mediated activation of phospholipase C in chemoattractant-activated cells (see Lew, 1989) and the stimulatory effects of PKC on actin polymerization (Zimmerman and Keller, 1988) suggest that the DAG/PKC pathway could be responsible for the morphological changes seen on stimulation in the absence of a Ca 2÷ spike (Fig. 7 B). Given the difference in affinities of these compounds for PKC and the InsP3 receptor, they would inhibit the InsP3/Ca 2+ and DAG/PKC pathways at high intracellular concentrations (Figs. 9 A and l0 A) but at low concentrations would block only the DAG/PKC pathway. To test this idea, cells from the same blood samples were injected with SPS at concentrations in the injectate of either 0.05 or 2 mg/ml. On stimulation, cells injected with the low dose flattened, polarized, and crawled normally and were morphologically indistinguishable from control cells. The three Ca 2+ images of such a cell in Fig. 9 C show that both resting [Ca2+]i and the initial spike produced by the chemoattractant stimulus were significantly higher than in control cells. The time-averaged [Ca2+]i during chemokinesis was slightly higher, although the difference was not significant, as shown by the bar graph in Fig. ll, which summarizes results from several experiments. Thus injecting cells with SPS to intracellular concentrations below their affinity for the InsP3 receptor inhibited a pathway, possibly leading to activation of PKC, that norreally limits the amplitude of the Ca: + spike. These results suggest that both InsP3 and PKC participate in chemoattractant stimulation and that PKC activity may suppress [Ca2+]i. Figure 9. Effects of sulfated polysaccharides on chemoattractant-induced changes in cell shape and [Ca2+]i. Phase-contrast images of a cell injected with heparan sulfate (intracellular concentration '~108/tg/ ml) and stimulated with chemoattractant are shown in the left column. Micrographs in the three double panels in the center show phase-contrast and [Ca2+]i images from a cell injected with de-N-sulfated hepa-

tin (de-NS) (intracellular concentration ,~104/~g/ml).
The time course of changes in average [Ca2+]i in each cell is shown in Fig. 10. The scale bar in the [Ca2+]i image at 540 s is 20/zm. The right column shows Ca 2+ images of a cell injected with pentosan polysulfate (intracellular concentration ~1.3 /~g/ml), and then activated with 10% newt serum. The images at time points flagged with an asterisk (0, 50 s) are displayed with the [Ca2+]i scale whose maximum value is 1/~M (50 s). The last image, 540 s after stimulation, is displayed with a [Ca2+]i scale whose maximum is 480 nM (140 s).

Injection of a Peptide Inhibitor of PKC Potentiates the Ca z+ Response to Chemoattractant
To demonstrate directly the effects of PKC inhibition, cells were injected with a pcptide that comprises the autoinhibitory domain of the enzyme. Chemoattractant-induced changes in [Ca:+]i are shown in Fig. 12. The cell in A was injected with the inhibitor peptide, the cell in B with an inactive control peptide. Both cells produced a Ca :+ spike within 30 s of chemoattractant application. However, the magnitude of the spike was more than three times larger in the cell injected with the inhibitor peptide, similar to the observations with low intracellular concentrations of SPS. No obvious differences were observed between the morphologi-A HEPARAN SULFATE 108 ~g/ml '°°I ~.   Effects of injecting the PKC inhibitor peptide into several cells are summarized in Fig. 13. The high standard error in peak [Ca'+]i in ceils injected with the peptide is probably due to the low frequency of image acquisition in these experiments (0.1 s-I). In many of these expe "rtments, [Ca2+]i reached its peak value within 10 s of stimulation, suggesting that the true maximal value was not recorded.

I~
The mean values from cells injected with control peptide include measurements from two ceils injected with a peptide inhibitor of cyclic AMP-dependent protein kinase. Although the intraceilular concentration of this peptide was 100x that required for complete inhibition of its target enzyme, neither the Ca 2÷ response nor the behavior of the cells was distinguishable of those cells injected with fura-2 alone or with the inactive analog of the PKC inhibitor. This result is consistent with an earlier observation (Brundage et al., 1993) that agents that increase intracellular cAMP (other than caffeine) have no detectable effect on cell behavior and support the conclusion that neither cAMP nor cAMPdependent protein kinase plays an important role in signal transduction in these cells. Furthermore, the results eliminate the possibility that the PKC inhibitor peptide had a nonspecific effect on cAMP-dependent kinase.

Activators of PKC Reduce [Ca2÷], and Cause Flattening and LameUipod Extension
If inhibition of PKC augments the chemoattractant-induced Ca 2+ response, agents that stimulate PKC activity should reduce the amplitude of the Ca 2÷ spike produced by chemoattractant and perhaps elicit changes in cell shape in its absence. To test this possibility, PMA and a diacyl glycerol (1,2-dioctanoyl glycerol, 1,2-DOG) were applied to cells before activation by chemoattractant, as shown in Figs. 14 and 15. Phase-contrast (Fig. 14) and [Ca2+]~ images of a resting cell in ACM were recorded for 30 s, and then the cell was Figure 14. Effects of diacyl glycerol on morphological responses to chemoattractant. Phase-contrast images of a cell exposed for 128 s to 1,2-dioctanoyl glycerol, and then to 1,2-DOG + 10% newt serum. Times are in seconds relative to 1,2-DOG treatment or chemoattractant stimulation (in parentheses). treated with 100 #M 1,2-DOG. Within 2 min lamellipod activity had increased, although, as shown by the graph in Fig.  15 C, [Ca2÷]~ remained low. The cell was next stimulated with 10% newt serum (containing 1,2-DOG). Almost 4 min later [Ca2÷]i increased slightly, to "-,250 nM. The cell never polarized and moved but instead situated its nucleus in the center and extended competing lamellipods from each end (Fig. 14). Fig. 15 shows changes in average [Ca2+], in three other cells, one (A) preincubated in 1,3-DOG, a diacyl glycerol not known to occur in live cells, another (B) preincubated in 1,2-DOG, and the fourth (D) exposed acutely to 50 nM PMA. All treatments except 1,3-DOG significantly suppressed and delayed the Ca 2+ response and stimulated lamellipod activity and some flattening but blocked polarization and locomotion.
A total of four 1,2-DOG-treated cells and four control cells were used in the experimental protocol illustrated in Figs. 14 and 15 C. As shown in the inset of Fig. 16, 1,2-DOG had no effect on resting [Ca2+]~ (p > 0.5 for paired observations). However, it did produce flattening and lamellipod activity in three of the four cells, while none of the control cells responded to replacing the medium. The cell shown in Fig. 14 exhibited the least flattening, but the most lamellipod activity, of the four cells treated acutely with 1,2-DOG. Shape changes induced by chemoattractant in cells pretreated with PKC activators were quite similar to those of Ca2+-depleted cells.
Seven other 1,2-DOG-treated cells and nine controls were used in the protocol illustrated in Fig. 15 B, in which cells were preincubated in medium with or without 1,2-DOG before recording began. As there were no differences in the Ca 2+ responses to chemoattractant in the 2-min and 10-min exposures to 1,2-DOG, results from experiments with the two protocols were pooled and are summarized in the large bar graph in Fig. 16. The suppression of the initial Ca 2+ spike by 1,2-DOG is highly significant and leads to the same conclusion as the effects of PKC inhibitors, that a chemoattractant-induced increase in DAG and PKC activity causes flattening and lamellipod extension and limits the magnitude of the initial Ca 2÷ spike. It is likely that other messenger pathways contribute to the response, as indicated by the additional morphological changes that occurred on chemoattractant exposure after pretreatment with 1,2-DOG and by the absence of effects on chemoattractant-induced changes in morphology of cells injected with PKC inhibitors.
In Dictyostelium, DAG has effects on the cytoskeleton that are not mimicked by other activators of PKC (Shariff and Luna, 1992). Evidence against this possibility was obtained by exposing four quiescent cells to 50 nM PMA for 2 min before stimulation by chemoattractant (see Fig. 15 D). All four cells flattened and extended lamellipods in response to PMA. None of the cells produced a Ca 2÷ response to stimulation, and none polarized and crawled. Morphologically these cells resembled those treated for long periods with EGTA to deplete Ca 2+ stores, as shown in Fig. 7. When cells were treated with PKC activators during chemokinesis, [Ca2+]i declined and locomotion ceased (Gilbert, S. H., K. Perry, and E S. Fay. 1991. Activators of protein kinase C paralyze locomotion and reduce [Ca++], in newt eosinophilic leukocytes. J. Cell Biol. 115:279a). However, the Ca 2÷ gradient was still apparent when average [Ca2+]~ had fallen to <50 nM, as shown by the images in Fig. 17. The cell was treated with octanoyl-acetyl-glyceride, and 2 min later [Ca2+]~ was <25 nM in the front of the cell and ~40 nM in the rear.

Properties of the Ca 2+ Response to Stimulation with Spatially Uniform Chemoattractant
On stimulation, spatially averaged [Ca2+]i in newt eosinophils rose immediately from ~100 nM to >~400 nM, and then declined and fluctuated around a mean value of 200 nM. The initial Ca 2÷ spike was insensitive to brief exposure to 5 mM EGTA applied before stimulation but was blocked by longer incubation in EGTA and by sulfated polysaccharides injected to intracellular concentrations shown to block InsP3 receptors in other systems (e.g., Ghosh et al., 1988;Hill et al., 1987;Worley et al., 1987;Tones et al., 1989). Active cells treated with 5 mM EGTA continued to produce Ca 2+ spikes for at least 2 min, but in some ceils [Ca2÷]~ subsequently ¢¢ ¢.9 < < 400 300 200 100 REST PEAK Figure 16. Effects of 1,2-DOG on resting and peak [Ca2+]~. Cells injected with fura-2 were exposed to ACM (control) or to ACM containing 100 #M 1,2-DOG for 2 or 10 min. Resting values of [Ca2+]i were obtained by averaging whole-cell [Ca2+]i for 1 rain before stimulation. The inset shows results obtained in ceils whose fluorescence intensity was being recorded when the medium was swapped either for ACM or ACM + 1,2-DOG. The slight difference in [Ca2+]i before and after DOG treatment of the resting ceils is not significant by a Student's t test for paired observations.
declined. The kinetics of the initial Ca 2÷ spike was slowed when fura-2 was injected to final intracellular concentrations above those required to measure [Ca:+]i. SPS injected to low intracellular concentrations, sufficient to block PKC activity in vitro (Herbert and Maffrand, 1991) but below the level required to interfere with InsP3 binding to its receptor (Tones et al., 1989), potentiated the Ca 2+ response. Injection of a peptide corresponding to the autoinhibitory domain of PKC also potentiated the early Ca 2+ spike, while preincubation with PKC activators blocked it and suppressed [Ca2+]i in activated cells. We conclude from these observations that the first Ca 2+ spike elicited by stimulation occurs by discharge of internal Ca 2÷ stores and does not require Ca 2+ influx across the plasma membrane. Inhibition of the response by high concentrations of SPS suggests that at least some of the stores are sensitive to InsP3. The existence of such stores in newt eosinophils is supported by preliminary evidence that [Ca2+]~ is increased by photolysis of caged InsP3 (Gilbert et al., 1994). In this respect newt eosinophils are similar to other electrically inexcitable cells (see Putney, 1993, and references therein).
The simplest interpretation of the results of experiments with activators and inhibitors of PKC is that activity of the enzyme reduces [Ca2÷]~ and attenuates the acute Ca 2+ response to chemoattractant. The postulate that PKC is activated by chemoattractant is suggested by augmentation of the first Ca 2÷ spike in cells injected with agents shown to inhibit PKC in vitro (House and Kemp, 1987;Malinow et al., 1989;Herbert and Maffrand, 1991). In this respect newt eosinophils are like human neutrophils, in which PKC activation inhibits discharge of internal Ca 2÷ stores and activates Ca 2÷ efflux (McCarthy et al., 1989). Alternative interpretations of the observations reported here cannot be ruled out. For example, it is possible that both PKC inhibitors and both PKC activators affect one or more unidentified pathways that influence Ca 2+ fluxes in opposite directions independently of PKC activation. The only way to rule out such an interpretation definitively is to develop methods of assaying PKC activity in live single cells. Figs. 1 and 9 illustrate that, once the cells were polarized, [Ca2+]i was always higher in the rear than in the front. The magnitude of the difference was largest at maximal average [Ca2+],, and a Ca ~+ gradient was apparent at peak average [Ca2+]t in these and all other control cells during chemokinesis. It also occurred in ceils injected with fura-2 to intracellular concentrations sufficient to damp the kinetics of changes in [Ca2+]~ (i.e., spikes did not occur) and persisted in active cells treated with EGTA, PMA, or diacyl glycerols until [Ca~+]i was <50 nM (Figs. 7 and 17).

Spatial Distribution of [CaZ+]t. Ca 2+ images in
A Ca 2+ gradient results from a polarized distribution of the relative levels of activity of Ca 2+ influx and efllux sites. This could result from a polarized distribution of messengers that control Ca 2+ fluxes or a polarized distribution of the influx and/or efflux sites themselves. The two mechanisms are not mutually exclusive, and, acting together, could reinforce the stability of the Ca 2+ gradient and possibly the direction of the cell, as discussed below. Observations reported here indicate that polarized messenger distribution is unlikely to be the sole determinant of the Ca 2+ gradient observed during chemokinesis, when the external stimulus is spatially uniform. For a Ca 2+ gradient to exist under these conditions, a polarized distribution of Ca2+-controlling messengers would have to result from a polarized distribution of responsive chemoattractant receptors (for which there is evidence in human neutrophils; Sullivan et al., 1984) and/or associated membrane enzymes that produce the messengers. If this were the only gradient-producing mechanism, however, then introducing uniformly into the medium PMA or diacyl glycerols, which suppress [Ca2+], might be expected to abolish the Ca 2+ gradient. This was not observed. Instead these agents caused [Ca2+]i to fall quickly in active cells during chemokinesis, but the Ca ~+ gradient persisted until average [Ca~+]i dropped to •50 nM, as shown in Fig. 17 (Gilbert, S. H., K. Perry and E S. Fay. 1991. Activators of protein kinase C paralyze locomotion and reduce [Ca++]i in newt eosinophilic leukocytes. J. Cell Biol. 115:279a). Furthermore, if the Ca 2+ suppressing action of these agents involved activation of PKC, then the Ca 2+ gradient should also be abolished by intracellular PKC inhibitors, and this also was not observed, as illustrated in Fig. 9 C If, on the other hand, the Ca 2+ gradient resulted from a polarized distribution of the targets of chemoattractantsensitive messengers, whether they were Ca ~+ stores or effiurdsequestration sites, then treatment with spatially homogeneous agents would not abolish the Ca 2+ gradient.
Preliminary observations from other recent experiments (Gilbert et al., 1994) support the notion that InsP3-sensitive Ca ~+ stores are more dense in the rear than in the front of the cell. Cells in which both cytoplasm and internal Ca 2+ stores were loaded with the acetoxymethyl ester of the low affinity indicator Magfura-2 show a higher ratio of fluorescence intensity at )~ 340 and 380 in the region near the nucleus and microtubule-organizing center than elsewhere. On the other hand, cells injected with the salt of the same probe show a lower and spatially uniform ratio. Thus there are internal stores of either Mg 2+ or Ca 2+ concentrated near the nucleus in newt eosinophils. In other experiments, spatially uniform photolysis of caged InsP3 produced an increase in [Ca2+]i that was highest in the perinuclear region, suggesting that the divalent cation detected in intracellular compartments of Magfura-2 loaded as the AM ester was more likely to have been Ca 2+ than Mg 2+. This is supported by previous observations of cells loaded with the AM ester of fura-2, revealing a membrane-bounded Ca 2+ store near the microtubule-organizing center (Brundage et al., 1993).

Effects of [CaZ+]~ and Other Messengers on Cell Morphology, Polarity, and Locomotion
Several methods of perturbing Ca 2+ homeostasis in these cells were identified by these experiments, and three of them blocked polarization and locomotion but had no effect on flattening and lamellipod formation elicited by chemoattractant (which were blocked by high concentrations of heparin, the fourth Ca2+-perturbing treatment). The three treatments were exposure to EGTA and PKC activators, which abolished or greatly attenuated the Ca 2+ spike, and injection of fura-2 to high intracellular concentrations, which significantly reduced its rate of rise. The results indicate that the early spike is not required for the initial morphological responses to chemoattractant but is necessary for polarization and locomotion. Furthermore, the abnormal morphological response of the cell injected with excess fura-2 (Figs. 7 C and 8 B) suggests that the kinetics of the Ca 2÷ response may be important for polarization and locomotion to progress normally. However, processes activated by the early spike are not sufficient for sustained polarity and locomotion in the presence of chemoattractant but low [Ca2+]i, as illustrated by the loss of polarity and motility in cells whose Ca 2+ responses to chemoattractant were suppressed by treatment with EGTA or PKC activators.
Two PKC inhibitors augmented the acute Ca 2+ response but had no readily detectable effect on the development of the Ca 2+ gradient and polarization and locomotion. These observations are consistent with the idea that a polarized distribution of Ca 2+ stores is sufficient to produce a Ca 2+ gradient, which in turn promotes polarization and locomotion. Cells whose [Ca2+]i, measured within 1 min of microinjection, was above resting levels flattened, polarized, and crawled normally for several minutes, suggesting that a Ca 2+ spike alone may be sufficient to initiate these processes in the absence of chemoattractant.
PKC activators, Ca 2+, and the cytoskeleton. Cells treated with 1,2-DOG or PMA in the absence of chemoattractant flattened and formed lamellipods, as did ceils stimulated with chemoattractant whose acute Ca 2+ responses had been blocked or altered. These observations suggest that the Ca2+-independent responses of the cytoskeleton to chemoattractant may be mediated by DAG and PKC. Unlike Ca 2+, however, this pathway is not sufficient to initiate polarization and locomotion, as indicated by the fact that cells treated with PKC activators did not polarize and crawl.
The experimental evidence suggests that Ca 2÷ homeostasis and morphological responses to chemoatwactant are partly controlled by the pathways summarized schematically in Fig. 18. Stimulation appears to release both InsP3 and DAG, and inhibition or potentiation of each of these pathways independently had effects consistent with the conclusion that  Figure 18. Schematic summary of components of the chemoattractant signal transduction pathway that participates in activation, polarization, and locomotion in newt eosinophils.
InsP3 through Ca 2+ activates polarization and locomotion, while DAG through PKC induces flattening and lamellipod formation. The InsP3/Ca 2+ pathway appears necessary for polarization and locomotion, and Ca 2+ alone may be sufficient to elicit these responses transiently. The DAG/PKC pathway alone is sufficient for flattening and lamellipod formarion but cannot produce polarization and locomotion in the absence of chemoattractant and may not be necessary for any of these processes to occur on stimulation. The augmentation of the early Ca 2+ spike by PKC inhibitors suggests that this pathway is activated by the stimulus, but the fact that flattening and lamellipod formation seem to have occurred normally in these cells suggests that as yet unidentified (and perhaps Ca~+-sensitive) pathways can also trigger these responses. The actin cytoskeleton and its accessory proteins are targets of both PKC and Ca 2+, and their interactions are complex. For example, the MARCKS protein (myristolated alanine-rich C kinase substrate; for a recent review see Aderem, 1992) binds to membranes, actin filaments, and the Ca2+-calmodulin complex and exhibits microfilament-bundling activity (Hartwig et al., 1992). Phosphorylation reduces its affinity for membranes, and association with the Ca2+-calmodulin complex, which is inhibited by phosphorylation, disperses microfilament bundles without dissociating MARCKS from individual microfilaments. Therefore MARCKS could respond to both PKC activation and increased [Ca2÷]i by disrupting the organization of microfilaments and their association with membranes and may play a role in converting local changes in [Ca2+]i and PKC activity to changes in the actin cytoskeleton and locomotion.
The myosins are also targets of Ca :+ and PKC. Phosphorylation of nonmuscle myosin 11, by PKC as well as the Ca2+-calmodulin/myosin light chain kinase pathway, appears to regulate its function in other systems (Higashihara et al., 1991;Pasternak et al., 1989;Murakami and Elzinga, 1993). Nonmuscle myosin II is regulated by myosin light chain kinase (Cande and Ezzell, 1986), and a bacterial-  Fig. 4 A of Brundage et al. (1991), in which a puffer pipette used as the source of chemoattractant to lead a newt eosinophil was moved from the front (F) to the rear (R) of the cell. Beneath the sketches of the cell are graphs showing hypothetical profiles of concentrations of chemoattractant, diacyl glycerol (DAG), activated PKC,InsP3,and [Ca2+i]. The proposed signal transduction pathway is shown schematically at the left. In the experiment reported by Brundage et al. (1991), the earliest response to reversing the stimulus gradient was reversal of the [Ca2+]i gradient. The concentration profiles of DAG, active PKC, and InsP3 are inferred from the effects of these agents on whole-cell [Ca2+]i and difference in partition coefficients between cytosol and membrane of InsP3 and DAG.
peptide chemoattractant has been shown to stimulate phosphorylation of the 20-kD myosin light chain in leukocytes (Fechheimer and Zigmond, 1983). Detailed studies of the genetics of type II myosins, especially those of Dictyostelium by Spudich and colleagues, show that they play a significant role in both polarization and normal locomotion. Mutants with truncated myosin II (incapable of forming minifilaments) flatten and extend lameUipods in all directions but can neither polarize nor move persistently in one direction (Fukui et al., 1990). Thus Ca2+'s effects on the state of myosin II and the contractile activity of acto-myosin II complexes may contribute to its role in polarization and locomotion.

The [Caz+]~ Gradient and Cell Polarity
The evidence that the DAG/PKC pathway is activated by the stimulus and has effects on both [Cae+]i and the cytoskeleton suggests that both PKC and Ca 2+ could play key roles in linking directional external signals to intracellular organization during chemotaxis. Furthermore, the possibility that PKC activity affects [Ca2+]i suggests a mechanism whereby this enzyme might contribute to the reorientation of the [Ca2+]~ gradient when the stimulus source is relocated (Brundage et al., 1991, Fig. 4 A), which cannot be explained by a polarized distribution of Ca 2* sources and sinks.
As shown by the cartoon in Fig. 19, these events might occur as a consequence of the difference in the spatial distributions of InsP3 and DAG, which have opposite effects on [Cae+]i. The intracellular mobilities and locations of these messengers are expected to be different because of their different partition coefficients between lipid membranes and aqueous cytoplasm (see Lew, 1989). When the stimulus source is moved from the front (F) to the rear (R) of the cell, both DAG and InsP3 would be produced by interaction of the chemoattractant with receptors present in the rear (R). InsP3 would rapidly equilibrate throughout the cell (Albritton et al., 1992), inducing Ca 2+ release wherever there are internal stores. DAG would remain in or near membranes close to the stimulus source in the rear. Activated PKC partitions into membranes in many cells (e.g., O'Flaherty et al., 1990;Ito et al., 1988) and could locally suppress [Ca2+]i (McCarthy et al., 1989), producing a [Ca:+]~ gradient oriented away from the repositioned stimulus (see Poenie et al., 1987).
Superficially, this mechanism might appear to contradict the experimental evidence that the Ca 2÷ gradient during chemokinesis is insensitive to spatially uniform activators and inhibitors of PKC. A fundamental and highly relevant issue, however, is the distinction between chemokinesis, when the stimulus is spatially uniform, and chemotaxis, when the stimulus is graded. We propose that the Ca 2÷ gradient observed during chemokinesis arises primarily from a polarized distribution of Ca 2÷ sources and/or efflux sites and persists in the face of exogenous, spatially uniform agents that suppress or elevate [Ca2+]~. During chemotaxis, however, the Ca 2+ gradient could reverse when the stimulus gradient is reversed because of a change in the intracellular distribution of stimulus-sensitive messengers that can affect Ca 2+ fluxes and override the spatial pattern resulting from the physical distribution of Ca :+ stores.
Both the [Ca2+]~ gradient and the spatially heterogeneous distribution of activated PKC could have profound effects on the cytoskeleton. Activation of PKC at R near the relocated pipette might destabilize the structure of cortical actin and myosin II, perhaps via MARCKS and myosin II phosphorylation. Also, low [Cae+]t in that region could inhibit myosin II activity and favor lamellipod formation and expansion. High [Ca2+]~ near the lamellipod at F could trigger depolymerization of actin microfilaments and destruction of the lamellipodial meshwork, perhaps again through Ca ~+sensitive proteins associated with or having effects on the actin cytoskeleton. Together these regional differences in the composition, structure, and activities of cytoskeletal elements would bring about reorientation of the cell. The existence of a mechanism for redistribution of messengers that control [Ca:÷]~ and PKC activity would thus provide the means for initiating reorientation of the cell in response to changes in the direction of the external stimulus. Redistribution of sites of Ca 2÷ influx and effiux initiated by regional changes in the state of the cytoskeleton could then reinforce the spatial gradient of subsequent Ca 2+ spikes, which would stabilize cytoskeletal organization and sustain the new direction of the cell.