ABSTRACT
Ca2+ responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in Dictyostelium D. discoideum mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faster fall rates. Correlations among these kinetic parameters and the response amplitudes suggested that key events in the Ca2+ response are autoregulated by the magnitude of the response itself, i.e., by cytosolic Ca2+ levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses: larger responses are faster in both mutant and wild-type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted D. discoideum proteome revealed three putative Ca2+ pumps and four putative Ca2+ channels. All but one contained sequence motifs for Ca2+- or calmodulin-binding sites, consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+ responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca2+ from the extracellular medium into the mutant cells was smaller. Compared to wild-type cells, Ca2+ release from the ER in the mutants thus contributes more to the total cytosolic Ca2+ response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca2+ from the ER contributes to cytosolic Ca2+ responses in D. discoideum.
Dictyostelium discoideum cells are responsive to various chemoattractants at different stages of their life cycle. In the growth phase Dictyostelium amoebae respond chemotactically to folate and other pterins and are believed to use this response to actively hunt bacteria that secrete these compounds. Upon starvation, the amoebae differentiate with the result that they begin to secrete and respond chemotactically to another chemoattractant, cyclic AMP (cAMP). Chemotactic aggregation in response to extracellular cAMP signals mediates the transition to the multicellular stages of the Dictyostelium life cycle.
Chemotactic responses to cAMP in Dictyostelium amoebae have been intensively studied: many of the molecular events in the signal transduction pathways involved have been identified so that this is perhaps the best understood example of chemotactic motility by amoeboid cells (for recent reviews see references 7, 40, and 55). The major receptor involved in cAMP chemotaxis is cAR1, one of four closely related seven-transmembrane domain cAMP receptors in D. discoideum, all of which couple to the heterotrimeric G-protein Gα2βγ. Downstream signaling molecules involved in chemotaxis include guanylyl cyclases that produce cGMP from GTP, the small GTP-binding protein RasC, phosphatidylinositol-3 kinases that phosphorylate phosphatidylinositol bisphosphate, converting it to phosphatidylinositol trisphosphate phosphatase PTEN, Pleckstrin Homology (PH) domain-containing proteins such as the docking protein PhdA, and the protein kinase Akt/PKB and other protein kinases such as PAKa and several myosin heavy- and light-chain kinases. Some of these molecules act at the leading edge of the cell where they activate pseudopod extension, while others act in the rear cortex of the cell to mediate retraction.
Not all of the intracellular signaling events activated by cAMP binding to cAR1 are G protein-dependent. The cAR1 receptor was the first G-protein-coupled receptor shown to elicit some downstream events in a G-protein-independent manner including an influx of extracellular Ca2+ (32, 33, 39, 45) and activation of the mitogen-activated protein kinase ERK2 (25). During chemotaxis in a spatiotemporal attractant gradient, the cytosolic Ca2+ elevation in response to an attractant occurs primarily in the rear cortex of the cell (59) where it may contribute to retraction of the rear and inhibition of inappropriate pseudopod extension in incorrect directions. The Ca2+ response is not essential for chemotaxis (53), but may play a role in its fine tuning, especially in shallow gradients.
Molecular genetic evidence indicates that there are two signaling pathways coupling the cAMP receptor to the Ca2+ channel responsible for Ca2+ influx, both of which are present in the early stages of differentiation (4 h differentiation) (39). One of these is G-protein-dependent and down regulated during early development, while a second is G-protein-independent and dominates responses in cells that are fully differentiated to aggregation competence (8 h differentiation).
Aside from the roles of the heterotrimeric G-proteins Gα4βγ (for responses to folate) and Gα2βγ (for responses to cAMP), little is known of the signaling events that connect the chemoattractant receptors to the Ca2+ influx channels. In the early, G-protein-dependent pathway, the unknown product of the stmF gene plays a role, since mutant alleles of this gene cause prolongation and enhancement of both cGMP and Ca2+ elevations in response to chemoattractant (23, 28, 39). Originally identified by chemical mutagenesis and classical genetics, stmF had been thought to encode the phosphodiesterase responsible for attenuating the cGMP responses to folate and cAMP. The prolonged G-protein-dependent Ca2+ responses of stmF mutants were therefore suggested to be caused by the prolonged cGMP responses. However this has recently been shown not to be the case.
A pdeD null mutant lacking the major cGMP-specific phosphodiesterase exhibits enhanced and prolonged cGMP responses like those of the stmF mutant (27). Yet in the early stages of differentiation (3 h) its Ca2+ responses are reduced rather than enhanced, consistent with the early suggestion that cGMP acts to inhibit Ca2+ influx (24). At later stages of development Ca2+ responses are unaltered in this (24) and another independent pdeD null strain as well as in several other mutants whose cGMP responses are dramatically altered by disruption of the guanylyl cyclase and cGMP phosphodiesterase genes (57). The stmF gene product thus appears to represent an upstream regulatory element in the G-protein-dependent pathway that restricts the duration of both the cGMP and Ca2+ responses.
Several observations led to the suggestion that, by analogy with hormone-stimulated mammalian cells, cAMP stimulation would elicit a rapid release of Ca2+ from the endoplasmic reticulum. These were reports that inositol (1, 4, 5)triphosphate (IP3) elicits release of Ca2+ from intracellular stores in permeabilized cells (12, 16), cAMP stimulates a rapid, transient elevation of IP3 in intact cells (peak within 5 seconds) (13, 56), and GTPγS stimulates IP3 synthesis in isolated membranes (14, 56).
However the predicted rapid Ca2+ response was not observed when measurements of cytoplasmic Ca2+ responses to chemoattractant were made in intact cells expressing the Ca2+-sensitive luminescent protein, aequorin (38). This assay has a temporal resolution of 20 ms and can detect changes in cytosolic Ca2+ levels of as little as 2 to 3 nM. The only observable intracellular Ca2+ response in wild-type cells occurs after the reported IP3 response has already subsided and it coincides with the influx of extracellular Ca2+. No intracellular Ca2+ transient is observed in the absence of extracellular calcium or in the presence of known Ca2+ channel blockers (38), while Ca2+ responses to chemoattractants are close to normal (39, 46) in a mutant in which the phospholipase C gene has been disrupted (10). This mutant remains able to synthesize IP3 from higher-order inositol polyphosphates through the action of a Ca2+-dependent phosphatase, but it exhibits IP3 responses only to cAMP concentrations several orders of magnitude higher than those required to elicit cytosolic Ca2+ responses (54).
Although chemoattractants do not stimulate an observable intracellular release of Ca2+ into the cytoplasm that is independent of the influx of Ca2+ from the medium, it remains possible that the influx is accompanied by and coupled to a release of Ca2+ from intracellular stores (38). By analogy with Ca2+-signaling systems in other organisms (3), there are two possible scenarios that are not mutually exclusive. One is that the Ca2+ influx causes and is accompanied by a Ca2+-induced release of Ca2+ from the endoplasmic reticulum. A second is that the Ca2+ influx after chemoattractant stimulation is activated by depletion of intracellular stores which elicits the opening of store-operated Ca2+ channels in the plasma membrane. This is referred to as capacitative Ca2+ entry. The store depletion that elicits it would result from the opening of Ca2+ channels in the endoplasmic reticulum (ER) by IP3 as described above or by other second messengers such as long-chain fatty acids. It has been shown that arachidonic acid elicits both an influx of Ca2+ into intact D. discoideum cells and a Ca2+ release from intracellular vesicles of permeabilized cells (46). This accords with the suggestion that chemoattractants such as cAMP may activate phospholipase A2, resulting in the production of long chain fatty acids and the release of Ca2+ from intracellular stores, depleting them and thereby activating influx (45).
To investigate these various possibilities, we have studied Ca2+ responses to chemoattractants by mutants lacking either or both of calreticulin and calnexin, two of the major Ca2+-binding proteins in the endoplasmic reticulum (30, 31, 35). All three knockout mutants exhibit qualitatively normal chemotactic responses and almost normal growth in liquid medium, while the double mutant (but not the single mutants) grows much more slowly on bacterial lawns because of a severe defect in phagocytosis (35). We report here that all three mutants exhibit enhanced cytosolic Ca2+ responses to chemoattractant. This is the first molecular genetic evidence that, in Dictyostelium cells, Ca2+ release from the endoplasmic reticulum contributes to the intracellular Ca2+ responses to chemoattractant stimuli.
MATERIALS AND METHODS
Dictyostelium strains and culture conditions.All work was done with Dictyostelium discoideum strain AX2 (2), with the mutant strains derived from it, HG1774 (calreticulin deficient), HG1770 (calnexin deficient), HG1772 (calreticulin and calnexin deficient) (35), and with aequorin-expressing transformants derived from them (HPF401 from AX2, HPF608 from HG1774, and HPF609 and HPF610 from HG1770). For axenic growth the amoebae were incubated as previously in shaken culture (150 rpm) at 23°C in axenic medium (45) or at 21°C in HL5 medium (38).
Measurement of intracellular Ca2+ responses using the aequorin method.Intracellular Ca2+ levels were measured in transformants of wild-type and mutant cells expressing recombinant aequorin as previously described (38). Cells were harvested from a growing axenic culture at a density of 1 × 106 to 2 × 106 cells/ml. For cAMP responses they were washed by centrifugation and suspended in morpholineethanesulfonic acid (MES)-DB buffer containing 0.416 μg/ml coelenterazine (the cofactor for aequorin) for development at 21°C with shaking for 6 h at a density of 2 × 107 cells/ml. For folate responses the washed cells were resuspended at a density of 2 × 107 cells/ml in fresh HL5 medium containing 0.416 μg/ml coelenterazine and incubated for 4 h at 21°C with shaking at 150 rpm. Prior to assay the cells were washed free of unbound coelenterazine by centrifugation and suspended at a density of 2 × 107 cells/ml in MES-DB for assay.
Aequorin light emission was measured with a New Brunswick Lumitran photometer whose output was digitized by a data logging card (PCI-20428W-1 Multifunction Board) from Intelligent Instrumentation Inc. installed in a personal computer. To assay intracellular Ca2+ concentrations in real time, a 500 μl aliquot of a 10−3 dilution of the cells (i.e., 104 cells) was first injected into 5 ml of lysis buffer containing an excess of Ca2+. This results in complete discharge of all active aequorin in the cells and the total light emission therefrom was measured for subsequent use in the recording software to calculate the fractional rate of light emission and Ca2+ concentration in real time. The Ca2+ responses were assayed in a gently stirred 5-ml suspension (i.e., 108 cells) in a plastic scintillation vial to which a 50-μl volume of attractant was added at the time of stimulation. Intracellular Ca2+ concentrations were saved to file and output graphically to the personal computer monitor in real time by the recording software. Data analysis was conducted on a SUN workstation in the S statistical and graphical programming environment.
Measurement of intracellular Ca2+ responses by fluorescence microscopy using fura2-dextran.Intracellular Ca2+ concentrations in individual cells were measured using the Ca2+-sensitive fluorescent indicator fura2 coupled to dextran as previously described (46, 50). Cells were harvested after 4 to 6 h of development, washed by centrifugation and resuspended in cold Sørensen phosphate buffer (17 mM Na+/K+-phosphate, pH 6.0). Amoebae were shaken at 2 × 107 cells/ml, 150 rpm, 23°C until use. The use of fura2-dextran prevents the rapid sequestration and extrusion of the dye that occurs when either fura2 is loaded or cells are incubated with the membrane-permeant dye fura2-AM (49). Cells were loaded with fura2-dextran by electroporation, after which nonviable cells (typically 20 to 30% of the cells) and extracellular fura2-dextran were removed by repeated washing in an Eppendorf centrifuge (50). Aliquots of washed amoebae in H5-buffer (5 mM HEPES, 5 mM KCl, pH 7.0; 2 to 5 μl) on glass coverslips were placed in a humid chamber until use. Under control conditions, 85 to 88 μl of H5 buffer containing 1 mM CaCl2 was added 15 min prior to the internal Ca2+ concentration ([Ca2+]i) imaging experiment.
Measurement of the influx of Ca2+ during Ca2+ responses.Calcium influxes were measured using a Ca2+-sensitive electrode to monitor extracellular Ca2+ concentrations as previously described (5, 45). Cells were harvested and washed in Tricine buffer (5 mM Tricine/5 mM KCl, pH 7.0) and resuspended at a density of 5 × 107 cells/ml in the presence of 5 or 10 μM extracellular Ca2+. Extracellular Ca2+ levels were assayed in a gently stirred, aerated 2 ml suspension with a Ca2+-sensitive electrode (ETH 1001; Möller, Zürich, Switzerland). The minimum extracellular Ca2+ concentration occurs 30 to 40 seconds following cAMP-stimulation. The maximal rate of decrease occurs at around 20 seconds (28), close to the time of maximal intracellular [Ca2+]i elevation.
RESULTS
Ca2+ responses to chemoattractants are larger and faster in calnexin/calreticulin-deficient mutants.To investigate a possible contribution by the ER to cytosolic Ca2+ responses, we studied responses in mutants deficient in calnexin or calreticulin, both of which are abundant Ca2+-binding proteins resident in the endoplasmic reticulum (ER), calreticulin in the lumen and calnexin anchored in the membrane by a single transmembrane domain near its C-terminal end (19, 30, 31, 35). Both proteins are believed to play roles in Ca2+ buffering in the ER, suggesting that their absence should impair Ca2+ homeostasis in the ER lumen and potentially alter any ER contribution to cytosolic Ca2+ signals.
Figure 1 shows an example of real-time recordings of cytosolic Ca2+ levels in aequorin-expressing transformants of either the calnexin-deficient or the calreticulin-deficient mutant stimulated with either folate or cAMP. For both attractants the magnitude of the ensuing Ca2+ responses was noticeably larger. To verify these findings and to determine if the calnexin/calreticulin-deficient double mutant showed similarly enhanced responses, we used cells loaded with fura2-dextran to assay the cytosolic Ca2+ responses in individual cells microscopically. The results in Table 1 showed that the percentage of responding cells was similar in the mutants and the wild type, but in the cells that did respond, the amplitude of the Ca2+ transient was greater in all three mutants. The basal calcium levels also appeared slightly elevated in the mutants. There was no evidence in the double mutant for an additive effect of the absence of the two proteins.
Cytosolic Ca2+ responses by calnexin- and calreticulin-deficient mutants derived from wild-type strain AX2. Cytosolic Ca2+ levels were monitored in real time by the aequorin method in a gently stirred suspension of 108 cells during responses to 1 μM folate (vegetative cells) or 1 μM cAMP (aggregation competent cells after 6 h starvation). Recording began at 0 s and stimuli were delivered 2 s later.
Calcium responses in individual cells of the wild-type strain and calreticulin- and calnexin-deficient mutantsa
Table 2 summarizes the responses to folate and cAMP observed in the single mutants using the aequorin method to measure Ca2+ responses in populations of cells in suspension in a large number of independent experiments. The resting cytosolic Ca2+ concentrations were found not to be significantly altered in the mutants and to fall within the range normally observed with wild-type cells (40 to 100 nM). However, when the cells were stimulated with either of the attractants, cytosolic Ca2+ levels began to rise sooner and peaked earlier at higher concentrations in both mutants than in the parental strain. This is shown graphically in Fig. 2 where the prestimulus Ca2+ levels 2 seconds before stimulation have been normalized to 70 nM, and the mean Ca2+ concentrations are plotted at the mean time of onset of the response, the mean time of the response peak, as well as at 60 seconds and 70 seconds after the onset of recording. We conclude that deficiencies in either calreticulin or calnexin result in larger, faster cytosolic Ca2+ responses to chemoattractant, indicating that the endoplasmic reticulum makes a contribution to the responses.
Normalized mean Ca2+ responses by calnexin- and calreticulin-deficient mutants compared to the wild-type strain AX2. The left panel shows response to folate and the right panel shows responses to cAMP. Points indicate the mean cytosolic Ca2+ concentrations at the mean time of onset of the response, the mean time of the response peak, and at the set times of 60 and 70 s after recording started. Stimuli were delivered 2 s after recording began. Mean responses were normalized to a prestimulus resting Ca2+ concentration of 70 nM. Vertical and horizontal bars indicate standard errors of the mean for [Ca2+] and for the onset and peak response times. In some cases the horizontal bars at the onset and peak times are too short (very small standard errors) to be clearly visible in the plot.
Calcium responses to chemoattractants by wild-type cells and calreticulin- and calnexin-deficient mutantsa
Magnitude and timing of Ca2+ responses to chemoattractants are coupled.Not only were there clear differences between the mutants and the wild-type strain, but all three strains exhibited smaller, slower responses to folate than to cAMP. Folate receptors are coupled to the Ca2+ channels by a G-protein-dependent pathway while cAMP receptors in the fully aggregation competent cells used here are coupled to the Ca2+ responses by a pathway that is almost completely independent of heterotrimeric G-proteins (38). That the Ca2+ responses to cAMP are faster than the responses to folate could therefore have been due to the fact that the receptors for these two attractants are coupled to the Ca2+ channels by different signaling pathways. However this hypothesis fails to explain why the larger responses by the mutants should also be faster for both attractants. The results suggest instead that the timing of the Ca2+ response is coupled in some way to the magnitude of the response itself.
To test this idea we plotted the magnitude of the response against the time of its onset and the time at which Ca2+ levels peaked. This was done for a large number of individual experiments with cells of all three strains stimulated with folate (vegetative cells) or cAMP (aggregation competent cells). For both the onset and the peak of the response, the timing and magnitude of individual responses to the different attractants by the different strains were scattered around the same quasihyperbolic line of best fit (Fig. 3). Since larger responses begin earlier, they would be expected to peak earlier even if the Ca2+ levels rise for the same length of time during the response. In this case the rise time would be constant regardless of the onset time.
Negative correlation between response magnitudes and the times of the onset and peak of cytosolic Ca2+ responses. Open circles represent individual experiments measuring the responses of aggregation competent cells to 1 μM cAMP. Squares represent individual experiments measuring the responses of vegetative cells to 1 μM folate. Vertical error bars show means and standard errors of response amplitudes for the calnexin- and calreticulin-deficient mutants and the wild-type cells. Horizontal bars show means and standard errors for the onset and peak response times. Response magnitudes were the difference between the cytosolic [Ca2+] at the onset and the peak of the response.
Figure 4 shows that this is not so, but that there is instead a strong positive correlation between the rise time and onset time. Thus, the earlier peak associated with larger responses is due not only to an earlier onset time but also a shorter rise time involving a faster net rate of entry of Ca2+ into the cytosol from intracellular stores and/or the extracellular medium. Furthermore the measurements from individual experiments for the different strains and attractants are again scattered around the same line of best fit. Clearly the differences in response kinetics among the strains and between the two attractants can be accounted for by the differences in the magnitudes of the responses. This indicates that the timing of both the onset of the Ca2+ response and its peak are coupled to the magnitude of the response and are controlled not by upstream signaling elements but by rate-limiting events common to the cAMP and folate signal transduction pathways after they converge.
Positive correlation between onset times and rise times for cytosolic Ca2+ responses. Circles represent individual experiments measuring the response of aggregation competent cells to 1 μM cAMP. Squares represent individual experiments measuring the responses of vegetative cells to 1 μM folate. Error bars indicate means and standard errors. From left to right, the error bars indicate in order the responses to cAMP by the calreticulin-deficient, calnexin-deficient, and wild-type cells and then the slower responses to folate by the calreticulin-deficient, calnexin-deficient, and wild-type cells. Cells were stimulated at 2 s.
Uptake of extracellular Ca2+ in response to chemoattractants is reduced in calnexin/calreticulin-deficient mutants.The simplest explanation for the observed coupling between response magnitudes and kinetics is that key events in the response are Ca2+-regulated. At the onset of the response, such events could include Ca2+-induced calcium release from the ER and the opening of store-operated channels in the plasma membrane. At the peak of the response when Ca2+ levels begin to decline back to prestimulus levels, they could include Ca2+-induced closure of plasma membrane Ca2+ channels and Ca2+-mediated activation of Ca2+ pumps in the plasma membrane or endoplasmic reticulum. If the mechanisms for terminating Ca2+ responses are indeed Ca2+ induced (either directly or indirectly), then larger responses resulting from earlier, more rapid rates of increase in cytosolic Ca2+ levels would also exhibit more rapid rates of decline in Ca2+ levels after the peak.
We therefore measured the rate of change of cytosolic Ca2+ levels during the calcium responses to both chemoattractants by the wild-type and mutant strains. Figure 5a illustrates this measurement for a typical response to cAMP. Using data from all three strains for both attractants, we found a strong correlation between the maximum rates of increase in the Ca2+ concentration in the rising phase of the response and the maximum rates of decrease in the falling phase of the response (Fig. 5b). The maximal increase rates ranged from about 2 nM/second to just over 60 nM/second (ca. 0.01 to 0.30 pmol/107 cells/second), while the maximal decrease rates were smaller in magnitude so that the return to resting levels of cytosolic Ca2+ took longer than the rise time. The observed correlation between the rise and fall rates is consistent with the Ca2+ response being self-limiting because of Ca2+-induced mechanisms for response termination and Ca2+ removal.
Rates of change of cytosolic Ca2+ concentrations during responses to chemoattractant. Panel a shows an example of measurement of the rates of change of [Ca2+] during a response to an attractant stimulus (in this case, 1 μM cAMP delivered to aggregation competent cells of the calnexin-deficient mutant 2 s after recording started). The maximum rates of [Ca2+] increase during the rising phase and of [Ca2+] decrease during the falling phase of the response were measured graphically as indicated using features of the S statistics and graphics package. Panel b shows the correlation between the maximum rise and fall rates during individual experiments measuring Ca2+ responses by vegetative (to 1 μM folate) and aggregation competent (to 1 μM cAMP) cells of the calreticulin- and calnexin-deficient mutants and wild-type cells. The error bars indicate the means and standard errors, in order from left to right, for the responses to folate by the wild-type, calnexin-deficient, and calreticulin-deficient cells and then the larger, faster responses to cAMP by the wild-type, calnexin-deficient, and calreticulin-deficient cells.
One of the mechanisms that may contribute to Ca2+-induced termination of Ca2+ responses is closure of the Ca2+ uptake channels. Earlier closure of the channels in the mutants could even result in smaller Ca2+ influxes from the extracellular medium, with a greater proportion of the cytosolic Ca2+ response being contributed by calcium release from intracellular stores, particularly the endoplasmic reticulum. We therefore compared the influx of Ca2+ in response to cAMP in the parental strain and the mutants, including the double mutant lacking both calnexin and calreticulin.
The results in Fig. 6 show that at two different extracellular calcium concentrations (5 and 10 μM), stimulating the cells with any of three different cAMP concentrations produced a smaller uptake of calcium by the mutants than the wild-type strain. Indeed the Ca2+ influx after a 1 μM cAMP stimulus was reduced by more than 50%. Clearly the additional cytosolic Ca2+ observed in the mutant responses comes from intracellular sources, not from the medium. This is consistent with enhanced intracellular release of Ca2+ from the endoplasmic reticulum producing larger cytosolic responses and either earlier closure of the influx channels or earlier activation of Ca2+ pumps in the plasma membrane.
Magnitude of the Ca2+ influxes elicited by cAMP stimuli in wild-type (AX2) cells and calreticulin-deficient (Crt−) and calnexin-deficient (Cnx−) single and double (Cnx/Crt−) mutants. Response amplitudes at the time of the minimum extracellular [Ca2+] (40 s after stimulation with 0.1, 1.0, and 10 μM cAMP) are shown for cells bathed in buffer containing either 5 μM or 10 μM Ca2+. Wild-type cells (eight experiments) were starved for 5 to 7 h, the single mutants Cnx− (five experiments) and Crt− (five experiments) for 6 to 8 h and the double mutant for 7 to 10 h (four experiments). In each panel the solid circles show wild-type responses and the open circles show responses by the indicated mutant. The wild-type responses are redrawn in each row to facilitate direct pairwise comparisons with each mutant. Responses were measured using a Ca2+-sensitive electrode. Error bars represent standard deviations.
Dictyostelium genome encodes at least three putative Ca2+ pumps and four putative Ca2+ channels that may be regulated by calcium.Because the Dictyostelium genome has been completely sequenced (11), we were able to search the predicted proteome for homologs to examples of each of the subunit families of known Ca2+ channels (6, 7, 20, 22, 41, 42) and pumps (51, 52, 61). The results (Table 3) revealed that D. discoideum has genes encoding at least four putative Ca2+ channels and at least three putative Ca2+ ATPases. The subcellular locations of these pumps and channels are unknown, except for the PatA ATPase, which resides in the membrane of the contractile vacuole (34).
Dictyostelium discoideum genes encoding putative Ca2+ channels and pumps
Based on the known subcellular location of their closest homologs (20, 37, 41-43, 52, 61), it is possible to predict the organelles to which most of the others are likely to be targeted. With three identifiable P-type Ca2+ ATPases, it seems likely that D. discoideum is provisioned with a single Ca2+ pump in each of the ER and vacuolar and plasma membranes. Of the Ca2+ channels, one could reside in each of the plasma membrane (polycystin-2 homolog), late endosomes/lysosomes (mucolipin homolog), the contractile vacuole (TPC, the two-pore Ca2+ channel homolog), and the ER (IPRL).
Which of these Ca2+ channels and pumps are likely to be regulated by calcium signals? Based on recognizable Ca2+- or calmodulin-binding sequence motifs and by analogy with their regulatory properties in other organisms, most if not all of them. We found putative calmodulin-binding domains in all three Ca2+ pumps and in IPRL, while the mucolipin and TPC homologs both contained putative EF-hand Ca2+-binding sites (Table 3). Polycystin-2 and the mucolipins are Ca2+-activatable channels belonging to the TRP (Transient Receptor Potential) family, various members of which are involved in vision, olfaction, osmoregulation, thermo- and mechanoreception (8). We were unable to detect a putative Ca2+-binding site in the sequence of the Dictyostelium polycystin-2 homolog, even though all other known examples of this little studied channel contain an EF-hand Ca2+-binding motif. We conclude that D. discoideum possesses at least six putative Ca2+ pumps and channels whose sequences contain Ca2+- or calmodulin-binding motifs. This is consistent with our observation that Ca2+ responses appear to be autoregulated by the magnitude of the cytosolic Ca2+ signal.
DISCUSSION
Calreticulin and calnexin reside in the ER, where they play roles both as Ca2+-sequestering proteins and as Ca2+-dependent chaperones of N-linked glycoproteins imported into the ER and destined for subsequent targeting to various cellular destinations (19, 30, 31, 35). Whereas calreticulin is found in the lumen of the ER, calnexin is anchored to the ER membrane by a single transmembrane domain and has a regulatory cytosolic domain as well as an ER-lumenal domain related to calreticulin. In this paper we have shown that cytosolic Ca2+ responses to chemoattractants are larger in mutants deficient in calreticulin and/or calnexin. Our results thus reveal that Ca2+ release from the endoplasmic reticulum contributes to the measured cytosolic Ca2+ responses in D. discoideum. This release from the ER coincides with and is coupled to the well-characterized influx of Ca2+ from the extracellular medium.
In other organisms, two kinds of coupling between intracellular Ca2+ release and Ca2+ influx have been observed, capacitative Ca2+ entry in which release of Ca2+ depletes intracellular stores and thereby opens store-operated channels in the plasma membrane, and Ca2+-induced calcium release in which influxes of Ca2+ cause Ca2+-sensitive Ca2+ channels in the ER to open (3). Enhanced responses in mutants deficient in calnexin or calreticulin could be consistent with both kinds of coupling.
In the case of capacitative Ca2+ entry, it is known that overexpression of calreticulin in fibroblasts (29, 36) and Xenopus laevis oocytes (58) increases total Ca2+ storage capacity and attenuates calcium influx through store-operated channels. Conversely, the absence of calreticulin reduces the total calcium storage capacity of the ER (36). Thus, in the D. discoideum mutants, the ER Ca2+ stores are expected to be depleted more rapidly and extensively after an attractant stimulus. This could trigger earlier and more extensive Ca2+ entry from the extracellular medium through store-operated channels so that cytosolic Ca2+ levels begin to increase earlier and more rapidly. This hypothesis is attractive but is not readily reconciled with the findings that the null mutants exhibit reduced rather than enhanced influxes of Ca2+ from the extracellular medium.
In the case of Ca2+-induced calcium release, the greater amplitudes of the mutant responses could result from higher resting levels of free Ca2+ in the lumen of the ER. Free calcium levels in the ER (40 to 600 μM when stores are filled) are substantially higher than in the cytoplasm (0.04 to 0.11 μM) in other organisms (31). However the concentrations of bound Ca2+ in the ER are an order of magnitude higher again (1 to 3 mM) because of the presence of major Ca2+-binding proteins, including calreticulin and calnexin. The absence of one or both of these two Ca2+-binding proteins could result in even higher free calcium levels in the ER, a steeper Ca2+ concentration gradient from the ER lumen to the cytosol and an increased flux of Ca2+ into the cytosol during responses to chemoattractant. Brini et al. (4) reported such a correlation between the resting [Ca2+]ER and the extent of IP3-induced release of Ca2+ into the cytosol.
The enhancement of the Ca2+ release as [Ca2+]ER increased was found to be saturable, so that beyond a certain threshold there was no further increase in the magnitude of the Ca2+ release. This was explained by the autoregulatory nature of Ca2+ responses; inhibition of the IP3 channel by cytosolic Ca2+ appeared to limit the magnitude of the responses. A similar phenomenon could explain why we observed no synergistic effect of the calnexin/calreticulin double deficiency. Ca2+ pumps and channels in the endoplasmic reticulum are regulated by both cytosolic and ER Ca2+ and thereby contribute to homeostatic control of Ca2+ levels in both compartments. Such mechanisms could prevent the absence of both calnexin and calreticulin from having additive effects on the free Ca2+ levels in the ER. It is noteworthy that the double mutant is much more severely impaired than the single mutants with respect to other phenotypes such as phagocytosis (35), suggesting that these phenotypes may not be explicable in terms of altered Ca2+ responses alone.
Steeper transmembrane calcium gradients across cellular membranes can also result from lower cytosolic Ca2+ levels such as are produced rapidly by EGTA treatment of cells (Z. Wilczynska and P. R. Fisher, unpublished data). Under the foregoing hypothesis, brief EGTA pretreatment should enhance subsequent responses to chemoattractant and this was recently reported to be the case (48). However this finding may also be interpreted as resulting from an indirect EGTA-mediated depletion of intracellular calcium stores (48).
Resting calcium levels in the ER have been reported to be unaffected by calnexin deficiency in a T-lymphoblastoid leukemia cell line (60), by calreticulin deficiency in fibroblasts (36), and by overexpression of calreticulin in both fibroblasts and in Xenopus oocytes (36, 58). However Arnaudeau et al. (1) reported that ectopic overexpression of calreticulin caused the basal levels of free Ca2+ in the ER of HEK-293 (HeLa) cells to double, while John et al. (21) observed either no change or a decrease in [Ca2+]ER as a result of calreticulin overexpression in Xenopus oocytes. The free Ca2+ levels in the ER are the outcome of a balance between sequestration and release from high capacity binding proteins and fluxes to and from the cytosol via Ca2+ pumps and channels. Depending on the relative rate constants for these processes and the autoregulatory mechanisms controlling them, the absence of one or more major classes of Ca2+ binding protein in the ER could have different effects on Ca2+ homeostasis in different cell types. It would clearly be valuable to assay free Ca2+ levels in the ER of Dictyostelium cells to further our understanding of the roles of ER Ca2+ in Dictyostelium responses to chemoattractant. However such experiments must await the development for D. discoideum of sensitive and accurate assays for ER Ca2+ comparable to those deployed in other organisms, for example, by targeting recombinant aequorin to the ER (4).
Calcium homeostasis and ligand-induced Ca2+ responses in other cell types are influenced not only by the Ca2+-binding capacities of calnexin and calreticulin, but also by their roles as Ca2+-dependent chaperones assisting the folding and regulating the function of other proteins (19, 30). For example, the bradykinin receptors of fibroblasts are improperly folded in the absence of calreticulin, so that Ca2+ responses to bradykinin are dramatically reduced in calreticulin-deficient cells (18, 36). In D. discoideum the cAMP and folate receptors and downstream signaling proteins clearly do not require calreticulin or calnexin for their proper folding, since both attractants elicit normal chemotaxis and large calcium responses in the mutants.
In animal cells, both calnexin (44) and calreticulin (21) bind to and regulate SERCA2b (Sarco/Endoplasmic Reticulum Ca2+ ATPase 2b) which pumps cytosolic Ca2+ into the ER. This Ca2+-dependent interaction provides an inhibitory feedback mechanism that contributes to Ca2+ homeostasis in several cell types and participates in regulating cytosolic Ca2+ oscillations in Xenopus oocytes. Overexpression of calreticulin or ectopic coexpression of calnexin with SERCA2b thus dampens the oscillations in Xenopus laevis, while the absence of either protein is expected to enhance them. If calnexin and calreticulin have similar regulatory roles in D. discoideum, their removal should enhance Ca2+ responses as observed.
Not only were the amplitudes of Ca2+ responses to chemoattractants greater in mutants lacking calnexin or calreticulin, but the responses were accelerated. Compared to the responses in control cells, those in the mutants began sooner, peaked earlier, and exhibited shorter rise times and faster rates of increase in the rising phase of the response as well as faster rates of decrease in the falling phase of the response. These kinetic changes in the mutants are explicable in terms of the observed relationship between the magnitudes and the kinetics of the responses; larger responses are faster, regardless of the chemoattractant used and the presence or absence of calnexin and calreticulin. This indicates that the timing of key events such as opening and closing of Ca2+ channels and activation of Ca2+ pumps is autoregulated by mechanisms common to the signaling pathways for both the folate and cAMP receptors. Furthermore these mechanisms serve to couple the kinetics and the amplitudes of the Ca2+ responses in such a way that deficiencies in calnexin or calreticulin can affect both.
How might this coupling between Ca2+ response amplitudes and kinetics occur? The larger responses in calnexin- or calreticulin deficient mutants occur in combination with an earlier onset and a greater net rate of Ca2+ entry into the cytosol during the rising phase of the response. This indicates that during the larger responses in the mutants, a higher proportion of the responsible Ca2+ channels in the ER and/or the plasma membrane are opened. Such would be the case if Ca2+ responses are autoactivatory, as occurs in Ca2+-induced calcium influx or release and in capacitative Ca2+ entry following calcium store depletion. The shorter rise times associated with larger responses indicate that the Ca2+ responses are also autoinhibitory, that the Ca2+ flux into the cytosol is terminated sooner by Ca2+-induced channel closure and/or balanced earlier by Ca2+ activation of calcium pumps.
Consistent with this apparent autoregulation of the Ca2+ signal in chemoattractant responses, we found that the Dictyostelium genome encodes at least four putative Ca2+ channels and at least three presumptive Ca2+ pumps, most or all of them likely to be regulated by Ca2+. The sequences for six of them included recognizable motifs for Ca2+- or calmodulin-binding sites. All three of the Ca2+ pumps were related to the highly conserved and heavily studied PMCA (Plasma Membrane Ca2+ATPase) (61) and SERCA (Sarcoendoplasmic Reticulum Ca2+ATPase) (52) families and contained a helical peptide, calmodulin-binding IQ motif. It is difficult to predict the subcellular localization of these three putative Ca2+ pumps from sequence similarities as they are all more closely related to the PMCAs than to the SERCAs. PatA is known to localize to the vacuolar membrane (34), so the other two are likely to reside in the ER and plasma membranes—one in each.
Of the four Dictyostelium Ca2+ channels, only IPRL belonged to a well-studied class, the ER-resident IP3 (41) and ryanodine (20) receptors. Although its subcellular location is unknown, IPRL seems the most likely candidate for an ER channel, and has been shown to play a role in cytosolic Ca2+ signaling in response to chemoattractants (53). In our search of the predicted Dictyostelium proteome we found no putative voltage-gated (6) or cyclic nucleotide-gated (22) channels and no homologs of the better known families (TRPC, TRPM, and TRPV) within the TRP (Transient Receptor Potential) superfamily of channels (8). Instead we found, in addition to IPRL, three channels that were homologs of mucolipins (43), polycystin-2 (37) and TPC (42). The first two belong to recently recognized groups within the TRP superfamily (mucolipins or TRPML and polycystins or TRPP) while TPCs (Two Pore Channels) are distantly related to the alpha subunits of the voltage-gated channels.
The best studied of the mucolipins, mucolipin-1, releases Ca2+ from the late endosome to facilitate the Ca2+-dependent interactions between endosomes and lysosomes (43). Mucolipin-1 mutations in humans cause a lysosomal storage disease, mucolipidosis type IV, which is associated with neurological and ophthalmological defects. Mucolipin-3 is found in vesicle membranes in cochlear hair cells and melanocytes—mutations in this protein in mice cause pigmentation defects, deafness and erratic circling behavior suggestive of inner ear pathology (9). By analogy with the animal mucolipins, the Dictyostelium homolog identified here is likely to be found associated with late endocytic vesicles and lysosomes.
Polycystin-2 (or PKD2) in animals is expressed in ciliated cells where it localizes to the plasma membrane of the ciliary shaft, interacts with the mechanoreceptor polycystin-1 and mediates Ca2+ signaling in response to mechanical stimuli (37). Mutant forms disrupt the mechanosensory function of ciliated epithelial cells—in the case of those lining the nephrons of the kidney this leads to polycystic kidney disease. Dictyostelium cells are not ciliated and we were unable to find a homolog of the polycystin-1 mechanoreceptor in the Dictyostelium genome. It is possible that the Dictyostelium polycystin-2 homolog is coupled instead to chemoreceptors and mediates Ca2+ influx through the plasma membrane in response to chemoattractant instead of mechanical stimuli.
TPC, the two-pore Ca2+ channel, is found in animals and in plants, but has been studied functionally only in the latter, where it is located in the vacuolar membrane and mediates Ca2+ release into the cytoplasm in response to hormonal and other stimuli (42). If it plays analogous roles in D. discoideum it could also participate in Ca2+ responses to chemoattractants, in which case the contractile vacuole would also contribute Ca2+ to the cytosolic Ca2+ signal.
Clearly any or all of these putative Ca2+ channels and pumps could participate in Ca2+ homeostasis and signaling in D. discoideum. Their existence reinforces the fact that our present results reveal only the contribution from the ER and that other intracellular organelles are likely also to participate in controlling cytosolic Ca2+ responses to chemoattractant. Understanding their roles will require functional studies using appropriately constructed mutants and accurate in vivo assays of Ca2+ concentrations in the various subcellular compartments.
Before accurate assays of cytosolic Ca2+ levels in D. discoideum became available, it was predicted that there would be two intracellular Ca2+ responses to chemoattractant—a rapid intracellular release accompanying the reported synthesis of IP3 and a slower influx which had been revealed by measuring the uptake of 45Ca2+. This would have paralleled the findings in other cells in which a rapid IP3-induced release of Ca2+ into the cytosol depleted the ER calcium stores and was followed by a slower store-operated influx of Ca2+ from the extracellular medium (see Fig. 4 of reference 58 for an example). Nebl and Fisher (38) showed that this prediction was not correct and that the only measurable cytosolic Ca2+ response coincided with the influx and was completely dependent on extracellular Ca2+. Nonetheless there is evidence that D. discoideum contains all of the elements necessary for intracellular Ca2+ release in response to chemoattractants (26, 46, 47).
In this paper we have provided the first molecular genetic evidence that the Dictyostelium ER does indeed contribute to the elevation of cytosolic Ca2+ after a chemoattractant stimulus. We cannot determine from our results whether this ER contribution is in the form of Ca2+-induced calcium release or of an attractant-stimulated depletion of intracellular Ca2+ stores or both. Positive autoregulatory mechanisms regulating the calcium responses are implicit in both mechanisms, i.e., Ca2+ influx induces release or vice versa. The coupling we observed between the kinetics of the calcium responses and their amplitudes indicates that both positive and negative autoregulatory elements participate in regulating the calcium responses. Further molecular genetic dissection is required to unravel these mechanisms that control chemoattractant-induced calcium responses in Dictyostelium.
ACKNOWLEDGMENTS
We are grateful to Frank Zucchetti for his expert technical assistance.
We are grateful to the Deutsche Forschungsgemeinschaft for financial support.
FOOTNOTES
- Received 19 April 2005.
- Accepted 17 June 2005.
- Copyright © 2005 American Society for Microbiology
REFERENCES
