Ca2+ regulation in the absence of the iplA gene product in Dictyostelium discoideum
© Schaloske et al; licensee BioMed Central Ltd. 2005
Received: 12 November 2004
Accepted: 11 March 2005
Published: 11 March 2005
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© Schaloske et al; licensee BioMed Central Ltd. 2005
Received: 12 November 2004
Accepted: 11 March 2005
Published: 11 March 2005
Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA - mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis.
We investigated Ca2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA - cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA - than in wild type amoebae.
These results support the view that [Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA - strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure.
Aggregation of Dictyostelium discoideum proceeds by an oriented migration of the amoebae towards a source of the attractant cAMP which is synthesized and released periodically by cells in the center of the aggregate. Stimulation with cAMP activates liberation of stored Ca2+ and extracellular Ca2+-entry  leading to a [Ca2+]i-transient [2–4]. Chemotaxis proceeds in the presence of extracellular EGTA but not in the presence of intracellular Ca2+ buffers, so a [Ca2+]i-elevation is necessary and release of stored Ca2+ is sufficient for oriented migration . On the other hand, the view that a [Ca2+]i-increase is essential for chemotaxis was called into question by analysis of a cell line where the iplA gene was disrupted by homologous recombination . The iplA gene is the only gene known in the Dicyostelium genome so far that shares homology with IP3-receptors of higher eukaryotes. However, whether its protein product indeed constitutes a functional IP3-receptor and its cellular localization are not known. The iplA - mutant was found to aggregate and to form fruiting bodies although neither cAMP-activated 45Ca2+-entry nor a [Ca2+]i-elevation were detected . From these results the authors concluded that the iplA gene product is central to the regulation of [Ca2+]i and that its presence and thus the presence of an agonist-activated [Ca2+]i-increase is not necessary for proper chemotaxis and development. However, agents that interfere with IP3-receptor mediated signaling such as XestosponginC  were found to influence not only cAMP-induced Ca2+-fluxes but also the chemotactic response and aggregation of Dictyostelium . In this study we aimed to clarify these conflicting findings and analyzed both, capacitative and chemoattractant-induced Ca2+-fluxes and the effect of their disturbance on chemotaxis and differentiation of the iplA - mutant. Mn2+-influx was used to determine the rates of ion fluxes into cells with filled and emptied stores and related to Ca2+-electrode recordings in cell suspensions. We found that ion fluxes, chemotaxis and differentiation were sensitive towards alteration of the Ca2+-homeostasis. Capacitative Ca2+-entry was normal in the mutant and upon stimulation with agonist Ca2+- and Mn2+-fluxes occurred, yet to a considerably reduced extent. Spontaneous motility and chemotactic performance of mutant amoebae was strongly impaired by the intracellular presence of a Ca2+-chelator.
Determination of Ca2+-fluxes in partially purified storage compartments of the iplA - mutant and of wild type. Ca2+-sequestering vesicles were prepared as outlined in Methods. Measurements were performed with the pellet and supernatant fraction. ATP-induced uptake and release activated by different agents is given as nmol Ca2+-uptake/min and mg of protein and pmol Ca2+-release/tube, respectively (mean ± s.d.). In release experiments 60–75 μl of pellet and 120–140 μl of supernatant fraction were used per tube. Numbers in brackets give number of experiments; n.d.: not determined.
1 mM ATP
1.96 ± 0.55 (4)
0.28 ± 0.15 (4)
1.87 ± 0.74 (3)
0.38 ± 0.16 (3)
10 μM AA
360 ± 227 (8)
761 ± 218 (6)
396 ± 122 (6)
961 ± 374 (2)
40 μM Thapsigargin
570 ± 250 (6)
170 ± 73 (6)
582 ± 123 (5)
265 ± 135 (2)
6 μM XestosponginC
201 ± 55 (4)
276 ± 46 (3)
2 μM Ionomycin
447 ± 147 (5)
147 ± 41 (4)
580± 173 (3)
193 ± 76 (2)
Rate of basal and cAMP-induced Mn2+-influx. Amoebae were challenged with 1 or 100 μM Mn2+ either with or without 1 μM cAMP. Cells were preincubated with 0.1 mM EGTA as outlined in Methods. Mn2+ quenching of fura-2-dextran fluorescence was tested in H5-buffer and is expressed as decrease in fluorescence units/sec (mean ± s.e.m.). Numbers in brackets give number of cells tested and number of experiments.
EGTA (0.1 mM)
1 μM Mn2+
0.37 ± 0.2 (142/3)
1 μM Mn2+/1 μM cAMP
1.4 ± 0.1 (207/4)
100 μM Mn2+
100 μM Mn2+/1 μM cAMP
1.0 ± 0.2 (292/9)
In principle, the iplA gene product could form a channel in the plasma membrane or in membranes of internal stores. The lack of the iplA gene product in the stores might impair their coupling to the plasma membrane. As we had observed capacitative Ca2+-entry in the mutant we asked whether manipulation of the filling state of the stores altered ion fluxes. First we tested the effect of emptying of stores on Mn2+-influx. When cells were preincubated with EGTA, the requirement for high doses of Mn2+ to quench fluorescence was abrogated. Now capacitative and also agonist-activated Mn2+-influx occurred at concentrations of MnCl2 comparable to those used under control conditions in wild type, in the range of 1–2 μM (Fig. 11 E, F). This result renders the possibility that the plasma membrane is altered in the mutant unlikely. Yet, the rate of Mn2+-influx observed in EGTA-treated mutant amoebae was still less than in wild type cells with respect to both basal and cAMP-activated fluxes (53 and 58% of wild type , respectively).
For stronger loading of Ca2+-stores, we preincubated cells with 1 mM CaCl2 for 4 h. After this treatment a [Ca2+]i-elevation upon addition of cAMP was detected in 60% of wild type amoebae even at low extracellular [Ca2+] levels, i.e. when the buffer used to wash the cells had been supplemented with only 1 μM CaCl2 (Fig. 12 C). Starting from a basal level of 48 ± 4 nM, the height of the [Ca2+]i-transient amounted to 23 ± 2 nM (mean ± s.e.m. of 12 determinations in 8 independent experiments). Again, these conditions were not effective in iplA - cells; rather, the sensitivity of the mutant was shifted to higher Ca2+ concentrations as had been found with Mn2+-quenching experiments. We preincubated iplA - amoebae with 20 mM CaCl2 for 3 h; after washing thoroughly, a cAMP-induced [Ca2+]i-elevation in the presence of 1 mM CaCl2 was observed (Fig. 12 D) in 28% of the cells; its average height amounted to 67 ± 11 nM (mean ± s.e.m. of 15 determinations in 4 independent experiments) starting from a basal level of 39 ± 2 nM. In the course of these experiments we once observed a response also under standard conditions, i.e. at 1 mM [Ca2+]e without prior incubation in 20 mM CaCl2; the height of the increase amounted to 54 ± 6 nM (mean ± s.e.m.). Yet, this was a rare event (once in 31 determinations).
The role of cAMP-activated [Ca2+]i-changes for chemotaxis has been questioned by Traynor et al.  who reported results obtained with the iplA - mutant cell line favouring insignificance of [Ca2+]i for the chemotactic response. The authors had observed formation of fruiting bodies even though neither 45Ca2+-fluxes nor an agonist induced [Ca2+]i-elevation were detectable. The discrepancy between our view that a [Ca2+]i-elevation is necessary for a proper chemotactic response  and the conclusion of Traynor et al. prompted us to analyze chemotaxis, differentiation and the [Ca2+]-regulation of the iplA - mutant in detail. In particular, we tested not only basal and cAMP-activated ion fluxes but also capacitative Ca2+-entry which is induced by emptying internal Ca2+-stores via preincubation of amoebae with EGTA .
Aggregation and development of wild type and mutant cells on agar plates was sensitive towards continuous emptying or loading of Ca2+-stores. These effects are not necessarily caused by altering chemotactic migration. It is conceivable that other Ca2+-dependent processes were affected, e.g. that the timing or pattern of gene expression or the establishment of cell contacts was altered. Although incubation of mutant amoebae for 2 h with 20 mM CaCl2 or of wild type with 1 mM CaCl2 for 4–5 h or with 1 mM EGTA for 1 h  did not significantly increase or lower basal [Ca2+]i it is possible that the continued presence of 10 mM EGTA or CaCl2 for many hours affects basal levels of [Ca2+]i which in turn might mediate effects on gene expression as was shown for prolonged incubation of cells with BHQ . During development Dictyostelium cells form Ca2+-dependent and EDTA/EGTA-sensitive cell-cell contacts that are mediated by gp24 and DdCAD-1 ([19, 20]; for review see ). Therefore, chelation of extracellular Ca2+ might also inhibit cell adhesion. However, mutant cells whose gene encoding DdCAD-1 had been disrupted show normal chemotaxis and cell streams. Furthermore, mound formation was accelerated and only culmination was delayed by about 6 hours . If only Ca2+-dependent cell adhesion was affected in our development assay in the presence of EGTA we would expect a similar phenotype. However, aggregation was clearly delayed. This argues for additional Ca2+-dependent processes during aggregation.
When we tested the influence of the presence of EGTA or CaCl2 on spontaneous motility and chemotaxis we found that in both strains motility in general was strongly impaired and that chemotaxis of the amoebae towards the cAMP-filled glass capillary was virtually abolished upon depletion of internal stores by the extracellular presence of EGTA. This effect is time dependent; after 30 min of incubation with 10 mM EGTA the behaviour of wild type amoebae was found to be unaltered and only after treatment for 0.5–1 h rounding and reduction of pseudopod elongation towards the capillary tip occurred . Prolonged incubation for more than 1 h as carried out in this study completely inhibited the chemotactic response. These results strengthen the view that Ca2+ has a necessary role in chemotaxis in wild type and in the mutant as well. When the cellular Ca2+ content falls below a critical value Ca2+-dependent cytoskeletal rearrangements [22, 23] that are necessary for both, random pseudopod extension during spontaneous motility and oriented pseudopod formation after chemotactic stimulation no longer take place correctly. On the other hand, the presence of 10 mM CaCl2 induced no alteration of basal cell motility in wild type or mutant amoebae. Yet, during chemotactic stimulation the average speed of migration towards the capillary tip was higher in mutant than in wild type cells. In this respect it is of importance that the basal level of [Ca2+]i was significantly lower in the former. At standard conditions the reduced basal [Ca2+]i does not impair the capacity of the mutant to chemotax. Therefore, this particular mutant strain represents the "minimal solution" with respect to the concentration of cytosolic Ca2+ necessary to accomplish cytoskeletal rearrangements and extrusion of a pseudopod correctly. However, in the presence of 10 mM extracellular Ca2+ during cAMP-stimulation Ca2+-fluxes are enhanced allowing more efficient formation of pseudopods. We had shown previously that a small global elevation of [Ca2+]i activates the extension of pseudopods all over the cell's circumference whereas a larger increase induces contraction of the amoebae . In our view, the strongest evidence that a [Ca2+]i-transient is necessary for the extension of pseudopods rests upon the experiment where a Ca2+-chelator was introduced into the cytosol of the amoebae. This treatment led to rounding of the amoebae and a general reduction of pseudopod formation (see also ). Upon stimulation with a cAMP-filled capillary, the extension of oriented pseudopods was greatly reduced and migration towards the capillary tip was abolished. As Speksnijder et al.  had pointed out the fact that the presence of a chelator has an effect shows that a [Ca2+]i-gradient is essential for a given response. In summary, these data support the notion that an elevation of [Ca2+]i is required to extend pseudopods; suppression of the [Ca2+]i-elevation inhibits motility in general. Upon chemotactic challenge with cAMP this [Ca2+]i-gradient has to be established in a locally restricted fashion in order to allow local, oriented pseudopod formation (see [2, 25, 26]); otherwise pseudopods would be extended in all directions (see above, ). Our results imply that in iplA - cells such a [Ca2+]i-gradient occurs as well, either nonrestricted allowing extension of pseudopods at random sites during spontaneous motility or restricted locally after chemotactic stimulation leading to oriented pseudopod formation. The fact that in the mutant cell line cAMP-activated [Ca2+]i-changes were practically undetectable under our standard condition argue for a [Ca2+]i-increase that is either smaller and/or more restricted to distinct domains within the cell than in wild type amoebae. Indeed, in only one out of roughly 30 determinations did we observe a cAMP-activated [Ca2+]i-transient under standard conditions. These results imply a crucial role but not an absolute necessity of the iplA gene product for the regulation of cAMP-induced [Ca2+]i-changes.
By using a Ca2+-sensitive electrode in cell suspensions, we analyzed which aspects of [Ca2+]i are controlled by the iplA gene product. Besides studying agonist-induced Ca2+-fluxes we also investigated capacitative Ca2+-entry and found that this type of influx was similar in mutant and wild type cell suspensions. We obtained equivalent results by testing Mn2+-quenching of Fura2-dextran fluorescence which showed that capacitative entry is independent of the iplA gene product.
On the other hand, using the Ca2+-sensitive electrode, we found that in the iplA - mutant the agonist cAMP and also AA did activate Ca2+-entry into intact cells. The difference between the data published by Traynor et al.  and our results is most likely due to the experimental conditions: the magnitude of the Ca2+-fluxes that we observed was considerably lower than in wild type cells and detectable at low extracellular [Ca2+] only. The 45Ca2+-flux studies had been performed at 100 μM external CaCl2; so the fraction of 45Ca2+ entering the cells was presumably too low to be detected reliably. Moreover, we found cAMP- and AA-induced Ca2+-release from stores in cells with permeabilized plasma membranes. These data show that cAMP-induced Ca2+-release from stores in iplA - cells is functional. However, much like the Ca2+-influx, agonist-activated liberation from stores was smaller than in wild type amoebae. In line with these results are the findings using Mn2+-quenching to assay ion fluxes in intact single cells: higher doses of Mn2+ were necessary to detect influx.
There are several interpretations for the results above. (i) There are two types of channels responsible for Ca2+-influx: one type being activated by emptying of the stores and sustaining capacitative Ca2+-entry which is unaffected in iplA - cells and the other one mediating agonist-induced Ca2+-fluxes, the latter being under the control of the iplA gene product. The view that there are two strictly separated ion channels seems unlikely as under conditions of emptied stores cAMP-activated Mn2+-quenching occurred in the mutant as well. (ii) The same channel(s) mediate capacitative and agonist-activated fluxes but upon stimulation with agonists it cannot be addressed properly when iplA is disrupted. This implies a role of the protein in the liberation of Ca2+ from the stores which is a prerequisite for the triggering of Ca2+-entry . In the mutant this cannot proceed normally so subsequent activation of Ca2+-influx and the generation of a full [Ca2+]i-increase is impaired. The results of the experiments where stores were strongly loaded with Ca2+ prior to stimulation support this notion. In this situation release from stores should be augmented. Indeed, in both, wild type and mutant cells, cAMP-activated [Ca2+]i-elevations occurred at an extracellular [Ca2+] (see Fig. 12) where without pretreatment no increase was observed. Presumably, release of Ca2+ from the filled stores contributed to the observed [Ca2+]i-increase to a greater extent than under standard conditions. The requirement for 20 fold higher concentrations of CaCl2 during preincubation to elicit an agonist-induced [Ca2+]i-elevation in iplA - cells are most likely due to the reduction in Ca2+-entry which necessitates a higher concentration gradient across the plasma membrane to fill the stores efficiently.
An as yet unresolved issue is the mechanism that induces Ca2+-entry upon liberation of Ca2+ from the stores. From our data we conclude that in Dictyostelium these signals are different when the stores are emptied by EGTA or by agonist-activated signaling cascades. Otherwise one cannot explain normal capacitative Ca2+- and Mn2+-influx induced by EGTA-treatment and a requirement for 100 fold higher ion concentrations to induce Mn2+-entry by cAMP. If indeed the iplA gene product constitutes an IP3-receptor like channel that is located on membranes of stores the physical coupling of the receptor to channels in the plasma membrane as a mechanism to activate extracellular Ca2+-entry  should be missing in the mutant. On the other hand, emptying of stores by EGTA-treatment influences not only the IP3-sensitive store but also other stores and thus exerts a much more general effect on the cells. Studies using microarrays should reveal whether the expression of other genes is affected by the absence of iplA and thus might give a clue how [Ca2+]i is regulated in the mutant although one type of Ca2+-store is malfunctional.
Our results show that Ca2+ fluxes and regulation of Ca2+ homeostasis take place in the iplA - mutant and that chemotaxis and development of the mutant are sensitive to disturbance of the Ca2+ homeostasis. In wild type cells and in cells lacking the iplA gene changes in [Ca2+]i are necessary to orient and to migrate chemotactically; their abolition causes loss of chemotaxis towards a cAMP source. The iplA gene product exerts a crucial role in the control of basal [Ca2+]i and of agonist induced Ca2+-fluxes. It is not required to activate capacitative Ca2+-influx. Thus the mechanisms responsible for capacitative and agonist-activated Ca2+-fluxes are different.
Fura2-dextran and Fura2 were purchased from MobiTec; cAMP was from Boehringer.
D. discoideum wild type strain Ax2 and the iplA - cell lines HM1049 and HM1038 (kindly provided by Dr. D. Traynor) were cultured as described  in the absence or presence of 10 μg/ml Blasticidin S, respectively. There was no difference between the two mutant strains with respect to the assays performed; therefore, results of measurements with either HM1038 or HM1049 are shown. Cells were washed by repeated centrifugation and resuspension in cold Sørensen phosphate buffer (17 mM Na+/K+-phosphate, pH 6.0). Amoebae were shaken at 2 × 107 cells/ml, 150 rpm and 23°C until use. The time, in hours, after induction of development is designated tx.
[Ca2+]e in cell suspensions was recorded as described elsewhere . Cells at t5–t8 were washed by repeated centrifugation and resupended at 5 × 107 cells/ml in 5 mM Tricine, 5 mM KCl, pH 7.0. Permeabilization was done by addition of filipin (15 μg/ml) to cell suspensions exactly as outlined in . Capacitative Ca2+-influx was analyzed in cells with emptied storage compartments : amoebae at t2–t4 were incubated with 5 mM EGTA for 30 min before washing in the above buffer.
Cells were loaded with Fura2-dextran (5 mg/ml + 1 mM CaCl2) at t4–t5 as described . Aliquots (2–5 μl) of washed cells in H5-buffer (5 mM Hepes, 5 mM KCl, pH 7.0) were placed on glass coverslips and incubated in a humid chamber. 10–15 min prior to the experiment, 85–88 μl of H5-buffer + 1 mM CaCl2 were added. In a series of experiments to load stores, wild type and iplA - cells were incubated with 1 mM CaCl2 for 4–5 h and with 20 mM CaCl2 for 2–3 h, respectively. Then they were thoroughly washed exactly as described previously  and incubated either in H5-buffer supplemented with 1 μM CaCl2 (wild type; free [Ca2+] in the solution was measured to be 2–2.5 μM, see also ) or in H5-buffer +1 mM CaCl2 (iplA -); final volume was 90 μl. Single cell [Ca2+]i-imaging was performed at t7–t8 as described ; stimulation was done by adding 10 μl of cAMP (10 μM). For Mn2+-quenching assays, washed cells were incubated in H5-buffer and challenged with Mn2+ or Mn2+/cAMP. In order to study fluxes in cells with partially emptied internal storage compartments cells were preincubated with EGTA (10 μl of H5-buffer plus 0.1 mM EGTA for 1–2 h). 10–15 min prior to the experiment this solution was carefully removed and 100 μl of H5-buffer was added. This was repeated three times; final volume was 90 μl. Fluorescence quenching was measured at 360 nm excitation; influx rates are given as decrease of fluorescence units/sec.
Analysis of vesicular Ca2+-fluxes was done as described . In brief, 3 ml of cells at t1–t6 (2 × 108 cells/ml) in 20 mM Hepes, pH 7.2, were lysed by passage through Nuclepore filters. A final concentration of 3 % sucrose, 50 mM KCl, 1 mM MgCl2, 20 μg/ml leupeptin, 1 μg/μl aprotinin, 2.5 mM dithiothreitol and 1 μM microcystin were added; unbroken cells were removed by centrifugation at 3000 g for 5 min. The supernatant was centrifuged again at 12000 g for 20 min. The sediment (P) was resuspended in 1 ml of the above buffer. The rate of uptake and release was determined in the pellet and supernatant fraction by measuring the extravesicular [Ca2+] with Fura2.
Cells were analyzed for chemotaxis towards a capillary filled with 0.1 mM cAMP . 250 μl of 1 × 105 cells/ml in H5-buffer were pipetted onto a glass coverslip and allowed to settle for 60 min. Chemotaxis was recorded on a video recorder for 30–45 min. Chemotaxis was also assayed in the presence of EGTA or CaCl2; then amoebae were incubated in the respective agents for 60 min before they were challenged with cAMP. Images were digitized and the behaviour of the cells was analyzed using a computer program written for this purpose. For determination of cell velocity, a square area of interest (AI) of variable size (usually roughly 1/3 of the area of the cell) was placed at the perimeter of the cell in the first image digitized at the beginning of the assay. In the next image (images were digitized at a 2–4 sec time interval) the program analyzed an area larger than the AI (this area was defined by adding a given number of pixels on each side of the AI) for a pattern that resembled that of the AI; when such a pattern was found then the AI was placed on this new spot. The difference between the position of the AI in the first image to that in the second image was expressed as a vector of a given length. The changes in cell shape during migration were compensated by updating the pattern within the AI for every consecutive image analyzed. Calibration of the system allowed to convert the sum of the vector lengths to the distance in μm that the cells had migrated at the end of the experiment and to calculate the velocity of the amoebae. To test the effect of the intracellular presence of a Ca2+-buffer on chemotaxis, cells were loaded with Fura2-dextran (5 mg/ml in the loading solution) by electroporation in the absence of added external Ca2+. The amount of indicator present in the cytosol is in the range of 2–5% of the concentration present during electroporation . 20 min after loading, cells were stimulated for 3–4 min by placing the cAMP-filled glass capillary at a distance of 10–20 μm of the cells and the number of cells that extended oriented pseudopods and thus elongated towards the capillary tip within this time period was counted. We had shown previously that loading of amoebae with FITC-dextran as a control does not alter chemotaxis as compared to untreated cells .
Time lapse recordings of the development of Ax2 and iplA - cells on 1.5 % agar in H5-buffer (H5-agar) were done by placing 4 × 106 cells each on one half of a petri dish (∅ 35 mm) at t1. The two populations were separated from each other by a thin plastic disc that had been inserted in the melted agar during cooling. Only after removal of fluid and slight drying of the plate the disc was removed which resulted in a thin rim separating the strains. Differentiation was recorded by capturing an image of the plate every 30 min using a stereo microscope (Stemi 2000, Zeiss) equipped with a CCD camera (AVT Horn) under the control of the AxioVision software package (Zeiss). In addition, development was assessed at various levels of extracellular CaCl2. Then H5-agar contained either 5–20 mM EGTA or 5–20 mM CaCl2.
Cytosolic free Ca2+ concentration
Area of interest
The authors wish to thank Dr. D. Traynor and Prof. R. Kay for generously providing the iplA - strain and Rupert Mutzel and Gerd Knoll for many helpful discussions and critical reading of the manuscript. We are indebted to Georg Heine and Williams Pascual from the Wissenschaftliche Werkstätten of the University of Konstanz for computer programming and to Katja Drews, Cyrus Nassiri and Frank Zucchetti for performing initial Ca2+-electrode measurements. This work was supported by the Deutsche Forschungsgemeinschaft.
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