Ca2+ regulation in the absence of the iplA gene product in Dictyostelium discoideum

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

iplA^- cells have an altered pattern of development. Differentiation of the mutant and the wild type strain was assayed in parallel on agar plates. Cells at different time points of development on H5-agar are shown. Wild type amoebae aggregated at t₇, whereas aggregation of the mutant strain was delayed and aggregation strands broke (t₁₀); therefore, smaller fruiting bodies were formed as compared to the wild type. The full width of the image corresponds to 12.5 mm.

Figure 2

Development of iplA^- cells is impaired by depletion of internal Ca²⁺-stores due to EGTA-treatment. Differentiation of the wild type and the mutant on plates containing 20 mM EGTA is shown. Aggregation was delayed in both strains till t₁₅ and t₁₈ in wild type and iplA^- cells, respectively. The size of the aggregates and the fruiting bodies were much smaller than under control conditions. The full width of the image corresponds to 12.5 mm.

Figure 3

In the presence of external Ca² aggregation is accelerated in iplA^- cells. Differentiation of the mutant and the wild type strain was assayed in parallel on agar plates supplemented with 20 mM CaCl₂. Aggregate formation occurred earlier in iplA^- cells (at t₇) than in wild type (starting at t₁₉) in the presence of Ca²⁺. The full width of the image corresponds to 12.5 mm.

Figure 4

General morphology of wild type and iplA^- cells under control conditions (A, B), in the presence of 10 mM EGTA for 60 min (C, D) or in the presence of 10 mM CaCl₂ for 80 min (E, F). In H5-buffer or in the presence of 10 mM CaCl₂ the morphology was not significantly different between wild type and mutant amoebae. However, in the presence of EGTA the cells of both strains were rounded. Photographs were taken at t₅. Basal motility under these conditions can be viewed in the accompanying movies.

Figure 5

Chemotaxis of wild type and mutant amoebae at different experimental conditions. The tracks of individual cells (in red) migrating during chemotactic stimulation (position of the tip of the cAMP-filled capillary: green star) are shown. In H5-buffer (A, B) both cell types migrated in an oriented manner towards the capillary tip, albeit not always in a straight line. After preincubation with 10 mM EGTA for 60 min and in its continued presence during the chemotaxis assay (C, D) the cells remained stationary with random pseudopod extension. Preincubation of amoebae with 10 mM CaCl₂ (E, F) did not impair chemotaxis; rather, the cells of both strains migrated towards the capillary tip. Chemotaxis experiments were done at t₆.

Figure 6

Chemotaxis of iplA^- cells is impaired in the intracellular presence of a Ca²⁺-buffer. Wild type and mutant amoebae were loaded with Fura2-dextran and their ability to protrude pseudopods towards a cAMP-filled glass capillary was compared to that of untreated cells. In both strains the presence of the chelator in the cytosol led to a decrease in the fraction of cells extending pseudopods and migrating towards the cAMP source.

Figure 7

Recordings of Ca²⁺-fluxes in iplA^- and wild type amoebae. [Ca²⁺]_e was measured in cell suspensions with a Ca²⁺-sensitive electrode. (A) Treatment of amoebae with 5 mM EGTA for 30 min activated capacitative Ca²⁺-influx (one out of 12/6 determinations in 4/3 independent experiments is shown for iplA^- and wild type, respectively). (B) Capacitative influx was blocked by the addition of 1 mM NaN₃ (one out of 5/4 determinations in 3/3 independent experiments). Measurements were done at t₃.

Figure 8

Agonist-activated Ca²⁺-fluxes in suspensions of iplA^- and wild type amoebae. cAMP elicited reversible Ca²⁺-influx in the mutant (A) and in wild type ((B) and see [14]); measurements were done at t₇–t_7.5. Note the different doses of CaCl₂ added for calibration. The time points of cAMP-addition (1 μM) and of AA-addition (6 μM) in the wild type are indicated by arrows.

Figure 9

Fatty acids activate Ca²⁺-fluxes in iplA^- amoebae. (A) 60 μM AA evoked a transient decrease in [Ca²⁺]_e representing Ca²⁺-influx; measurement was done at t₆ (B) After preincubating amoebae with the SERCA-type Ca²⁺-ATPase blocker BHQ (100 μM) for 20 min to inhibit uptake of Ca²⁺ into internal storage compartments, the AA-activated response was absent; measurement was done at t_7.5. Results of measurements with wild type (C, D) stimulated with 6 μM AA are shown for comparison.

Figure 10

cAMP and arachidonic acid elicit Ca²⁺-release from internal stores. (A) [Ca²⁺]_e was recorded at t₇ in iplA^- cells with permeabilized plasma membranes. Amoebae were challenged with 1 μM cAMP and 3 μM AA, respectively. (B) The response of permeabilized wild type stimulated with 1 μM cAMP at t₆ is shown for comparison.

Figure 11

Basal and cAMP-induced Mn²⁺-influx. Influx was assayed by quenching of Fura2-dextran fluorescence. (A, B) The response of wild type amoebae is shown for comparison; 1 μM Mn²⁺ ± 1 μM cAMP was added. iplA^- cells in nominally Ca²⁺-free buffer were challenged with 100 μM Mn²⁺ ± 1 μM cAMP at t₇ (closed symbols); when 1 μM Mn²⁺ was added (open symbols) no influx was detected (C, D). After preincubation with EGTA influx was observed at 1–2 μM Mn²⁺ ± 1 μM cAMP (E, F). Fluorescence intensity at 360 nm excitation is shown as mean ± s.e.m

Figure 12

[Ca²⁺]_i-recordings in cells preincubated with CaCl₂ in order to load stores. (A) The response of wild type upon stimulation with 1 μM cAMP (arrow) at standard conditions, i.e. after preincubation with 1 mM CaCl₂ for 10–15 min and stimulated in the presence of 1 mM CaCl₂ is shown. Values give mean ± s.e.m. of 7 cells. (B) When iplA^- cells were stimulated with 1 μM cAMP at standard conditions (as outlined in (A)), no response was observed. Mean ± s.e.m. of 6 cells is shown. (C) Wild type was preincubated with 1 mM CaCl₂ for 4–5 h; after washing cells were incubated in H5-buffer supplemented with 1 μM CaCl₂ and challenged with 1 μM cAMP (arrow). Values give mean ± s.e.m. of 16 cells. (D) iplA^- incubated for 3 h with 20 mM CaCl₂ were washed and subsequently [Ca²⁺]_i-imaging was done in buffer containing 1 mM CaCl₂. Arrow indicates the time point when 1 μM cAMP was added. Mean ± s.e.m. of 10 cells is shown.