The effects of extracellular calcium on motility, pseudopod and uropod formation, chemotaxis, and the cortical localization of myosin II in Dictyostelium discoideum

Abstract

Extracellular Ca(++), a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity, and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca(++) is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca(++) only affects turning, but in combination with extracellular Ca(++), enhances the effects of extracellular Ca(++). Potassium, at 40 mM, can partially substitute for Ca(++). Mg(++), Mn(++), Zn(++), and Na(+) cannot. Extracellular Ca(++), or K(+), also induce the cortical localization of myosin II in a polar fashion. The effects of Ca(++), K(+) or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca(++). These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1

Extracellular calcium regulates velocity and turning in the absence of chemoattractant. Cells were analyzed in a Sykes-Moore perfusion chamber. All test solutions were made in tricine buffer (TB) which contained 5 mM K⁺, pH 7.0. Test solutions tested were tricine buffer plus 5 mM EGTA (EGTA), or tricine buffer containing 0 to 40 mM CaCl₂. Error bars represent the standard deviation of the means. At least 10 cells were analyzed in each of three independent experiments and the data pooled.

Figure 2

Extracellular calcium regulates the length of perimeter tracks and cell shape in the absence of chemoattractant. See legend to Figure 1 for details. Cell perimeters were obtained at 4 sec intervals for 10 min and smoothed with Tukey windows (Soll, 1995). Outlines were drawn every 12 sec. Insets in the lower right of each panel show shapes at the end of the track for the representative cells. The numbers refer to the individual cells monitored. Arrows indicate average direction of transduction. TB, tricine buffer.

Figure 3

3D reconstructions using 3D-DIAS software reveal that extracellular calcium regulates cell shape, pseudopod dynamics, uropod formation, adhesion to the substratum and uropod formation, in the absence of chemoattractant and in a gradient of cAMP gradient. A through D. Representative 3D reconstructions in test solutions in the absence of chemoattractant. E through H. Representative 3D reconstructions in test solutions in the presence of a spatial gradient of cAMP. In each case the cell is viewed at two time points and three different angles (90°, 15°, 0°). Blue represents cell body; yellow represents pseudopods; s, seconds. Black arrows represent the direction of the cAMP gradient in panels E through H.

Figure 4

Extracellular calcium regulates lateral pseudopod formation in the absence of cAMP (A) and in a spatial gradient of cAMP (B). Pseudopods were identified and counted as described in Methods section. The mean is presented for 10 cells at each concentration. The standard deviation was less than 50% of the mean for all data points ≥ 2.4, and no greater than the mean for all data points ≤ 1.3.

Figure 5

Extracellular calcium regulates velocity, turning and the efficiency of chemotaxis in a spatial gradient of cAMP. Cells were analyzed in a spatial gradient chamber. See legend to Figure 1 for details of basic test solutions. Error bars represent the standard deviations of the means. At least ten cells were analyzed in each of three independent experiments and the data pooled.

Figure 6

Extracellular calcium regulates localization of myosin II in the cell cortex. Cells were stained with anti-myosin II antibody, and a confocal microscope projection image derived from the center five sections analyzed for pixel intensity through a zig-zag scan. The anterior and posterior ends of amorphous cells were identified by the location of tail probes (Heid et al., 2005). Percent maximum intensity was computed from the most intense point (100%) in the scan. Data for the anterior half of the cell is dark green, and that for the posterior half light green. A through D. Scans for representative cells in test solution in the absence of cAMP. The zigzag track of the scan is presented in the dark box and the percent maximum intensity is plotted along the zigzag (“pixel”) scan for each analyzed cell. E. Average staining intensities of the cortex of the anterior and posterior halves of five test cells as a function of CaCl₂ concentration in TB. F. Percent maximum intensities of the cortex of the anterior and posterior halves of five test cells as a function of CaCl₂ concentration in TB. G. Ratio of the percent maximum intensity of posterior to anterior half as a function of CaCl₂ concentration in TB. Error bars represent standard deviations of the means.

Figure 7

Extracellular potassium substitutes for extracellular calcium in the presence of a spatial gradient of cAMP. A through D. A comparison of the effects of the buffered salt solution BSS, and TB containing 0 and 20 mM CaCl₂, 20, 40 and 60 mM KCl and 20 mM NaCl₂. The main cation in BSS is 40 mM K⁺, it contains no added Ca⁺⁺. E through H. A comparison of the effects of divalent cations on behavior and chemotaxis. With the exception of BSS, all test solutions but BSS contained TB plus the noted concentration of each monovalent or divalent cation. I through L. Analysis of the effects of pH. pH was adjusted in TB buffer in the presence of 10 mM CaCl₂. Cells were analyzed in a spatial gradient of cAMP. Error bars represent standard deviations of the means.

Figure 8

Extracellular CaCl₂, KCl and a spatial gradient of cAMP induce increases in free cytosolic Ca⁺⁺. Cells were loaded with Fura-2-dextran then analyzed by fluorescent imaging methods in test solutions. All data points represent the mean of the relative free cytosolic Ca⁺⁺ normalized to the highest data point obtained at 10 or 20 mM CaCl₂ in each of five independent experiments. The error bars represent the standard deviation of the means for the five experiments. A. Relative free cytosolic Ca⁺⁺ in TB containing 0 to 40 mM CaCl₂. B. Relative free cytosolic Ca⁺⁺ in TB containing 40 mM KCl. C. Relative free cytosolic Ca⁺⁺ in a spatial gradient of cAMP (open circles), and in the absence of cAMP (closed circles) in 0 and 10 mM CaCl₂.