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. 2012 Apr 1;125(Pt 7):1770-83.
doi: 10.1242/jcs.098301. Epub 2012 Feb 28.

The IplA Ca2+ channel of Dictyostelium discoideum is necessary for chemotaxis mediated through Ca2+, but not through cAMP, and has a fundamental role in natural aggregation

Daniel F Lusche  1 , Deborah Wessels, Amanda Scherer, Karla Daniels, Spencer Kuhl, David R Soll
Affiliations

The IplA Ca2+ channel of Dictyostelium discoideum is necessary for chemotaxis mediated through Ca2+, but not through cAMP, and has a fundamental role in natural aggregation

Daniel F Lusche et al. J Cell Sci. .

Abstract

During aggregation of Dictyostelium discoideum, nondissipating, symmetrical, outwardly moving waves of cAMP direct cells towards aggregation centers. It has been assumed that the spatial and temporal characteristics of the front and back of each cAMP wave regulate both chemokinesis and chemotaxis. However, during the period preceding aggregation, cells acquire not only the capacity to chemotax in a spatial gradient of cAMP, but also in a spatial gradient of Ca(2+). The null mutant of the putative IplA Ca(2+) channel gene, iplA(-), undergoes normal chemotaxis in spatial gradients of cAMP and normal chemokinetic responses to increasing temporal gradients of cAMP, both generated in vitro. However, iplA(-) cells lose the capacity to undergo chemotaxis in response to a spatial gradient of Ca(2+), suggesting that IplA is either the Ca(2+) chemotaxis receptor or an essential component of the Ca(2+) chemotaxis regulatory pathway. In response to natural chemotactic waves generated by wild-type cells, the chemokinetic response of iplA(-) cells to the temporal dynamics of the cAMP wave is intact, but the capacity to reorient in the direction of the aggregation center at the onset of each wave is lost. These results suggest that transient Ca(2+) gradients formed between cells at the onset of each natural cAMP wave augment reorientation towards the aggregation center. If this hypothesis proves correct, it will provide a more complex contextual framework for interpreting D. discoideum chemotaxis.

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Figures

Fig. 1.
Fig. 1.
IplA, a putative Ca2+ channel, containing six transmembrane domains, is involved in Ca2+ binding and is distributed at high levels in cytoplasmic vesicles and very low levels in the plasma membrane. (A) A general model for IplA in a membrane, based on models developed for inositol trisphosphate receptors in higher eukaryotes (Furuichi et al., 1989; Joseph, 1996; Foskett et al., 2007; Foskett, 2010; Mignery and Sudhof, 1990). IP3, inositol trisphosphate. (B) The iplA− mutant binds less extracellular 45Ca2+ than parental Ax2 cells. (C,D) Fluorescence and differential interference contrast microscopy (DIC) images, respectively, of a representative live iplA− cell, showing no fluorescence. (E–H) Fluorescence (E,G) and DIC (D,H) images of two live iplA−/iplA+-GFP cells, showing vesicular staining throughout the cytoplasm. (I,J) Fluorescence and DIC images, respectively, of a representative iplA− plasma membrane ghost, showing no fluorescence upon image enhancement. (K–N) Fluorescent (K,M) and DIC (L,N) images of representative iplA−/iplA+-GFP plasma membrane ghosts, showing low-level fluorescence upon image enhancement. Note that the fluorescence of whole live cells was far higher than that of the plasma membrane, and that the iplA− images were taken with the same settings or with the same enhancement as iplA−/iplA+-GFP images. Scale bar: 5 μm.
Fig. 2.
Fig. 2.
Three different chambers were used to assess cell behavior. (A) The Sykes–Moore chamber. (B) The Zigmond gradient chamber. (C) The microfluidic chamber. Chambers are described in the Results section. Shear force and the capacity to generate a chemotactic gradient are noted at the bottom of panels.
Fig. 3.
Fig. 3.
Mutant iplA− cells exhibit selective defects in the facilitation of velocity by Ca2+. The behavior of mutant iplA− cells was compared with that of parental Ax2 and complemented iplA−/iplA+ cells in a Sykes–Moore chamber perfused with TB containing different concentrations of Ca2+, in the absence of cAMP. (A) Motility parameters (means ± standard deviation) at different concentrations of Ca2+. (B) Instantaneous velocity plotted as a function of Ca2+ concentration. (C) Proportion of cells with instantaneous velocities ≥9 μm/minute plotted as a function of Ca2+ concentration. (D) Positive flow plotted as a function of Ca2+ concentration. (E) Directional persistence plotted as a function of Ca2+ concentration. (F,G) Perimeter tracks of representative parental Ax2 and iplA− cells, respectively, translocating in TB containing 10 mM Ca2+. Insets in F and G reveal no differences in shape. (H) Motility parameters of Ax2 and iplA− cells in a Sykes–Moore chamber perfused with TB containing 40 mM K+ in the absence of cAMP.
Fig. 4.
Fig. 4.
Mutant iplA− cells exhibit selective defects in Ca2+ facilitation of velocity, but undergo normal chemotaxis in a spatial gradient of cAMP. Studies were performed in spatial gradients of cAMP generated in TB containing different Ca2+ concentrations in a Zigmond chamber. (A) Motility parameters (means ± standard deviation) at different concentrations of Ca2+. (B) Instantaneous velocity as a function of Ca2+ concentration. (C) Proportion of cells with instantaneous velocities ≥9 μm/minute as a function of Ca2+ concentration. (D) Directional persistence as a function of time. (E) Chemotactic index as a function of time. (F,G) Perimeter tracks of Ax2 and iplA− cells, respectively, in cAMP gradients generated in 10 mM Ca2+. Insets are for shape comparisons. (H) Motility parameters of Ax2 and iplA− in spatial gradients generated in TB containing 40 mM K+ in TB.
Fig. 5.
Fig. 5.
Mutant iplA− cells undergo increases in instantaneous velocity in the increasing phase (f, blue zone) of a series of temporal waves of cAMP generated in a Sykes–Moor chamber. The temporal waves of cAMP approximate the temporal dynamics of natural cAMP waves in the absence of a spatial gradient. These experiments were performed in a buffered salt solution containing 40 mM K+ as the facilitating cation.
Fig. 6.
Fig. 6.
Mutant iplA− cells lose the capacity to undergo chemotaxis in a spatial gradient of Ca2+ generated in a microfluidic chamber. (A) Computing rightward directionality (RD). x, net distance a cell moves to the right, the direction of flow; y, total distance a cell moves. (B) Rightward movement of representative parental Ax2 cells in response to the rightward shear force in the absence of a chemotactic gradient, in uniform 10 mM Ca2+ in TB. (C) The absence of rightward movement of representative mutant iplA− cells in response to rightward shear force in the absence of a chemotactic gradient in uniform 10 mM Ca2+. (D) Motility parameters in the absence of a chemotactic gradient in the microfluidic chamber in uniform 10 mM Ca2+ in TB. (E) Chemotaxis up a cAMP gradient by parental Ax2 cells. The cAMP gradient was generated in 10 mM Ca2+ in TB. (F) Chemotaxis up a cAMP gradient by iplA− cells. The cAMP gradient was generated in 10 mM Ca2+ in TB. (G) Chemotaxis up a Ca2+ gradient by parental Ax2 cells. The Ca2+ gradient was generated in TB. (H) Random movement in a Ca2+ gradient by iplA− cells. The Ca2+ gradient was generated in TB. (I) Motility and chemotaxis parameters in a cAMP or Ca2+ gradient generated in a microfluidic chamber.
Fig. 7.
Fig. 7.
Mutant iplA− cells do not reorient in the front of natural waves generated by parental Ax2 cells. To assess how iplA− cells respond to outwardly moving, nondissipating relayed waves of chemoattractant, DiI-labeled iplA− cells and unlabeled parental Ax2 cells were mixed in a 1:9 ratio and allowed to aggregate. Before streaming, the behavior of individual iplA− and Ax2 cells was analyzed. (A) 2D model of naturally relayed, outwardly moving, nondissipating waves of cAMP in an aggregation territory. (B) 1D model of naturally relayed waves. (C,E) Velocity plots from two independent experiments of individual majority parental Ax2 (black dots) and neighboring minority mutant iplA− (red dots) cells responding to natural waves. The waves diagrammed at top, were deduced from Ax2 cell behavior. (D,F) Centroid tracks from two independent experiments, of representative Ax2 cells (black dots) and iplA− cells (red dots). Agg. center, aggregation center. W1, W2, W3. Waves 1, 2 and 3. P, velocity peaks.
Fig. 8.
Fig. 8.
Measurements of defective reorientation in the front of natural chemotactic waves. Mutant iplA− cells and parental Ax2 cells were mixed at a 1:9 ratio and allowed to aggregate. Orientation angles (<) were computed in the front of each relayed wave. (A,B) Method for measuring orientation angle. In A, the angle demonstrates orientation in the general direction (~30°) of the aggregation center, the source of the wave. In B, the angle (~90°) suggests random direction. (C) Measurements of angles for parental Ax2 (WT) and iplA− cells. Average angle ± standard deviation is calculated for each cell in four to five successive waves. (D) Model in which a short-lived Ca2+ gradient (red line) is generated between cells at the onset of each cAMP wave, the latter relayed through the population.
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