Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium discoideum

Abstract

The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two- and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 perpendicular RegA perpendicular [cAMP] --> PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateral-pseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.

FIG. 1.

Model describing cell behavior in the different phases of the natural wave and the relationship of the wave to the basic motile behavior of a cell. The wave is separated into four phases, A, B, C, and D. Descriptions of the behavior of cells in each phase and the characteristics of the wave responsible for these behaviors were derived from previous studies (29, 37, 38, 40, 41, 47, 49). Vertical arrows represent regulatory pathways emanating from the different phases of the wave which target machinery involved in basic motile behavior, leading to the cell behaviors specific to each phase of the wave (29).

FIG. 2.

pkaR mutant cells are defective in natural aggregation. The behavior of wild-type (Ax4) (A) and pkaR mutant (108d3) (B) cells in monolayers on nonnutrient agar was analyzed. For each cell type, a representative field of cells was videorecorded for 120 min and their motion was analyzed with the 2D-DIAS vector flow program (19, 28). For each cell type, representative video images at 20, 60, and 100 min are shown. In the vector flow plots, the magnitudes of the vector components parallel to the selected direction of the aggregation center for Ax4 cells and parallel to an arbitrary direction for 108d3 cells were averaged and plotted over time. The x axis represents time, and the y axis represents the direction (+ or −) and extent of the displacement of cells. Note that while Ax4 cells form streams, pkaR mutant cells do not and that while Ax4 cells exhibit cyclic surges toward the aggregation center, pkaR mutant cells do not.

FIG. 3.

pkaR mutant cells are impaired in their response to natural cAMP waves. The behavior of individual wild-type (Ax4) and pkaR mutant (DG1075 [1075] and 108d3) cells in aggregation territories composed of 90% Ax4 cells and 10% mutant cells was analyzed. In the top of each panel, a plot of the instantaneous velocity of the representative cell under analysis is shown, and at the bottom of each panel a centroid track is presented. The instantaneous velocity plots were smoothed 10 times with Tukey windows of 10, 20, 40, 20, and 10. The arrows denote the direction toward the aggregation center assessed from the behavior of the predominant Ax4 cells. For each pair (A and B and C and D), the Ax4 cell was the closest neighbor to the analyzed pkaR mutant cell. per, average period of velocity peaks in minutes; pk, average peak velocity in micrometers per minute; tr, average trough velocity in micrometers per minute; Inst. Vel., instantaneous velocity; Agg. Center, aggregation center.

FIG. 4.

Computer-generated perimeter tracks reveal that pkaR mutant cells translocate at lower velocities and turn more often than Ax4 cells in a spatial gradient of cAMP. Tracks of the three Ax4 cells (A) and the three pkaR mutant cells (B and C) with the highest chemotactic indices are shown. Cell perimeters were drawn every 4 s. 1075, DG1075.

FIG. 5.

pkaR mutant cells behave aberrantly in the front of temporal waves of cAMP. The instantaneous velocities (Inst. Vel.) of a representative Ax4 cell (A), a representative 108d3 cell (B), and a representative DG1075 (1075) cell (C) during four simulated temporal waves of cAMP generated in a perfusion chamber are plotted as a function of time. The concentration of cAMP (cAMP Conc.; top lines), estimated in dye experiments, is presented as a function of time through the four waves. Instantaneous velocity plots were smoothed 10 times with Tukey windows of 10, 20, 40, 20, and 10. Note that, unlike the Ax4 cell, pkaR mutant cells do not exhibit a velocity surge in the front of waves 2, 3, and 4.

FIG. 6.

3D reconstructions of a representative Ax4 cell in the front (A), at the peak (B), and in the back (C) of a simulated temporal wave of cAMP generated in a perfusion chamber. Nonparticulate pseudopodial zones are demarcated in red. The cell is viewed at each time point at angles of 15 and 60° from the surface. Note that the Ax4 cell is elongate along the substratum in the front of the wave, rounds up and retracts the dominant pseudopod at the peak of the wave, and resumes pseudopod formation but in all directions and without cell elongation in the back of the wave. The behavior of this cell is representative of that of nine additional Ax4 cells reconstructed in 3D in a similar fashion.

FIG. 7.

3D reconstructions of a representative pkaR mutant cell in the front (A), at the peak (B), and in the back (C) of a simulated temporal wave of cAMP generated in a perfusion chamber. Nonparticulate pseudopodial zones are demarcated in red. The cell is viewed at each time point at angles of 15 and 60° from the surface. Note that pkaR mutant cells remain ovoid throughout the three phases of the wave. Note also that pkaR mutant cells retract the dominant pseudopod at the peak of the wave and resume apolar pseudopod formation in the back of a wave. The behavior of this cell is representative of that of nine additional pkaR mutant cells reconstructed in 3D in a similar fashion.

FIG. 8.

pkaR mutant cells in buffer respond in an apparently normal fashion to the rapid addition of 1 μM cAMP. Centroid tracks of three representative Ax4 (A) and three representative pkaR mutant (108d3) (B) cells responding to the rapid addition of 1 μM cAMP are shown. The total track, that portion of the track before the addition of cAMP (in buffer), and that portion of the track after the addition of cAMP are shown for each representative cell. Inst. Vel., instantaneous velocity; Rnd., roundness parameter.

FIG. 9.

The organization of the cytoskeletons of pkaR mutant cells appears normal in buffer and in the front of a temporal wave of cAMP. Representative Ax4 and pkaR mutant (108d3) cells in buffer and in the front of the third in a series of four temporal waves of cAMP were stained for F-actin (A to H), myosin II (I to P), and tubulin (Q to W). a, anterior end; u, uropod. Bars, 10 μm.

FIG. 10.

Model of the regulatory circuitry for PKA activation during normal Dictyostelium chemotaxis. When the cAMP receptor cAR1 is occupied in the increasing phase of the wave, the mitogen-activated protein kinase ERK2 and ACA are activated (4, 5, 15, 19). ERK2 inhibits the internal phosphodiesterase RegA (7, 22, 23, 34), which allows cAMP, synthesized by ACA, to accumulate. As the internal concentration of cAMP increases, so does the activity of PKA. When cAR1 occupancy decreases in the back of the wave, both ERK2 and ACA are deactivated, resulting in an increase in RegA activity, a decrease in the internal concentration of cAMP, and a decrease in PKA activity. PKA also inhibits ERK2 (4, 5), noted by a dashed line. Hence, when PKA activity increases, it begins to shut down ERK2 activity, resulting in an increase in RegA activity and a decrease in the intracellular cAMP concentration. In the pkaR mutant, PKA activity is uncoupled from this circuit and will remain constitutively high under all test conditions. R, regulatory subunit; C, catalytic subunit.