PLA2 and PI3K/PTEN pathways act in parallel to mediate chemotaxis

Abstract

Directed cell migration involves signaling events that lead to local accumulation of PI(3,4,5)P(3), but additional pathways act in parallel. A genetic screen in Dictyostelium discoideum to identify redundant pathways revealed a gene with homology to patatin-like phospholipase A(2). Loss of this gene did not alter PI(3,4,5)P(3) regulation, but chemotaxis became sensitive to reductions in PI3K activity. Likewise, cells deficient in PI3K activity were more sensitive to inhibition of PLA(2) activity. Deletion of the PLA(2) homolog and two PI3Ks caused a strong defect in chemotaxis and a reduction in receptor-mediated actin polymerization. In wild-type cells, chemoattractants stimulated a rapid burst in an arachidonic acid derivative. This response was absent in cells lacking the PLA(2) homolog, and exogenous arachidonic acid reduced their dependence on PI3K signaling. We propose that PLA(2) and PI3K signaling act in concert to mediate chemotaxis, and metabolites of PLA(2) may be important mediators of the response.

Figure 1

(A) A model for regulation of chemotaxis. The dashed line shows a putative pathway that acts in parallel with the PI3K/PTEN pathway. (B) Scheme of the genetic screen. After mutagenesis, individual phenotypically wild-type colonies were separately plated in 96-well format, replicated and induced to differentiate in non-nutrient buffer containing different concentrations of LY. Mutants with altered sensitivity to LY are scored based on their ability to aggregate. Phenotypes of wild type, plaA⁻, pi3k1^-/2^- and plaA⁻/pi3k1^-/2^- cells on bacteria lawn (C), non-nutrient agar (D), and in under-buffer assay (E).

Figure 2

PLA₂A and PI3K actin parallel to mediate aggregation. (A, B) Under-buffer assay of indicated cells in the absence or presence of 60 μM LY or 5 μM BEL are shown. (C) Complementation of plaA⁻ cells by GFP tagged wild type and inactive (S61A) version of PLA₂A protein. The phenotype of cells in the under-buffer assay in the presence of 20 μM LY is shown. (D) Fluorescence images of wild type and plaA⁻ cells expressing PLA₂A-GFP. (E) Immuno-blot shows expression of camp receptor, cAR1, in wild type, plaA⁻, pi3k1^-/2^- and plaA⁻/pi3k1^-/2^- cells during development. Bands are 40 kD. All experiments were repeated at least 3 times.

Figure 3

PI(3,4,5)P₃ accumulation in wild type, plaA⁻, pi3k1^-/2^- and plaA⁻/pi3k1^-/2^- cells. (A) Chemoattractant-induced translocation of PH_crac-GFP to the plasma membrane is observed by epifluorescence microscopy. Images were captured at the indicated time points after uniform stimulation of 1 μM cAMP. When indicated, 30 μM of LY was added 10 minutes before stimulation. (B) Immuno-blotting shows amounts of membrane-associated PH_crac-GFP (arrow) at the indicated time points after addition of 1 μM cAMP. (C) Quantification of membrane-bound PH_crac-GFP was accomplished by densitometry and NIH image. The integrated amount of membrane-bound PH_crac-GFP above the prestimulated level was determined. Normalized values relative to controls in the absence of LY are shown. Experiment was repeated 3 times. Means and standard deviations are shown.

Figure 4

Chemotactic responses towards a micropipette filled with 10 μM cAMP. Cells were observed at 10-second intervals for 10 minutes. There was a random distribution of cells at the onset and images at 10 minutes are shown. (A) Chemotaxis of wild type and plaA⁻ cells was observed in the absence or presence of 30 μM LY using phase-contrast microscopy. LY was added as indicated 10 minutes before the start of the experiments. (B) Chemotactic responses of wild type, plaA⁻, pi3k1^-/2^-, plaA⁻/pi3k1^-/2^- cells were observed by DIC microscopy. (C) The speed (S), calculated from the distance a cell covered divided by the experimental time, and chemotactic index (CI), defined as the cosine of the angle formed by the line between the cell start point and the micropipette tip and the line from the cell start to ending point (Iijima and Devreotes, 2002; Chen et al., 2003), of wild type (S = 4.38 ± 1.94; CI = 0.96 ± .03), plaA⁻ (S = 3.24 ± 1.82; CI = 0.94 ± 0.10), pi3k1^-/2^- (S = 5.60 ± 2.58; CI = 0.92 ± 0.17), plaA⁻/pi3k1^-/2^- (S = 4.66 ± 2.14; CI =; 0.55 ± 0.57) cells were calculated and plotted (n= 15, 37, 37, 49, respectively). Three independent experiments were quantified. (D) Chemotaxis of wild type and pi3k1^-/2^- cells was observed in the absence or presence of BEL by phase-contrast microscopy. Cells were treated with BEL (5 μM) for 10 minutes, then the inhibitor was washed away. Cells were allowed to recover for 5 minutes before the start of the experiments.

Figure 5

Chemoattractant-induced actin polymerization assay. (A) Wild type (diamonds) and plaA⁻ cells (pink squares). (B) plaA⁻ cells in the absence (blue diamonds) or presence of 30 μM LY (pink squares). (C) pi3k1^-/2^- (blue diamonds) and plaA⁻/pi3k1^-/2^- cells in the absence (pink squares) or presence of 50 μM LY (green squares). For each experiment, all values were normalized to the amount of F-actin at time 0, which was taken before the addition of 100 nM cAMP. When indicated, LY was added 10 minutes before experiments. Experiments were repeated at least 3 times. Values are Means ± standard deviation.

Figure 6

Conversion of phosphatidylcholine to arachidonic acid by purified PLA₂A-FLAG. (A) PLA₂A-FLAG was affinity purified. Coomassie Blue stained gel (CB) and immuno-blot (IB) shows cell lysate and eluted fractions prepared from PLA₂A-FLAG expressing cells or wild type cells. The major eluted band from PLA₂A-FLAG expressing cells is at the predicted size of 44 kD, as indicated by arrow. A similar-sized non-specific band was eluted from wild type cells but not recognized by Flag antibodies. Another non-specific band (~ 70 kD) recognized by FLAG antibody is present in the cell lysate, but not in the purified fractions. (B) Thin layer chromatography (TLC) of ³H-arachidonic acid (³H-AA) released from ³H-phosphatidylcholine (³H-PC) by purified PLA₂A-FLAG. The eluted fractions were incubated with ³H-PC in buffer containing 5 mM Ca²⁺ or 5 mM EGTA for 60 minutes at 25°C. ³H-AA generated by a snake venom sPLA₂ (Sigma P7778) was used as positive control. The eluted fraction from wild-type cells was used as negative control. The experiment was repeated at least five times.

Figure 7