Ca2+ influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages

Abstract

In migrating eukaryotic cells, phosphatidylinositol 3-kinase (PI3K), filamentous actin (F-actin), and monomeric Rho GTPases are key components of a complex positive-feedback system that maintains and amplifies a phosphatidylinositol-3,4,5-trisphosphate signal at the leading edge of the cell. This lipid signal is required for cell polarization and movement. In leukocytes and Dictyostelium, activation or inhibition of any one of these components leads to the activation or inhibition, respectively, of the others via undefined feedback interactions. The role of Ca(2+) signals in migrating leukocytes is controversial, and there has been no indication that Ca(2+) participates in positive feedback. Here, we demonstrate that an extracellular Ca(2+) influx is required for positive feedback at the leading edge of spontaneously polarized macrophages. Inhibition of extracellular Ca(2+) influx leads to loss of leading-edge PI3K activity, disassembly of F-actin, cessation of ruffling, and decay of chemoattractant signals. Conversely, increasing cytosolic Ca(2+) enhances membrane ruffling, PI3K activity, and F-actin accumulation. Overall, these findings demonstrate that an extracellular Ca(2+) influx is an essential component, together with PI3K and F-actin, of the positive-feedback cycle that maintains leading-edge structure and ruffling activity and that supports the chemoattractant response. Strikingly, the Ca(2+)-sensitive enzyme protein kinase Calpha (PKCalpha) is enriched at the leading edge, and its enrichment is sensitive to blockade of Ca(2+) influx, to inhibition of PI3K activity, and to F-actin depolymerization. These findings support the working hypothesis that a local, leading-edge Ca(2+) signal recruits PKCalpha as a central player in the positive-feedback loop.

Fig. 1.

Characterization of polarized RAW cells. (A) Wild-type, polarized RAW cells imaged at 5-s intervals as visualized by Nomarski optics, illustrating the pronounced, ruffling leading edge. (B and C) Time-lapse fluorescence images of spontaneously polarized RAW cells expressing GFP-Akt1PH (Upper) or GFP-actin (Lower) treated with 500 nM wortmannin (WORT) (B) or 10 μg ml⁻¹ latrunculin B (LatB) (C). Images show the cell at the time indicated relative to drug addition, illustrating the redistribution of fluorescent protein from the leading-edge region to the cell body (defined as the regions to the left and right of the vertical bar, respectively) after treatment. For each time series, the intensity scaling was the same for all images. Images are representative of at least five experiments. (D) Plots show the integrated GFP-Akt1PH fluorescence versus time for the leading-edge and cell body regions before and after wortmannin (Left) or LatB (Right) addition, thereby quantitating the transfer of fluorescent protein from the leading edge to the cell body after treatment. See also SI Movies 1, 3, and 5–7 for A, B, and C.

Fig. 2.

Inhibition of extracellular Ca²⁺ influx results in loss of PI3K activity, F-actin, and ruffling from the leading edge. (A and B) Time-lapse fluorescence images of RAW cells expressing GFP-Akt1PH (A) or GFP-actin (B) treated with 3 mM EGTA (Top), 1 mM LaCl₃ (Middle), or 20 μM SKF (Bottom). Indicated times are relative to drug addition. The images show the transfer of fluorescent protein from the leading-edge region to the cell body after drug addition, as do the accompanying plots of integrated GFP-Akt1PH fluorescence. (C) Nomarski images of wild-type cell treated with 3 mM EGTA at the indicated time, illustrating the loss of ruffling and retraction of the leading edge after treatment. For each time series, the intensity scaling was the same for all images. Images are representative of at least five experiments. See also SI Movies 8–12 for A, B, and C.

Fig. 3.

Recruitment of PKCα to the leading edge is sensitive to extracellular Ca²⁺ influx, PI3K activity, and F-actin polymerization. (A) Time-lapse fluorescence images of polarized RAW cells expressing YFP-PKCα treated with 3 mM EGTA (Upper) or 1 mM LaCl₃ (Lower). Plots show the integrated YFP-PKCα fluorescence versus time for the leading-edge and cell body regions before and after drug addition. Both the images and plots reveal a significant transfer of YFP-PKCα from the leading edge to the cell body after blockade of Ca²⁺ influx. (B) Time-lapse fluorescence images of polarized RAW cells expressing YFP-PKCα treated with 500 nM wortmannin (WORT) (Upper) or 10 μg ml⁻¹ latrunculin B (LatB) (Lower). Plots show the integrated YFP-PKCα fluorescence versus time for the leading-edge and cell-body regions before and after drug addition. Both the images and plots illustrate the redistribution of YFP-PKCα from the leading edge to the cell body after treatment. (C) Time-lapse fluorescence images of a ruffling RAW cells expressing YFP-PKCα treated with global addition of 25 μM ATP, showing a large increase in fluorescence at the leading-edge plasma membrane as well as a global targeting to plasma membrane throughout the cell body. Indicated times are relative to addition of drug. For each time series, the intensity scaling was the same for all images. Images are representative of at least five experiments. See also SI Movies 18–22 for A, B, and C. Both wortmannin and LatB cause dissociation of YFP-PKCα from the leading edge; in addition, LatB causes loss of YFP-PKCα through internalization of plasma membrane, as best seen in SI Movie 21.

Fig. 4.

Inhibition of extracellular Ca²⁺ influx or PI3K reverses the accumulation of PI(3,4,5)P₃ at the plasma membrane in LPS-treated cells. Time-lapse fluorescence images of nonpolarized RAW cells plated on fibronectin and expressing GFP-Akt1PH. Shown are cells before and after treatment with 10 μM LPS at 10 s, followed by treatment at 70 s with 0.1% DMSO (Top), 20 μM SKF (Middle), or 500 nM wortmannin (Bottom). Plots show average pixel intensity of Akt1PH fluorescence in the cytosol before and after drug additions. Indicated times are relative to drug addition. Both images and plots indicate that the carrier (DMSO) has no effect, whereas the Ca²⁺ channel blocker (SKF) and PI3K inhibitor [wortmannin (WORT)] both inhibit the PI3K activity in attractant-stimulated cells. For each time series, the intensity scaling was the same for all images. Vertical dotted lines indicate addition of LPS (Left) and either SKF, wortmannin, or DMSO (Right). Images are representative of at least three experiments. See also SI Movie 23.