G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton

Abstract

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein beta subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type beta subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In beta subunit-null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein-actin transfected cells showed that beta subunit- null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

Figure 7

Phagocytosis and actin dynamics in GFP–actin-transfected wild-type (A and B) and β-null (C–F) cells. Each panel (A–F) shows a series of images recorded at intervals of 34 s. Phase-contrast and corresponding GFP–actin fluorescence images are shown on alternate rows. In the fluorescence images, TRITC-labeled yeast particles are shown in red, whereas increasing intensities of GFP–actin are pseudocolored from dark green to light yellow. A–C show successful phagocytosis in AX2 (A), AX3 (B), and LW6 (C). In all cases, an actin-enriched layer surrounds the particle during uptake and disappears upon engulfment. In B, two cells compete for a particle at the left-hand side of the frames. The particle is first attached to the cell on top, and induces a rim of accumulated actin (fourth panel). Simultaneously, the cell on the bottom forms a phagocytic cup and within half a minute succeeds in enclosing the particle, forming a continuous rim of actin. Afterwards, the rim disassembles, beginning at the proximal side of the phagosome, while a new actin-rich leading edge is formed at the extreme right end of the cell (sixth and seventh panels). D–F illustrate gβ⁻cells that fail to incorporate attached particles. (D) A half cup enriched with actin is formed, followed by actin disassembly and detachment of the particle. (E) Actin-rich protrusions of the cell are formed to the left and right of an attached particle, but no phagocytic cup is formed. (F) A particle adheres to the cell over a period of 7 min without being taken up. Bars, 10 μm.

Figure 1

G protein β subunit stimulates phagocytosis, but not pinocytosis. Phagocytosis of (A) FITC-labeled E. coli B/r or (B) 1-μm-diam latex beads were assayed by incubating cells under shaking with a 1,000- or a 200-fold excess of bacteria or latex beads, respectively. At the indicated times, cells were centrifuged free of particles and then lysed with Triton X-100 to determine the fluorescence of ingested bacteria or turbidity of ingested latex beads. (C) Pinocytosis of FITC-labeled dextran dissolved in axenic medium was tested under similar conditions. In both cases, 30-min starving cells were used. AX2, wild-type; LW 6 and LW 14, gβ⁻clones; LW 18 and LW 20, rescue clones. Results are the average of duplicate determinations in parallel experiments performed with the same cell culture. SD varied between ±4 and 20% of the values shown. Similar results were obtained in a total of (A) six, (B) five, and (C) two independent experiments, respectively. For further details, refer to Materials and Methods.

Figure 1

G protein β subunit stimulates phagocytosis, but not pinocytosis. Phagocytosis of (A) FITC-labeled E. coli B/r or (B) 1-μm-diam latex beads were assayed by incubating cells under shaking with a 1,000- or a 200-fold excess of bacteria or latex beads, respectively. At the indicated times, cells were centrifuged free of particles and then lysed with Triton X-100 to determine the fluorescence of ingested bacteria or turbidity of ingested latex beads. (C) Pinocytosis of FITC-labeled dextran dissolved in axenic medium was tested under similar conditions. In both cases, 30-min starving cells were used. AX2, wild-type; LW 6 and LW 14, gβ⁻clones; LW 18 and LW 20, rescue clones. Results are the average of duplicate determinations in parallel experiments performed with the same cell culture. SD varied between ±4 and 20% of the values shown. Similar results were obtained in a total of (A) six, (B) five, and (C) two independent experiments, respectively. For further details, refer to Materials and Methods.

Figure 2

G protein β subunit is required for chemoattractant-induced actin polymerization. F-actin formation in suspended cell cultures following addition of: (A) supernatants from bacterial growth medium or (B) 10⁻⁶ M cAMP. Closed triangles, LW20; open circles, LW6. (A) 1- or (B) 4-h starving cells were used. The mean value of triplicate determinations is shown. Similar results were obtained in five independent experiments. For experimental details refer to Materials and Methods. Bacterial-conditioned medium was prepared by clarification of a stationary phase culture of K. aerogenes grown at 22°C. Medium was used as a stimulus at a dilution of 1:100.

Figure 3

Phagocytosis correlates with rates of F-actin formation in response to chemoattractants. The data reported in Table I, columns 6 and 7 for gβ⁻cells, point mutants, rescue cells, and wild-type were plotted to determine the regression curve (R^{− 2}).

Figure 4

G protein β subunit mutants are impaired in yeast particle uptake. Phagocytosis of TRITC-labeled heat-killed yeast particles by AX2 (closed circle) or LW6 (open circle) was measured by incubating cells with a fivefold excess of particles for the indicated times. At the end of the incubation, the cells were diluted 10-fold with cold buffer containing trypan blue for quenching the fluorescence of noningested particles. The fluorescence of ingested yeasts was determined in a spectrofluorimeter as described in Materials and Methods. Mean values of three duplicate experiments ±SD are shown.

Figure 5

Dynamics of yeast particle uptake by wild type and β subunit–null cells. Serial light microscopy images of AX2 or LW6 cells incubated on a solid substratum with yeast particles. A total of 10⁶ WT (AX2) or gβ⁻ (LW6) cells in 5 ml of Soerensen phosphate buffer, pH 6.0, were incubated with 10⁷ heat-killed yeast particles in 60-mm Petriperm dishes. Phagocytosis by single cells was followed for 2 h by time-lapse videomicroscopy on a Zeiss Axiovert 35 microscope equipped with a 100× Neofluar objective and a Zeiss ZVS-47DE charge-coupled device videocamera connected to a Panasonic 6050 videorecorder. Images were recorded at 0.5-s intervals. Selected images were captured with the Apple video player and mounted using Adobe Photoshop. Numbers indicate time in s.

Figure 6

Actin localization in wild-type and β subunit–null cells during phagocytosis of yeast particles. WT (AX2), rescue (LW20) and gβ⁻ (LW6) cells incubated with TRITC-labeled yeast particles on glass coverslip. Phase-contrast and corresponding FITC-phalloidin fluorescence images are shown on alternate lines. For LW20 and LW6, single cells showing membrane projections enriched with actin are shown in addition to cells engulfing yeasts. Cells were incubated with yeast particles on a glass coverslip for 30 min followed by fixation and labeling with FITC-phalloidin.