RacG regulates morphology, phagocytosis, and chemotaxis

Abstract

RacG is an unusual member of the complex family of Rho GTPases in Dictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 microM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPgammaS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or more Dictyostelium Rho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

FIG. 1.

Expression of endogenous and GFP-tagged RacG. Western blot analysis of AX2 and strains overexpressing GFP fusions of WT, constitutively active (RacG-V12), and dominant negative (RacG-N17) forms of RacG. Total homogenates of 4 × 10⁵ cells were resolved in 12% polyacrylamide gels and blotted onto nitrocellulose membranes. Blots were incubated with a polyclonal antiserum raised against RacG.

FIG. 2.

Subcellular distribution of RacG. (A) Images of living Dictyostelium cells expressing GFP fusions of RacG mutations. GFP fluorescence was recorded. For RacG-WT, an average projection of 18 confocal sections 488 nm apart is shown, along with the corresponding phase-contrast image. A single confocal section of RacG-V12, RacG-N17, or RacG-V12Δins is presented. A phase-contrast image of AX2 cells is also shown. Bar, 10 μm (20 μm for RacG-V12). (B) Fractionation of Dictyostelium cells overexpressing GFP fusions of RacG-WT, RacG-V12, RacG-N17, and RacG-V12Δins. Cells were lysed by sonication, and cytosolic (C) and particulate (P) fractions were separated by ultracentrifugation. Samples were resolved in 12% polyacrylamide gels and blotted onto nitrocellulose membranes. Blots were incubated with anti-GFP MAb K3-184-2. The left half of the panel shows one representative sample of each strain. The right half of the panel shows the average ± the standard deviation of the densitometric quantitation of three independent fractionations.

FIG. 3.

Cell morphology and F-actin organization of RacG mutants. Cells were grown overnight on coverslips in axenic medium, fixed with picric acid-paraformaldehyde, and stained with actin-specific MAb Act1-7, followed by Cy3-labeled anti-mouse immunoglobulin G. In AX2, RacG-KO, and RacG-N17 cells, actin predominates at cortical crown-like structures. In RacG-WT and RacG-V12 cells, long, actin-rich filopods are abundant. The top three panels on the right are closer views of cells of the strains in the left panels. Pictures are the maximum projection of 20 confocal sections 400 nm apart. Bars, 10 μm.

FIG. 4.

Localization of RacG during phagocytosis of yeast cells. (A) Time series showing the dynamics of GFP-RacG redistribution on uptake of a yeast cell. Dictyostelium cells expressing GFP-RacG-WT were allowed to sit on glass coverslips and then challenged with TRITC-labeled yeast cells. Images were taken with a confocal laser scanning microscope. Images from green and red channels were independently assigned color codes (green for GFP and red for TRITC) and superimposed. RacG detaches from the phagosome shortly after internalization of the yeast particle. The elapsed time in seconds is shown at the top right corner of each image. (B) RacG accumulates at the rim of the nascent phagosome. Images were obtained as in panel A with the difference that the signal corresponding to GFP-RacG was assigned a glow-over look-up table to better reveal intensity differences. Pixels with maximum intensity appear blue. (C) Colocalization of RacG with actin at the phagosome. Confocal section of a cell expressing GFP-RacG-WT during uptake of an unlabeled yeast particle. Cells were allowed to sit on glass coverslips, incubated for 20 min with heat-killed yeast cells, and fixed and stained as described in the legend to Fig. 3. From left to right, images correspond to GFP-RacG (green), actin (red), overlay, and phase-contrast. Note the accumulation of GFP-RacG and actin at the rim of the phagocytic cup (arrows). Bars, 5 μm.

FIG. 5.

Phagocytosis, fluid-phase uptake, and exocytosis of RacG mutants. (A) Phagocytosis of TRITC-labeled yeast cells. Dictyostelium cells were resuspended at 2 × 10⁶/ml in fresh axenic medium and challenged with a fivefold excess of fluorescent yeast cells. Fluorescence from internalized yeast cells was measured at the designated time points. (B) Fluid-phase endocytosis of TRITC-dextran. Cells were resuspended in fresh axenic medium at 5 × 10⁶/ml in the presence of 2 mg/ml TRITC-dextran. Fluorescence from the internalized marker was measured at selected time points. (C) Fluid-phase exocytosis of TRITC-dextran. Cells were pulsed with TRITC-dextran (2 mg/ml) for 3 h, washed, and resuspended in fresh axenic medium. Fluorescence from the marker remaining in the cell was measured. Data are presented as relative fluorescence, that of AX2 being considered 100%. For clarity, error bars extend only in one direction.

FIG. 6.

Effect of the PI3 kinase inhibitor LY294002 on phagocytosis and morphology. (A) Phagocytosis of TRITC-labeled yeast cells. AX2 and RacG-WT cells were assayed as described in the legend to Fig. 5, in the absence or presence of 50 μM LY294002. (B) Morphology of RacG-WT cells in the presence of 50 μM LY294002. Axenically growing cells were washed with Soerensen buffer and allowed to sit on a glass coverslip. The inhibitor was then added, and images were taken with a conventional epifluorescence microscope. The corresponding phase-contrast images are shown on the right. Bar, 10 μm.

FIG. 7.

Chemotactic movement of aggregation-competent RacG mutant cells to a micropipette containing cAMP. Cells were starved for 6 h, allowed to sit on a glass coverslip, and stimulated with a micropipette filled with 0.1 mM cAMP. Images of cells exhibiting chemotaxis were captured every 30 s. Cell movement was analyzed with the DIAS software. In RacG-V12 and RacG-N17 cells, migration is severely impaired.

FIG. 8.

cAMP-dependent processes in aggregation-competent RacG mutant cells. Cells were starved for 6 h prior to the determinations as specified in Materials and Methods. (A) Actin polymerization responses upon cAMP stimulation. Relative F-actin content was determined by TRITC-phalloidin staining of cells fixed at the indicated time points after stimulation with 1 μM cAMP. The amount of F-actin was normalized relative to the F-actin level of unstimulated cells. For simplicity, error bars are shown only for the 5-s time point. (B) RacG translocates to the detergent-insoluble F-actin pellet upon cAMP stimulation. Samples of AX2 cells were processed as for panel A, with the difference that phalloidin was omitted and the F-actin pellet was processed for Western blotting. Blots were incubated with a polyclonal antiserum specific for RacG or with MAb 33-294-17,which is specific for contact site A (an adhesion membrane protein present in Triton-insoluble lipid rafts) as a control for loading. The blot shown is representative of two independent experiments. (C) Activation of Rac1 in RacG mutants upon cAMP stimulation. Rac1-GTP was separated with glutathione-Sepharose beads with the GST-fused CRIB domain of Dictyostelium WASP and analyzed by immunoblot assay with a Rac1-specific MAb. A representative blot of each strain is shown on the left. Data on the right were derived from quantitation of at least four independent pull-down experiments.

FIG. 9.

RacG induces actin polymerization. (A) Comparison of the actin polymerization responses of lysates of AX2, RacG-V12, and RacG-V12Δins cells. Relative F-actin content was determined by TRITC-phalloidin staining of samples fixed at the indicated time points after induction with 100 μM GTPγS. The amount of F-actin was normalized relative to the F-actin level of the corresponding uninduced lysate. (B) Specificity of the actin polymerization response induced by RacG. Samples were preincubated for 30 min with RacG-specific polyclonal antiserum or with nonimmune serum (NIS) at a 1:100 dilution prior to GTPγS addition. (C) Recombinant RacG induces actin polymerization. Lysates of AX2 cells were processed as in panel A after addition of GTPγS or 0.4 μM GTPγS-charged recombinant RacG. (D) RacG induces actin polymerization through activation of the Arp2/3 complex. Lysates of AX2 and RacG-V12 cells were processed as in panel A after addition of GTPγS. Where appropriate, samples were preincubated for 30 min with Arp3-specific purified polyclonal antibody at a dilution equivalent to a 1:20 dilution of serum.