PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells

Abstract

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.

Figure 1.

Expression and localization of PRG in dHL60 cells. (A) Lysates from nondifferentiated dHL60 cells (ND) or cells differentiated for 7 d (D7) were immunoblotted for PRG protein. RhoA was used as a loading control. (B) DIC and fluorescent time-lapse images of a live dHL60 cell transfected with PRG-YFP. Time indicates elapsed time after uniform addition of 100 nM fMLP at 0:00. (C) DIC and fluorescent images of a representative polarized cell showing PRG-YFP (green) and actin (red). Cells transfected with PRG-YFP were stimulated with 100 nM fMLP for 3 min, fixed, and stained for F-actin. Arrowheads outline pseudopod; arrow depicts the cell's back. Bar = 10 μm.

Figure 2.

PRG depletion disrupts fMLP-induced polarity. DIC time-lapse images of dHL60 cells expressing luciferase-targeted control or PRG-targeted shRNA. (Right) DIC and fluorescent overlays of control and PRG KD cells processed as in Fig. 1 C. Time indicates elapsed time after uniform stimulation with 100 nM fMLP. Different arrowhead colors indicate separate pseudopod. Bars = 10 μm.

Figure 3.

PRG mediates fMLP-induced RhoA activation and consolidation of front and back. (A) fMLP-induced membrane association of RhoA. Control or KD cells were stimulated with or without fMLP (100 nM, 1 min) as indicated. RhoA associated with the particulate fraction of cell extracts was assessed as described. A representative immunoblot and quantification of immunoblotted bands are shown. For each immunoblot, background signal was subtracted and the level of RhoA normalized to that of transferrin receptor in the particulate fraction; all values were further normalized to the signal detected in the unstimulated control, set at 1.0. Bar represents the average of four independent experiments, with different symbols representing the actual values. P values are shown. **, P ≤ 0.05. (B) DIC and fluorescent images of representative cells expressing control or PRG-targeted shRNA with or without a Myc-tagged rat PRG processed as in Fig. 1 C and stained for actin (green), p-MLC2 (red), and Myc (blue). Bars = 10 μm.

Figure 4.

Spatial distribution of PRG depends on G12/13 and myosin II. (A) dHL60 cells transiently transfected with PRG-YFP alone, or together with DN or CA G12 and G13 were stimulated with 100 nM fMLP, fixed, and imaged. Representative DIC and fluorescent images of each group are shown. Cells expressing PRG-YFP alone with or without 45 min pretreatment with 100 μM blebbistatin were processed and imaged as above. Percent of cells with mislocalized PRG-YFP was plotted for control (n = 60), KD cells expressing rat PRG (rescued; n = 36), cells coexpressing DN G12/13 (n = 33), CA G12/13 (n = 28), or CA RhoA (n = 31) and cells treated for 45 min with 100 μM blebbistatin (n = 46), 10 μM Y-27632 (n = 29), 1 μM PIK-90 (n = 37), or 25 μM nocodazole (n = 47). (B) Quantification of mean FRET/CFP ratio in RhoA biosensor cells treated with or without Y-27632 (10 μM, 45 min), stimulated and processed as above. Mean FRET/CFP ratios ± SEM (error bar) in unstimulated (n = 23) and stimulated (n = 26) control, unstimulated (n = 24) and stimulated (n = 27) Y-27632-treated cells are 1 ± 0.03, 1.3 ± 0.02, 1.17 ± 0.03, and 1.22 ± 0.03, respectively. P values are as indicated. Similar results were observed in three independent experiments. **, P ≤ 0.05. (C) Representative DIC, CFP, and FRET/CFP ratio images of control and Y-27632–treated cells. Ratio images were scaled relative to each other. Warm color = high value, cold color = low value. Different color of arrowheads marks different pseudopod. Bars = 10 μm.