Reorganization of actin cytoskeleton by the phosphoinositide metabolite glycerophosphoinositol 4-phosphate

Abstract

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.

Figure 1

GroPIns-4P– and EGF-induced modifications of the actin cytoskeleton in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells (A) were treated with either 50 μM GroPIns-4P (B and C) or 20 ng/ml EGF (D and E) for 2 and 12 min, respectively. Cells were fixed and stained with TRITC-labeled phalloidin as described under MATERIALS AND METHODS. The results shown are representative of at least 30 independent experiments performed in triplicate. (F and G) Higher magnification of the boxed areas in B and E, respectively. Bar, 20 μm.

Figure 2

Effects of the inactivation of Cdc42, Rac, and Rho on the GroPIns-4P–induced actin cytoskeleton reorganization. Serum-starved Swiss 3T3 cells microinjected with expression constructs encoding a Myc-tagged version of N17Cdc42 (A–D), N17Rac1 (E–H), or with C. botulinum C3 transferase and fluorescein isothiocyanate-dextran (I and L) were treated with 50 μM GroPIns-4P for 2 min (A and E) or 12 min (C, G, and I). Cells were fixed and costained either for the Myc-tag by using an anti-Myc specific mAb (9E10) (B, D, F, and H) or for filamentous actin by using TRITC-labeled phalloidin (A, C, E, G, and I) or revealed for the presence of fluorescein-dextran staining (L). The N17Rac1 and N17Cdc42 constructs were microinjected at 0.5 μg/μl 4 h before the addition of GroPIns-4P. C. botulinum C3 transferase was microinjected at 160 μg/ml 15 min before the addition of GroPIns-4P. The results shown are representative of three to five independent experiments performed in triplicate. Bar, 20 μm.

Figure 3

GroPIns-4P rescues the EGF-induced effects on the actin cytoskeleton in Swiss 3T3 cells pretreated with MAFP or wortmannin. Serum-starved Swiss 3T3 cells without pretreatment (A and D) or pretreated with MAFP (15 μM for 15 min) (G and H) or wortmannin (100 nM for 10 min) (B, C, E, and F) were stimulated with EGF (20 ng/ml) alone or in the presence of GroPIns-4P (50 μM) for 2 (C) and 12 min (F and H). Cells were fixed and stained with TRITC-labeled phalloidin as described under MATERIALS AND METHODS. The results are representative of at least three independent experiments performed in triplicate. Bar, 20 μm.

Figure 4

Comparison between the stress fiber formation induced by leukotriene E₄ and by GroPIns-4P. Serum-starved Swiss 3T3 cells were treated with either leukotriene E₄ (5 μM) (A) or GroPIns-4P (50 μM) (B) for 12 min. Cells were fixed and stained with TRITC-labeled phalloidin as described under MATERIALS AND METHODS. The results are representative of three independent experiments performed in triplicate. Bar, 20 μm.

Figure 5

Localization of Rac and actin in GroPIns-4P-induced membrane ruffles. Subconfluent Swiss 3T3 cells were transfected with the pEGFP-Rac construct as described under MATERIALS AND METHODS. Twenty-four hours after transfection, cells were treated with GroPIns-4P (50 μM) for 2 min (D–F) or 12 min (G–I), fixed, and stained with TRITC-labeled phalloidin. Unstimulated control cells are shown in A–C. Arrowheads show areas of extensive membrane ruffles where Rac colocalizes with F-actin. The results are representative of three independent experiments performed in triplicate. Bar, 20 μm.

Figure 6

Regulation of Rac activity by GroPIns-4P. (A) Serum-starved Swiss 3T3 cells were treated with 50 μM GroPIns-4P for 2 min. Lysates from unstimulated and stimulated cells were processed as indicated under MATERIALS AND METHODS and analyzed by Western blotting with a mAb against Rac. Lysates from control cells were incubated with GTPγS (100 μM) or GDP (1 mM) for positive and negative controls, respectively. Rac-relative activation was calculated as the amount of PBD-bound Rac in stimulated cells, and normalized vs. the amount of Rac in unstimulated cells. (B) Western blots from one of the representative experiments showing activation of Rac. (C) Time course of Rac activation by GroPIns, GroPIns-4P, and GroPIns-4,5P₂. Serum-starved Swiss 3T3 cells were treated with these stimuli (50 μM) for the indicated times. Rac-relative activation was calculated as the amount of PBD-bound Rac in stimulated cells, and normalized vs. the amount of Rac in unstimulated cells. Data are means ± SE of three independent experiments. *p < 0.05, with respect to unstimulated cells, as analyzed by paired Student's t test.

Figure 7

Dose response of GroPIns, GroPIns-4P, and GroPIns-4,5P₂ on Rac activation. (A) Serum-starved Swiss 3T3 cells were treated with the indicated amounts of GroPIns, GroPIns-4P, or GroPIns-4,5P₂ for 5 min; lysates from unstimulated and stimulated cells were processed as indicated under MATERIALS AND METHODS and analyzed by Western blotting with a mAb against Rac. Rac-relative activation was calculated as the amount of PBD-bound Rac in stimulated cells, and normalized vs. the amount of Rac in unstimulated cells. Data are means ± SE of three experiments. *p < 0,05, with respect to unstimulated cells, as analyzed by paired Student's t test. (B) Western blots from one of the representative experiments showing the amount of activated Rac present in the affinity precipitations and the constant level of endogenously expressed Rac in the different cell lysates.

Figure 8

Nucleotide exchange and GTPase activity of Rac1. (A) Time course of the [³H]GDP/GTP exchange reaction on bacterially expressed GST-Rac1. The exchange reactions were performed in a GEF buffer as described under MATERIALS AND METHODS, in the absence (empty circles) or presence (filled circles) of 50 μM GroPIns-4P. The reactions were terminated by nitrocellulose filtration at the indicated times. The results are expressed as percentage of the [³H]GDP remaining bound at each time point vs. the amount of radioactivity at time 0 (100%). (B) Intrinsic GTPase activity of Rac1 was determined by a filter binding assay (see MATERIALS AND METHODS) in the absence (empty circles) or presence (filled circles) of 50 μM GroPIns-4P. The same reactions were performed in a GAP buffer containing 300 μg of lysates from Chinese hamster ovary cells in the absence (empty squares) or presence (filled squares) of 50 μM GroPIns-4P. Data in A are the averages of two independent experiments, expressed as means ± SE (n = 5) and in B they are from representative experiments expressed as means ± SE (n = 4).