Palmitoylation regulates vesicular trafficking of R-Ras to membrane ruffles and effects on ruffling and cell spreading

Abstract

In this study we investigated the dynamics of R-Ras intracellular trafficking and its contributions to the unique roles of R-Ras in membrane ruffling and cell spreading. Wild type and constitutively active R-Ras localized to membranes of both Rab11- and transferrin-positive and -negative vesicles, which trafficked anterograde to the leading edge in migrating cells. H-Ras also co-localized with R-Ras in many of these vesicles in the vicinity of the Golgi, but R-Ras and H-Ras vesicles segregated proximal to the leading edge, in a manner dictated by the C-terminal membrane-targeting sequences. These segregated vesicle trafficking patterns corresponded to distinct modes of targeting to membrane ruffles at the leading edge. Geranylgeranylation was required for membrane anchorage of R-Ras, whereas palmitoylation was required for exit from the Golgi in post-Golgi vesicle membranes and trafficking to the plasma membrane. R-Ras vesicle membranes did not contain phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)), whereas R-Ras co-localized with PtdIns(3,4,5)P(3) in membrane ruffles. Finally, palmitoylation-deficient R-Ras blocked membrane ruffling, R-Ras/PI3-kinase interaction, enrichment of PtdIns(3,4,5)P(3) at the plasma membrane, and R-Ras-dependent cell spreading. Thus, lipid modification of R-Ras dictates its vesicle trafficking, targeting to membrane ruffles, and its unique roles in localizing PtdIns(3,4,5)P(3) to ruffles and promoting cell spreading.

Figure 1. R-Ras traffics to membrane ruffles in recycling vesicles. (A) R-Ras and H-Ras constructs used in this study, expressed as red or green fluorescent protein (RFP or GFP) fusions. G38V and G12V (starred) are constitutively active mutants of R-Ras and H-Ras, respectively. HVR C-terminal sequences are shown with post-translational modification sites indicated: palmitoyl, blue; geranylgeranyl, green; farnesyl, purple; mutations are shown in red. R-Ras(G38V/203)H-Ras(175–189) is activated R-Ras, residues 1–203, deleted in the HVR and replaced with the farnesyl-specific HVR of H-Ras. R-Ras(S43N) is constitutively inactive. (B) R-Ras and Rab11 trafficking in live, spread cells. RFP-R-Ras(G38V) (red) and GFP-Rab11 (green) were tracked in live NIH 3T3 cells by confocal microscopy. Images were acquired every 30 sec to facilitate vesicle tracking; representative images are shown. Left panels (1) show R-Ras anterograde transport with recycling endosomes. R-Ras transport vesicles included Rab11-containing RE and Rab11-negative puncta. Right panels (2) show retrograde R-Ras transport from membrane ruffles that do not contain Rab11. Arrowheads point to individual R-Ras/Rab11 vesicles tracked across images. The lower panel shows a representative image of the whole cell used for imaging, at lower magnification, with zones 1 and 2 indicated by white boxes. (C) R-Ras and H-Ras (wt) partially traffic in transferrin (Tf)-containing RE. Cells expressing GFP-R-Ras or -H-Ras (green) were labeled with Alexa546-Tf (red) by a pulse-chase scheme (see Materials and Methods) and viewed by live confocal microscopy with images acquired every 30 sec. Areas of co-localization are seen as yellow. Inset areas in white boxes are shown at 2x. Arrowheads point to Tf-negative R-Ras or H-Ras vesicles. Bars, 10 μm.

Figure 2. R-Ras and H-Ras segregate into distinct vesicles near the leading edge in a HVR-dependent manner. (A) Cells co-transfected with GFP-H-Ras(G12V) and RFP-R-Ras(G38V) were seeded at low density to allow for random migration, then fixed and imaged by confocal microscopy; merged images are shown at the bottom. The two boxed zones are shown at high magnification. H-Ras and R-Ras co-localized in vesicles proximal to the nucleus (1), but occupied distinct compartments (2) closer to the leading edge of the cell (dotted white line). (B) GFP-R-Ras(G38V) (green) and RFP-R-Ras(G38V/203)H-Ras(175–189) were co-expressed and imaged in live, randomly migrating cells. Merged images are shown at the bottom. White arrowheads point to selected vesicles containing either R-Ras(G38V) or R-Ras(G38V/203)/H-Ras(175–189). Bars, 7.5 μm.

Figure 3. Fast R-Ras trafficking to ruffles requires the R-Ras HVR, and is distinct from slow H-Ras trafficking. (A) Fluorescence recovery after photobleaching (FRAP) was assessed in a ~100 μm² boxed region at the leading edge of migrating cells expressing GFP-R-Ras, -H-Ras or -Rab11 fusions as indicated. GFP fluorescence intensities were measured for the selected area before (set to a normalized value of 100%) and after bleaching at 5 sec for the first minute, followed by 10 sec intervals for 3 min. Intensities were normalized with an adjacent, non-bleached zone, and are shown as percent recovery ± SD. At least 15 cells per type were measured; representative of three independent experiments. (B) Time of recovery (t_1/2) and mobile fraction (M_f) for fluorescence in leading edge membrane ruffles are shown ± S.D, calculated from exponential curve fits with a minimum R² of 0.95.

Figure 4. R-Ras palmitoylation is required for localization in vesicles and at the leading edge. (A) Cells co-expressing GFP-R-Ras(G38V) (green) with RFP-R-Ras(G38V) (red) harboring C-terminal site mutations were seeded in chamber slides and imaged live by confocal microscopy. Merged images are shown at the bottom. (B) RFP-R-Ras variants (red) co-expressed with Golgi marker GFP-GM130 (green). R-Ras(wt) partially localized to the Golgi (overlap seen as yellow). The C213S mutant was sequestered in the Golgi, whereas Golgi association was blocked by C215S mutation. Bars, 10 μm.

Figure 5. H-Ras vesicular targeting does not require R-Ras trafficking. RFP alone or fused to R-Ras variants as indicated (red) was co-expressed with GFP-H-Ras(wt) (green) and live cells were imaged by confocal microscopy. R-Ras Cysteine mutations altered R-Ras localization and blocked membrane ruffling but did not prevent H-Ras vesicular targeting. Bar, 10 μm.

Figure 6. Cell spreading and ruffling regulated by R-Ras lipid modification. Cells were transiently transfected with RFP vector alone (RFP), RFP-R-Ras fusions or RFP plus R-Ras shRNA as indicated. After 24 h, cells were seeded onto fibronectin-coated surfaces and membrane ruffling (A) and cell spreading (B) in RFP-positive cells were determined as described in Materials and Methods. (A) Percentages of transfected cells displaying edge ruffles for at least 100 cells per sample are shown + SEM *, p < 0.001. (B) Average cell areas for at least 100 cells per sample are shown + SEM *, p < 0.05; **, p < 0.004; ***, p < 0.003. Data are from three independent experiments each.

Figure 7. R-Ras vesicles are PI(3,4,5)P₃-negative, whereas R-Ras co-localizes with PI(3,4,5)P₃ in membrane ruffles from which R-Ras is recycled. RFP-R-Ras (red) was co-transfected with GFP-PH-Akt (green), as a marker for PI(3,4,5)P₃. (A) Dynamics of R-Ras(G38V) and PH-Akt monitored in a migrating cell. Images were acquired every 30 sec; 1 min intervals are shown. R-Ras was in perinuclear vesicles which trafficked toward the plasma membrane (e.g., white arrowheads), and in retrograde membrane ruffles. In contrast, PH-Akt was restricted to the retrograde ruffles, where it co-localized with R-Ras. Both proteins moved in retrograde fashion in the ruffles. In some cases R-Ras recycled from ruffles through vesicular structures (e.g., yellow arrowheads) that lacked PH-Akt. (B) RFP or RFP-R-Ras fusions as indicated, co-expressed with GFP-PH-Akt and imaged in live cells by confocal microscopy. Bars, 7.5 μm.

Figure 8. PtdIns(3,4,5)P₃ in membrane ruffles requires R-Ras lipid modification. (A) GFP-R-Ras fusions as indicated were imaged along with PtdIns(3,4,5)P₃ antibody staining in fixed cells. White arrowheads point to PtdIns(3,4,5)P₃ in ruffles, co-localized with R-Ras. Bar, 7.5 μm. (B) Cells transfected with the indicated GFP-R-Ras fusions were serum-starved (0.5%) for 2 d and either fed with serum (10%) for 30 min (+ FBS) or kept in starvation medium (-) before being lysed. Lysates were subjected to western blotting with antibodies to GFP, total Akt (Akt) or Akt phosphorylated at Ser473 (pAkt). Densitometric ratios of pAkt:Akt are shown as fold change relative to the starved cell ratio. Representative of three independent experiments. (C) GFP or GFP-R-Ras fusions as indicated were transfected, then immunoprecipitated from cell extracts using GFP antibodies. GFP and R-Ras fusions, and endogenous p110α subunit of PI3K were detected by immunoblotting the immunoprecipitate (IP) or whole cell lysate (WCL) fractions using GFP and p110α-specific antibodies. Representative of four independent experiments.