R-Ras regulates beta1-integrin trafficking via effects on membrane ruffling and endocytosis

Abstract

Background: Integrin-mediated cell adhesion and spreading is dramatically enhanced by activation of the small GTPase, R-Ras. Moreover, R-Ras localizes to the leading edge of migrating cells, and regulates membrane protrusion. The exact mechanisms by which R-Ras regulates integrin function are not fully known. Nor is much known about the spatiotemporal relationship between these two molecules, an understanding of which may provide insight into R-Ras regulation of integrins.

Results: GFP-R-Ras localized to the plasma membrane, most specifically in membrane ruffles, in Cos-7 cells. GFP-R-Ras was endocytosed from these ruffles, and trafficked via multiple pathways, one of which involved large, acidic vesicles that were positive for Rab11. Cells transfected with a dominant negative form of GFP-R-Ras did not form ruffles, had decreased cell spreading, and contained numerous, non-trafficking small vesicles. Conversely, cells transfected with the constitutively active form of GFP-R-Ras contained a greater number of ruffles and large vesicles compared to wild-type transfected cells. Ruffle formation was inhibited by knock-down of endogenous R-Ras with siRNA, suggesting that activated R-Ras is not just a component of, but also an architect of ruffle formation. Importantly, beta1-integrin co-localized with endogenous R-Ras in ruffles and endocytosed vesicles. Expression of dominant negative R-Ras or knock down of R-Ras by siRNA prevented integrin accumulation into ruffles, impaired endocytosis of beta1-integrin, and decreased beta1-integrin-mediated adhesion. Knock-down of R-Ras also perturbed the dynamics of another membrane-localized protein, GFP-VSVG, suggesting a more global role for R-Ras on membrane dynamics. However, while R-Ras co-internalized with integrins, it did not traffic with VSVG, which instead moved laterally out of ruffles within the plane of the membrane, suggesting multiple levels of regulation of and by R-Ras.

Conclusions: Our results suggest that integrin function involves integrin trafficking via a cycle of membrane protrusion, ruffling, and endocytosis regulated by R-Ras, providing a novel mechanism by which integrins are linked to R-Ras through control of membrane dynamics.

Figure 1

Endogenous R-Ras and β₁-integrin localize to membrane ruffles. (A) The localization of endogenous R-Ras in Cos7 cells was determined by immunocytochemistry using antibody specific for R-Ras, which assembled into ruffles (arrows). (B) Immunofluorescence of a cell containing fluorescently labeled siRNA directed against R-Ras shows a loss of R-Ras from the plasma membrane, which occurred in 30/30 cells imaged. Perinuclear staining with this antibody in both A and B is non-specific, as determined by peptide competition experiments (not shown). (C) Western blot analysis to demonstrate R-Ras knock-down by transfection of either of two siRNA sequences targeting R-Ras (20 nM). (D-E) Imaging of fluorescent cholera toxin B-stained cells shows localization to ruffles. This localization was lost in 13/15 cells upon knockdown of R-Ras with siRNA.

Figure 2

GFP-R-Ras localizes to discrete plasma membrane domains. Cos-7 cells were transfected with GFP-R-Ras constructs, and the localization of each to the plasma membrane determined. Plasma membrane localization was noted in 79% of GFP-R-Ras(wt) (A, n≥100 cells for each) and in 91% of constitutively active GFP-R-Ras(38V) (B) but not in cells transfected with dominant negative GFP-R-Ras(41A) (C, < 5% of cells). In all three transfectants, there was a variable amount of perinuclear fluorescence which included small, dot-like vesicles positive for GFP-R-Ras (arrows in C). In addition, both wt and 38V-expressing cells contained medium- and large-sized vesicles (≥0.4 μm). (D) 3× zoomed view of dashed box in (A) highlights the appearance of plasma membrane fluorescence (arrowheads) and the assortment of vesicle and endosomal compartment sizes (arrows). (E) A cell transfected with GFP-R-Ras(38V) was co-stained with 4 μg/mL Alexa Fluor-555 cholera toxin B (top panel) to reveal co-localization of R-Ras with ganglioside-GM1 in ruffles (arrowheads). The lack of ruffle formation in GFP-R-Ras(41A) cells was independently confirmed by co-staining with cholera toxin B where ruffles were observed in 0 of the 26 cells imaged. (F) Western blot for GFP-R-Ras shows that protein levels were the same in GFP-R-Ras-(wt), -(38V) and -(41A) transfected cells in spite of the dramatic differences in localization of each isoform. (G) The number of endocytic compartments per cell was measured in >100 cells for each of the transfected constructs (bars). The percentage of cells that contained GFP-R-Ras localized to ruffles was quantified (triangles).

Figure 3

GFP-R-Ras colocalizes with Rab11, but not Rab5- or Rab4-positive endosomal compartments. Cos7 cells were transfected with GFP-R-Ras-(wt), -(38V), or -(41A) and then fixed and stained with antibodies against: (A-F) Rab11; (G-I) Rab5; or (J-L) Rab4. The largest vesicle compartments in 85% of -(wt) and 92% of -(38V) transfected cells were positive for Rab11 (arrows, for -(wt) n = 13 cells and -(38V), n = 12), but not Rab5 or Rab4 (<5%, n = 15 each). Intracellular GFP-R-Ras localized to smaller compartments and PM fluorescence lacked correlation with Rab fluorescence. Scale bar is 20 μm. (D-F) Insets of cells shown in A-C are shown at 3× zoom to highlight features.

Figure 4

R-Ras regulates integrin localization in the plasma membrane. (A) Immunofluorescence of fixed Cos7 cells using antibodies against R-Ras and β₁-integrin reveal colocalization of endogenous R-Ras and β₁-integrin at ruffles. Scale bar = 20 μm. (B) R-Ras activation enhances integrin localization to low-density lipid fractions. Lysates from T47D cells stably expressing R-Ras(38V), R-Ras(41A) or control (vector only) were fractionated on an Optiprep™ gradient, and analyzed by subsequent SDS-PAGE and immunoblotting. Activated R-Ras increased the amount of β₁-integrin (determined by antibody to the α₂ subunit) at the plasma membrane, specifically in the low-density microdomain that contains Src, which itself was not increased in this membrane fraction by activation of R-Ras. Integrin was not found in the bulk plasma membrane, identified by Rack1. (C) Cos7 cell adhesion on increasing fibronectin (FN) concentrations was reduced following the transfection of 20 nM siRNA directed against R-Ras.

Figure 5

β₁-integrin is endocytosed from ruffles in an R-Ras dependent manner. Alexa Fluor555 (red)-labeled β₁-integrin antibody was applied to live Cos7 cells expressing: (A-B) GFP-R-Ras-(wt), (C) -(38V), or (D) -(41A). There was significant overlap between β₁-integrin and R-Ras (yellow) at ruffles and in vesicles in GFP-R-Ras(wt) and GFP-R-Ras(38V) cells, which was absent in GFP-R-Ras(41A) cells. Note the uniform distribution of β₁-integrin in cells expressing GFP-R-Ras(41A) (D). Scale bar is 20 μm. (E-F) Cells were transfected with the following constructs; lanes 1, 6, and 7- GFP-R-Ras(wt), lane 2- GFP-R-Ras(38V); lane 3- GFP-R-Ras(41A), lane 4- GFP, lane 5- untransfected. Following a 30 min incubation with β₁-integrin antibody (with the exception of lane 6 as a control, where cells were exposed to antibody for <1 min), cells were biotinylated (with the exception of lane 7, as a control), washed, then lysed and cleared of the biotinylated antibody using strep-avidin beads. The presence of any remaining antibody would be a result of endocytosis during the incubation period, and was quantified by western blot. Densitometry of bands was normalized to lane 7 (wt with no biotinylation) and averaged data (± SEM) from three separate experiments is shown in E. Panel F is data from two experiments where whole cell lysate was probed for β₁-integrin as a loading control. Exemplar blots are shown above the bar graphs.

Figure 6

β₁-integrin traffics through R-Ras-positive vesicles. Time course images of (A) a GFP-R-Ras(wt) transfected Cos7 cell incubated in Alexa Fluor555 (red) labeled β₁-integrin antibody solution for 30 mins reveal the interplay of R-Ras and β₁-integrin (See Additional file 8, Movie S5). (B, C) The inset of images from the boxed region in A acquired at one minute intervals show that R-Ras and β₁-integrin traffic together to large endosomal vesicles. (D) Endocytosed components are then repackaged together and bud off from these organelles. Scale bar is 20 μm.

Figure 7

Plasma membrane ruffling is dependent on R-Ras. NMuMG cells stably transfected with GFP-β₁-integrin (A, a) contained many ruffles (in 23/23 cells imaged) whose retraction from the cell edge can be observed in the accompanying kymograph (lowercase letters, corresponding to each panel). Cells transiently transfected with two different siRNA sequences targeting R-Ras did not have ruffles in 16/16 cells imaged (B, b) even though β₁-integrin could be seen at thecell edge in portions of many cells. However kymography analysis of these areas reveals that the β₁-integrin is dynamic in cells replete with endogenous R-Ras. (C, c and D, d) GFP-VSVG localizes to the bulk plasma membrane, suggesting that R-Ras has a more general effect on membrane ruffling. Ruffling that was observed in 15/15 cells imaged was abolished in 15/15 cells that were transfected with R-Ras siRNA. Scale bars = 20 μm (A, B, C, D) or 10 mins (a, b, c, d).