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First published online 14 March 2006
doi: 10.1242/jcs.02835

Journal of Cell Science 119, 1307-1319 (2006)
Published by The Company of Biologists 2006
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The small GTPase R-Ras regulates organization of actin and drives membrane protrusions through the activity of PLC{epsilon}

Aude S. Ada-Nguema1, Harry Xenias2, Michael P. Sheetz2 and Patricia J. Keely1,*

1 Department of Pharmacology, University of Wisconsin-Madison, Madison, WI 53706, USA
2 Department of Biological Sciences, Columbia University, New York, NY 10027, USA


Figure 1
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  		Fig. 1. Cells expressing constitutively activate R-Ras spread by continuously extending lamellipodia with few retraction events. (A) Total area of spread MCF10A cells comparing cells expressing vector alone with those expressing R-Ras38V. Bar graph represents the average total spread area of 23 R-Ras cells or 16 control cells ± s.e.m. Note that R-Ras-expressing cells are more than twice as spread as control cells. (B) TIRF images of an individual control and R-Ras38V-expressing cell during spreading on 10 µg/ml fibronectin. Brighter regions represent areas of greater contact with the ECM. (See Movies 1 and 2, in supplementary material.) (C) Analysis of protrusion and retraction events for control and R-Ras38V-expressing cells. Images such as those shown in B were analyzed as described (Giannone et al., 2004Go) to characterize cell-edge dynamics. Top traces show the increase in total cell area over time for a representative control and R-Ras38V cell. Radial edge velocity maps of the entire membrane periphery plotted as a function of time and arc length are shown below, where arc length denotes the separation between two polar coordinates along the periphery of the cell. Membrane activity was determined and expressed as the velocity of protrusion events (warm colors) or retraction events (cool colors). Dotted lines delineate the phases in cell spreading.

 

Figure 2
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  		Fig. 2. Expression of constitutively active or dominant-negative R-Ras regulates the organization of the peripheral actin network and formation of filopodia. (A) Stable expression of constitutively active R-Ras promotes a strong, branched peripheral actin network. MCF10A cells stably transfected with control vector or constitutively active R-Ras38V were plated on collagen I (30 µg/ml), fibronectin (20 µg/ml) or poly-L-lysine (0.01%) for 30 minutes. The cells were then fixed and stained with TRITC-phalloidin to visualize F-actin. Note the dense peripheral actin and loss of filopodia in R-Ras38V-expressing cells. A bubble is present in the lower right corner of control p-lysine cells. (B) Dominant-negative R-Ras enhances filopodia formation in MCF10A cells. MCF10A cells were transiently co-transfected with pCMV-R-Ras41A and GFP to identify transfected cells. 48 hours post-transfection, the cells were assayed for their ability to spread on fibronectin-coated coverslips for 45 minutes. Arrow indicates a cell transfected with R-Ras41A. (C) R-Ras regulates membrane protrusion and filopodia formation. Cos7 cells were transiently transfected with GFP:R-RasWt, GFP:R-Ras38V or GFP:R-Ras41A. 48 hours post-transfection, cells were detached and were allowed to adhere and spread on fibronectin (30 µg/ml) for 1 hour. Cells were then fixed and stained for F-actin. Overlay images of F-actin (red) and GFP-R-Ras (green) are shown. Note that GFP-R-Raswt localizes to leading edges, whereas GFP-R-Ras41A is not found on the plasma membrane, and GFP-R-Ras38V is localized more uniformly around the cells at the ruffling lamellipod. Representative phenotypes for each transfection are shown, and are labeled A, B and C. (D) Quantification of phenotypes shown in C. Note that expression of constitutively active R-Ras38V dramatically enhances phenotype C, which has a strong peripheral actin network and no filopodia, whereas expression of dominant-negative R-Ras41A promotes phenotype B, which has enhanced filopodia formation. Values represent the mean of three experiments ± s.d. Bars, 10 µm (A-C).

 

Figure 3
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  		Fig. 3. Endogenous R-Ras contributes to cell spreading. MCF10A cells were transfected with siRNA oligonucleotides of a random sequence (control) or targeted against R-Ras. (A) siRNAs directed against R-Ras are effective. Half of the transfected cells were lysed, and equal cell numbers for control and R-Ras siRNA separated by SDS-PAGE. Transfers were immunoblotted with antibodies against R-Ras to demonstrate a significant knockdown with the anti-R-Ras siRNA. (B) Cell spreading is diminished by R-Ras knockdown. The other half of the cells transfected were detached and allowed to spread on 20 µg/ml fibronectin for 45 minutes, fixed, and cell area was analyzed as described in the Materials and Methods using Image J software. Results for 20 cells are graphed in a box-and-whisker plot such that the horizontal line represents the mean, the box represents the 25% and 75% confidence intervals, and the error bars represent the s.d. ***P<0.001 compared with cell area of control cells using a two-tailed unpaired t-test.

 

Figure 4
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  		Fig. 4. The formation of the ruffling lamellipod in R-Ras-expressing cells depends on PLC activity. (A) Pharmacological inhibitors of intracellular Ca2+ and PLC inhibit R-Ras-enhanced adhesion. To assess adhesion, control and R-Ras38V-expressing cells were loaded with 20 µg/ml calcein-AM for 20 minutes. The cells were then pretreated with the indicated pharmacological agent (DMSO, EGTA, BAPTA or the broad-specificity PLC inhibitor, U73122) for 30 minutes, and allowed to adhere onto 96-well plates coated with 20 µg/ml fibronectin for 15 and 50 minutes. The number of adherent cells was evaluated with a fluorescent plate reader, using a standard curve to determine cell number from calcein fluorescence. (B) Inhibition of phospholipase C reverts the strong peripheral actin ring induced by expression of R-Ras38V. Control and R-Ras38V cells were pretreated with 1 µM of the phospholipase C inhibitor, U73122, for 30 minutes followed by adhesion onto 20 µg/ml fibronectin for 30 minutes. The cells were stained with Alexa-phalloidin to visualize F-actin. (C) Inhibition of phospholipase C diminishes cell spreading. The area of cells prepared as in B was analyzed with Image J, as in Fig. 3. Treatment of both control and R-Ras38V-expressing cells with U73122 inhibits the measured area of cell spreading. Data represent measurements of >20 cells and are plotted as a box-and-whisker plots ± s.d. ***P<0.001.

 

Figure 5
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  		Fig. 5. Spreading and the strong peripheral actin ring induced by R-Ras is dependent on intracellular, but not extracellular Ca2+. (A) The depletion of cytosolic Ca2+, but not extracellular Ca2+ reduces the peripheral actin ring and rescues filopodia formation in R-Ras38V-expressing cells. Control and R-Ras38V cells were pretreated for 20 minutes with 10 µM BAPTA-AM for 20 minutes to chelate intracellular Ca2+. The cells were then plated on 20 µg/ml fibronectin-coated coverslips for 30 minutes in the presence of 10 µM BAPTA-AM (pre-adhesion) then fixed and stained for F-actin. Alternatively, untreated Control and R-Ras38V cells were plated on 20 µg/ml fibronectin until they were adherent followed by treatment with 10 µM BAPTA-AM for another 30 minutes (post-adhesion). To determine the role of external Ca2+, cells were pretreated with 2 mM EGTA for 20 minutes and allowed to spread on 20 µg/ml fibronectin (pre-adhesion), then fixed and stained for F-actin. Alternatively, cells were allowed to adhere to fibronectin until they were adherent before the addition of 2 mM EGTA for another 30 minutes. Results are representative of three such experiments. Bar, 11 µm. (B) Chelation of intracellular Ca2+ reduces cell spreading. The spread area of more than 20 cells from the experiment shown in A was quantified and presented as described for Fig. 3. Treatment with BAPTA-AM significantly diminishes cell spreading in both control and R-Ras38V-expressing cells. ***P<0.001; ns, not significant. (C) Endoplasmic reticulum Ca2+ stores are depleted in R-Ras cells. Control and R-Ras38V cells were loaded with 20 µg/ml of the Ca2+ indicator Indo-1-AM for 20 minutes at 37°C. The cells were then stimulated with 4 µM thapsigargin (TG) or 60 µM ionomycin for the duration of the measurement and Ca2+ flux was measured by flow cytometry. TG- and ionomycin-induced Ca2+ flux are diminished in cells expressing R-Ras38V.

 

Figure 6
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  		Fig. 6. R-Ras interacts with PLC{epsilon}. (A) R-Ras co-immunoprecipitates with PLC{epsilon} in Cos7 cells. Cos7 cells were transfected with Flag-PLC{epsilon} and control vector (lane 1), R-RasWT (lane 2) or R-Ras38V (lane 3), lysed and PLC{epsilon} immunoprecipiated with anti-Flag antibody. Association with R-Ras was determined by immunoblotting with anti-R-Ras antibody. (B-D) Active R-Ras interacts with the Ras-association domain, RA2, of PLC{epsilon}. A pulldown assay was performed by incubating GST-RA2 with lysates from MCF10A cells expressing-R-Ras38V (B). Alternatively, lysates were obtained from control MCF10A cells that were stimulated with 8-CTP-2Me-cAMP for 30 minutes (C) or stimulated by adhesion to fibronectin for 45 minutes to activate endogenous R-Ras. In B-D, lysates were incubated with GST (negative control), GST-Raf-RDB (a robust, positive control), and PLC{epsilon}-RA2 domain for 2 hours at 4°C. Often endogenous R-Ras is noted as a doublet band, as seen here. (E) Constitutively active R-Ras activates PLC{epsilon} activity. Cos7 cells were transiently transfected with PLC{epsilon} and pCMV vector, pCMV-R-Ras41A or pCMV-R-Ras38V and a PLC activity assay was performed 48 hours post transfection. Data represent the means of two separate experiments each performed in duplicate. The minimum value of the y-axis is 100 cpm, which represents the background count. (F) Immunoblot for the PLC assay shown in E, demonstrating expression of Flag-PLC{epsilon} as well as R-Ras41A and R-Ras38V in transfected, unlabelled Cos7 cells.

 

Figure 7
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  		Fig. 7. PLC{epsilon} mediates R-Ras signaling to membrane protrusions. MCF10A cells stably expressing either a control vector or R-Ras38V were transfected with control siRNA, or 50 nM of pooled siRNA directed against PLC{epsilon}. (A) Cells were harvested 72 hours later, normalized by cell number and evaluated for PLC{epsilon} knockdown by immunoblotting with anti-PLC{epsilon}. (B) PLC{epsilon} siRNA diminishes the measured area of spreading. Although the effect is not statistically significant, it does extend the mean and range of cell areas measured downward as shown by the box-and-whisker plots. (C) MCF10A cells treated with siRNA against PLC{epsilon} were allowed to adhere onto 20 µg/ml fibronectin for 45 minutes, fixed, and then stained with Alexa-phalloidin to evaluate F-actin. Arrows indicate four representative phenotypes (phenotypes A-D) observed and quantified in D. Two different fields are shown for PLC{epsilon} siRNA to show the range of phenotypes observed. Phenotypes A, B and C are similar to those quantified in Fig. 2. Note the appearance of a new phenotype, D, characterized by multiple membrane protrusions. (D) Quantification of the phenotypes labeled in B for control (pZIP) or R-Ras38V-expressing cells.

 

Figure 8
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  		Fig. 8. Proposed signaling pathway by which R-Ras promotes strong lamellipodia formation and cell spreading. We propose that activated R-Ras directly binds to PLC{epsilon} at the plasma membrane and activates the integrin-PLC-Ca2+-signaling pathway that is required for the initiation of cell protrusion following integrin activation. The stimulation of PLC mobilizes Ca2+ from the endoplasmic reticulum. High cytoplasmic Ca2+ levels then activate Ca2+-dependent actin-binding proteins, such as capping proteins, causing an increase in actin polymerization. Additionally, Ca2+ release may also enhance the recruitment and accumulation of actin and/or adhesion components to the plasma membrane. The overall outcome is the formation of a strong ruffling lamellipod that drives leading-edge protrusion, enhancing cell spreading and adhesion.

 

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© The Company of Biologists Ltd 2006