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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.
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