Fig. 9. Targeting of GFP-Rac and -Cdc42 fusion proteins to EC intracellular vacuole membranes during morphogenesis in three-dimensional collagen matrices. (A) Western blot analysis showing production of GFP-Rac1 and GFP-Cdc42 fusion proteins. (B) ECs expressing GFP alone were cultured in collagen matrices (24 hours) in the presence of carboxyrhodamine in the culture media to label EC lumens after rinsing out free dye. (i) Left panel: a photograph with a rhodamine filter; middle panel: a photograph with a fluorescein filter; right panel: merged image. (ii) Dual images indicating rhodamine labeling of vacuoles and lumens of ECs expressing GFP. (C) ECs expressing GFP-Rac1V12, GFP-Rac1wt and GFP-Cdc42wt were cultured in collagen matrices for 8-24 hours in the presence of carboxyrhodamine to label pinocytic intracellular vacuoles. After washing, ECs were photographed with the rhodamine or fluorescein filters. Note the labeling of vacuoles with GFP-Rac1 or GFP-Cdc42 constructs. ECs expressing either GFP-Rac1V12 (D) or GFP-Cdc42 wt (E) were cultured in collagen matrices and photographed to indicate the targeting of Cdc42 and Rac to vacuole membranes (D,E). Arrowheads indicate labeling of vacuolar membranes. Bar=20µm in panel B, 30 µm in panels C-E.

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