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The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices

Department of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, College Station, Texas, 77843-1114, USA

View larger version (58K): [in a new window]   Fig. 1. (A) Time course of EC morphogenesis illustrating the formation of vacuoles and lumens in three-dimensional collagen matrices. Arrowheads indicate vacuole structures (8 hr) and nuclei (48 hr); arrows indicate lumen structures. Bar=50 µm. (B) Electron micrographic images of ECs undergoing morphogenesis in three-dimensional collagen matrices. Bar=5 µm.

View larger version (62K): [in a new window]   Fig. 2. C. difficile toxin B completely inhibits EC morphogenesis in both collagen and fibrin matrices. (A) Photographs of three-dimensional cultures fixed and stained with toluidine blue. ECs were allowed to undergo morphogenesis in collagen and fibrin matrices for 8, 24 or 48 hours as indicated. Arrowheads indicate vacuoles; arrows indicate developing lumins. Cells pretreated with toxin B are shown in lower panel. Note the complete lack of vacuoles and lumenal structures in toxin B group. Quantification of cells forming vacuoles and lumens in three-dimensional collagen (B) and fibrin matrices (C). Cultures were fixed and stained at various time points following pretreatment with toxin B and C3 ribosyltransferase (collagen only). Data are expressed as a percentage of cells forming vacuoles±s.d. (n=3). Bar=30 µm.

View larger version (135K): [in a new window]   Fig. 3. C. difficile toxin B completely inhibits pinocytic uptake into intracellular vacuoles during EC morphogenesis. Three-dimensional EC cultures were allowed to develop in the presence of carboxytetramethyl rhodamine added to culture media (2 µg/ml). After 4 hours, collagen matrix was digested with collagenase for 10 minutes at 37°C in M199. Cells were plated onto pronectin-F coated coverslips and allowed to attach before rinsing off the free dye. Coverslips were mounted onto culture media and photographed live under phase contrast (A,C) and fluorescence (B,D) microscopy. The control group (A,B) are shown with toxin-B-treated cells (C,D). Bar=50 µm.

View larger version (12K): [in a new window]   Fig. 4. Adenoviruses expressing mutant forms of Rho GTPases induce specific expression of each protein in ECs. ECs were infected with either DN or CA Rho, Rac or Cdc42 adenoviruses. Cells were allowed 24 hours to express proteins before placing them in three-dimensional collagen matrices. Cultures were allowed to proceed for 24 hours prior to preparing extracts. Samples were loaded onto 12% SDS-PAGE gels, blotted to PVDF and probed with GTPase-specific antibodies. Infection with GFP control adenovirus showed no signal (not shown).

View larger version (18K): [in a new window]   Fig. 5. Influence of Rho GTPase mutants on EC vacuole and lumen formation in three-dimensional collagen matrices. Endothelial cells were infected for 24 hours prior to resuspension in three-dimensional collagen matrices. Cultures were allowed to proceed for 24 (A,B) or 48 (C,D) hours in the presence (A,C) or absence (B,D) of TPA before fixation in paraformaldehyde. Quantitation was carried out using fluorescence microscopy. The average vacuole formation expressed as a percentage of cells forming vacuoles is shown±s.d. (200 cells counted per group; n=3).

View larger version (29K): [in a new window]   Fig. 6. The Cdc42 and Rac1 GTPases are required for EC morphogenesis in three-dimensional extracellular matrices. Recombinant adenoviruses were used to express either dominant-negative (DN) or constitutively active (CA) Rho GTPases in ECs. ECs were cultured in either collagen or fibrin matrices, and were quantified by counting the percentage of fluorescent ECs with vacuoles over time. (A) Cultures formed with phorbol ester; (B) Cultures formed without phorbol ester. The average vacuole formation expressed as a percentage of cells forming vacuoles is shown±s.d. (200 cells counted per group; n=3).

View larger version (62K): [in a new window]   Fig. 7. Expression of constitutively active Cdc42 blocks EC vacuole formation in three-dimensional collagen matrices. Upper panel: ECs were photographed using phase contrast (A,C) and fluorescence microscopy (B,D-F). ECs were treated with control GFP adenovirus (A,B,E) or Cdc42 CA adenovirus (C,D,F). Arrowheads indicate the presence of EC vacuoles. Shown in A-D are ECs digested from collagen assays (4 hours) and plated onto coverslips. Arrowheads indicate the presence of EC vacuoles. (E,F) Fluorescent images of intact cultures fixed at 24 hours of morphogenesis. Arrowheads indicate vacuole and lumen formation. Lower panel: magnified images of intact cultures using ECs infected with GFP, CA Rac and CA Cdc42 adenovirus at 24 hours in three-dimensional collagen matrices.

View larger version (17K): [in a new window]   Fig. 8. Induction of Cdc42 protein during EC morphogenesis in three-dimensional collagen matrices. ECs were cultured in collagen matrices for various times and extracts were prepared at the time points indicated. Samples were run on SDS-PAGE gels, blotted to PVDF membranes and probed with monoclonal antibodies specific to either Cdc42 or control G3PDH.

View larger version (57K): [in a new window]   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.

View larger version (101K): [in a new window]   Fig. 10. Confocal analysis of GFP-Rac constructs targeting to vacuolar and lumenal membranes. (A) GFP-Racwt and (B) GFP-Rac1V12 localize to vacuolar EC membranes (arrowheads) as well as lumenal membranes (open arrows). Vacuolar fusion events (arrows) appear to occur between vacuoles and the lumenal membrane. Serial sections are 1 µm thick. Bar=20 µm.

View larger version (88K): [in a new window]   Fig. 11. Confocal analysis of GFP-Cdc42wt localization to EC vacuolar membranes and lumenal structures. (A) Targeting of GFP-Cdc42wt to vacuolar (arrowheads) and lumenal membranes (arrows). (B) Three-dimensional reconstruction of a multicellular structure containing a lumen (L). Left: propidium iodide (PI) staining of EC cultures indicating multiple cells interconnect to form a lumenal compartment. Nuclei are indicated with arrowheads. Right: GFP-Cdc42wt (shown in red) localizes to lumenal membranes (arrows).

View larger version (13K): [in a new window]   Fig. 12. Expression of a GFP fusion protein containing the VCA domain of N-WASP interferes with EC vacuole formation. (A) ECs were induced to express GFP control or GFP-VCA (a C-terminal domain of N-WASP that activates the Arp 2/3 complex). Extracts were made and samples were run on SDS-PAGE, blotted to PVDF membranes and probed with anti-GFP antibodies. (B) Quantitation of the effects of GFP-VCA chimera on EC vacuole formation at 24 hours of EC morphogenesis. (C) ECs expressing GFP-VCA were cultured in collagen matrices and photographs were taken after 24 hours of culture. Images show ECs in three-dimensional collagen expressing GFP-VCA that are unable to form lumenal structures but form numerous processes. Bar=25 µm.