Rac regulates endothelial morphogenesis and capillary assembly - PubMed (original) (raw)

Rac regulates endothelial morphogenesis and capillary assembly

John O Connolly et al. Mol Biol Cell. 2002 Jul.

Abstract

Endothelial cells undergo branching morphogenesis to form capillary tubes. We have utilized an in vitro Matrigel overlay assay to analyze the role of the cytoskeleton and Rho GTPases during this process. The addition of matrix first induces changes in cell morphology characterized by the formation of dynamic cellular protrusions and the assembly of discrete aggregates or cords of aligned cells resembling primitive capillary-like structures, but without a recognizable lumen. This is followed by cell migration leading to the formation of a complex interconnecting network of capillary tubes with readily identifiable lumens. Inhibition of actin polymerization or actin-myosin contraction inhibits cell migration but has no effect on the initial changes in endothelial cell morphology. However, inhibition of microtubule dynamics prevents both the initial cell shape changes as well as cell migration. We find that the small GTPase Rac is essential for the matrix-induced changes in endothelial cell morphology, whereas p21-activated kinase, an effector of Rac, is required for cell motility. We conclude that Rac integrates signaling through both the actin and microtubule cytoskeletons to promote capillary tube assembly.

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Figures

Figure 1

Figure 1

Phase contrast and electron micrographs of capillary tube formation. (A) Endothelial cells overlaid with Matrigel form a network of capillary tube-like structures composed of multiple cells with intercellular spaces or lumens. Cells are bipolar and are aligned in the axis of the tube. (B) High resolution phase contrast of a 1-μm transverse section through capillary-like tubes demonstrating lumen formation (arrows). (C) Electron micrograph demonstrating lumen in capillary tube. Lumens frequently contain debris or matrix-like material. (D) Some capillary tubes are composed of single endothelial cells containing a large vacuole or lumen. (E) Apoptotic cell surrounded by three cells and detached from matrix. (F) Modulation contrast image of capillary tube with vacuoles (arrows) and intercellular spaces (arrowheads). Bar, 100 μm in A, and 1 μm in C-E.

Figure 2

Figure 2

Morphological changes during capillary tube formation. (A) Phase contrast micrographs showing cell morphology changes during capillary tube formation. Final panel is a higher resolution view of mature tubules. Bar, 100 μm. (B) Endothelial cells were fixed and labeled with rhodamine phalloidin (i and ii), anti-VE cadherin antibody (iii), and anti-tyr-tubulin antibody (iv). Capillary tube structures had little organized filamentous actin structures and formed branching networks, with some cells extending protrusions along one another (arrow in iii). Cells formed adherens junctions (iii), and microtubules aligned along the long axis of the tubule (iv). Bar, 50 μm.

Figure 3

Figure 3

Rho is not required for capillary tube formation. (A) Cells were treated with the indicated concentration of toxin B 10463 or 1470 and were overlaid with Matrigel. (B) Capillary tube formation was inhibited in a dose-dependent manner. Results represent mean ± SD from at least three independent experiments performed in duplicate. *P < 0.01, **P < 0.001 in unpaired t test. Bar, 100 μm. (C) Cells were treated with the Rho kinase inhibitor Y-27632 (10 μM). Capillary tube formation was not affected, and intercellular spaces (arrows) resembling lumens were readily seen (upper panels). Cells were labeled with rhodamine phalloidin after overnight treatment with Y-27361and after tube formation in the presence of the drug. Lamellipodia formation was not affected, but actin stress fiber formation was inhibited (lower panels). Bar, 50 μm. (D) Cells were treated with C3 exoenzyme (3 μg/ml) or Y27632 (10 μM) and were overlaid with Matrigel. Tube formation was assessed after 24 h. Data represents mean ± SD from experiments performed in duplicate at least four times. No significant differences by unpaired t test.

Figure 4

Figure 4

Rac is required for capillary morphogenesis. (A and C) Cells were injected with the indicated constructs and GFP vector. Injected cells incorporated into capillary-like structures composed of at least three cells were scored as positive. Cells injected with N17 Rac were not incorporated into capillary tubes. Results (mean ± SD) from at least four independent experiments performed in duplicate are shown. **P < 0.001 in unpaired t test. Bar, 100 μM. (B and D) Cells were microinjected with N17 Rac and GFP or GFP expression vector alone, overlaid with Matrigel, and fixed and stained with rhodamine phalloidin after 3 h. A cell without Matrigel overlay is shown for comparison (upper panel). Bar, 10 μm. (D) Cells were injected with the indicated constructs and the percentage that formed protrusions after Matrigel overlay is shown. Results show mean ± SD from least four experiments performed in duplicate. *P < 0.05 in unpaired t test.

Figure 5

Figure 5

The actin cytoskeleton is not required for the early morphological response to Matrigel. (A and B) Cells were treated with Cytochalasin D (100 nM), ML7 (10 nM), and BDM (10 mM). Capillary tube formation was quantified 24 h after Matrigel overlay and results represent the mean ± SD from at least four independent experiments performed in duplicate. **P < 0.001 by unpaired t test. Bar 100, μm. Lower panel in A shows vacuole formation (arrows) still present in cells treated with cytochalasin D. (C) Cells treated with cytochalasin D and labeled with rhodamine phalloidin and antitubulin antibody. Protrusion formation was not affected by treatment with cytochalasin D. Bar, 10 μm.

Figure 6

Figure 6

The microtubule cytoskeleton is required for morphological response to Matrigel. (A**)** Cells treated with low-dose taxol (0.05 μM) do not form capillary-like structures. (B) Cells were labeled with antitubulin antibody and rhodamine phalloidin. Top panel shows growing HUVECs. Bottom panels show cells 3 h after Matrigel overlay either untreated or treated with taxol (0.05 μM) or Nocodazole (0.1 μM). Bar, 10 μM. (C) Taxol-treated cells injected with L61 Rac and overlaid with Matrigel. L61 Rac restored membrane ruffling but not protrusion formation. Bar, 10 μm in B and C.

Figure 7

Figure 7

Pak is required for endothelial motility but not the morphological response to Matrigel. (A) Cells were microinjected with the Pak inhibitory fragment (amino acids 83–149), kinase dead full-length Pak, or control vector and E-GFPC1. The number of injected cells incorporated into capillary structures is shown in and represents mean ± SD from at least three experiments performed in duplicate. No significant difference was detected (unpaired t test). (B) Cells were injected with Pak inhibitory fragment and photographed 24 h after Matrigel overlay (upper panels) or were fixed and stained with rhodamine phalloidin to demonstrate cell morphology (lower panels). Injected cells were incorporated into capillary-like structures and adopted an elongated bipolar morphology. Bar, 100 μm in upper panels and 10 μm in lower panels.

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