Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors

Abstract

This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2-hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4-green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2-hybrid and glutathione-S-transferase pulldown analyses show Robo4 binding to a Wiskott-Aldrich syndrome protein (WASP), neural Wiskott-Aldrich syndrome protein, and WASP-interacting protein actin-nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4-mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation-promoting factors to promote endothelial cell migration.

Figure 1

Robo4 siRNA inhibits wound healing and Matrigel tube formation. A) Immunoblot of 1 nM R4(1) siRNA in HUVECs at different time points. B) qPCR analysis of Robo4 and ISG20 mRNA levels after transfection of 1 nM Robo4 siRNA oligos. Results normalized to flotilin2 loading control. C) Wound healing assay with Robo4 siRNA-transfected HUVECs. D) Measurements of the distance covered by each wound relative to the mock control. ***P < 0.001 vs. scrambled control. E) Matrigel with Robo4 siRNA-transfected HUVECs. Scale bars = 200 μm. F) Measurement of the average number of nodes present in Matrigel assay under the different conditions. ***P < 0.001 vs. mock control. Error bars ± sd. M, mock transfected; S, scrambled negative control duplex; R4(1), Robo4 duplex 1 siRNA; R4(2), Robo4 duplex 2 siRNA.

Figure 2

Robo4-GFP induces filopodia formation in HUVECs. A) FL-Robo4-GFP and GFP alone expressed in HUVECs and stained with TRITC phalloidin. Arrows indicate filopodia. Scale bars = 10 μm. B) FACS scan with a monoclonal antibody that recognizes the extracellular domain of Robo4 (MR7). Green, MR7; blue, isotyped IgG2a negative control. C) Quantitative analysis of filopodia numbers after expression of GFP alone or FL-Robo4-GFP (***P<0.001). Error bars ± se.

Figure 3

WASP and NWASP are expressed in HUVECs. A) Domain structure of full-length Robo4, the Robo4 yeast 2-hybrid bait, and intracellular Robo4-HA-GST constructs. Ig, immunoglobulin-like domain; Fn3, fibronectin type 3 domain; Tm, transmembrane domain; CC0/2, cytoplasmic conserved domains 0 and 2. B) Immunoblot analysis of WASP and NWASP expression in a variety of cell lines and HUVECs.

Figure 4

Robo4 binds WASP, NWASP, and Mena in GST pulldown analyses. A) Immunoblots of HA-GST alone and FL-WASP-HA-GST pulldowns with full-length Robo4-myc and a WIP-myc positive control. B) Immunoblots of HA-GST alone and Int-Robo4-HA-GST pulldowns with NWASP-myc, WASP-GFP, and a Mena-myc positive control.

Figure 5

Robo1 siRNA inhibits in vitro angiogenesis. A) Immunoblot analysis of Robo4 and Robo1 protein expression across a panel of lysates. B) Coimmunoprecipitation analysis of Robo1-Robo4 binding. C) Titration analysis determining the optimal concentration of siRNA duplexes [R1(1) and R1(2)] required for maximum Robo1 knockdown. D) Matrigel assay with R1(1) and R1(2) siRNA-transfected HUVECs. Scale bar = 200 μm. E) Measurement of the number of nodes present in Matrigel assay under the different conditions. **P < 0.05 vs. mock control. Error bars ± sd.

Figure 6

Robo1 and Robo4 cooperate in filopodia formation. A) Quantification of the number of filopodia in mock transfected or Robo1 siRNA-treated HUVECs expressing GFP alone or FL-Robo4-GFP. B) Modified Boyden chamber assay analyzing the migration of HUVECs to different stimuli. ***P < 0.001. Error bars ± se.