MT1-MMP activity controls Cdc42 activation as well as EC lumen and vascular guidance tunnel formation. (A) ECs were infected with the indicated recombinant adenoviruses, which were GFP control, MT1-MMP full-length (FL), MT1-MMP FL with the EA mutant which makes the enzyme catalytically inactive, MT1-MMP without its cytoplasmic tail (tailless or ΔCT), and MT1-MMP EA mutant without its cytoplasmic tail. ECs were seeded in 3D collagen matrices, and real-time video analysis was performed to quantitatively measure lumen formation over the indicated time course. EC lumen areas were determined at each time point (n = 10, ± SD). (B) Cultures at 24 hours were fixed with paraformaldehyde and stained with anti–collagen type I antibodies to assess vascular guidance tunnel formation. Arrows indicate vascular guidance tunnels. Bar equals 100 μm. (C) ECs were infected with the indicated recombinant adenoviruses and cultures were established in 3D collagen matrices. After 16 hours, detergent lysates were prepared and incubated with S-protein agarose beads as described¹⁶  to assess whether the indicated components of the lumen signaling complex could be found to be coassociated with MT1-MMP. In addition, the lysates were incubated with GST-PAK-PBD beads to assess the degree of Cdc42 activation. In each case, the starting material lysates were examined for total Cdc42 levels.