MT1-MMP activity and the Jam-B and Jam-C cytoplasmic tails are necessary for Cdc42 activation in 3D collagen matrices but not on 2D collagen surfaces. (A) EC cultures were established on 2D collagen substrates or within 3D collagen matrices in the presence or absence of 5μM GM6001 and otherwise were cultured under the same conditions. The ECs on 2D collagen substrates were seeded in a subconfluent state to mimic the lack of cell-cell contacts observed in our assays that mimic vasculogenesis in 3D collagen matrices. Cell lysates were prepared at 16 hours in such a manner that the volume of lysis buffer used in relation to total cell number was identical in both cases. Lysates were incubated from the different culture conditions with either GST-PAK-PBD (to bind Cdc42-GTP) or Rhotekin beads (to bind RhoA-GTP) to assess the level of either activated Cdc42 or RhoA. Starting material lysates were also probed on Western blots for either Cdc42 or RhoA. (B) ECs were infected with the indicated recombinant adenoviruses carrying GFP-Cdc42, S-GFP-Cdc42, MT1-MMP, MT1-MMP-S, Jam-B wt, and Jam-B wt-S. ECs were then cultured in 3D collagen matrices for 16 hours. At this time point, detergent lysates from the 3D cultures were prepared and incubated with S-protein agarose beads to selectively bind S-tagged recombinant proteins and their associated proteins as described.16 Eluates were probed on Western blots for the indicated proteins. EC starting lysates were from control 3D cultures at 16 hours to assess whether the proteins examined were present at this stage of tube formation. (C) ECs were infected with the indicated wild-type and mutant Jam viruses as well as control GFP virus, and the cells were seeded on 2D collagen surfaces or within 3D collagen matrices for 16 hours. At this time, detergent lysates were prepared and incubated with GST-PAK-PBD beads to assess the degree of Cdc42 activation. Eluates were probed on Western blots for Cdc42 and starting lysates were similarly examined for Cdc42 levels.
