EB1, p150Glued, and Clasp1 control accumulation of acetylated and detyrosinated tubulins in a subapical distribution during EC tubulogenesis in 3D collagen matrices. (A) ECs were treated with control (luciferase), p150Glued, Clasp1, or EB1 siRNAs and suspended within 3D collagen matrices for 24 hours. After fixation, cultures were immunostained with antibodies to the indicated tubulins and modified tubulins. Cultures were costained with AlexaFluorX-conjugated phalloidin to label F-actin and Dapi to label nuclei. Bar equals 20 μm. (B) Day 5 embryonic quail chorioallantoic membranes were stained with the indicated antibodies to label tubulins, with phalloidin to label F-actin, and Dapi to label nuclei within endothelial cells. A subapical distribution of modified tubulins is noted in the developing embryonic quail vasculature along with a basal distribution for F-actin; this recapitulates what we observe using our in vitro EC tubulogenesis model (A). L indicates lumen space. Bar equals 20 μm.
