Posttranslationally modified tubulins, including acetylated and detyrosinated tubulins, accumulate in a subapical membrane location during EC tubulogenesis in 3D extracellular matrices, while F-actin is predominantly observed in a basal distribution. (A) ECs were cultured in 3D collagen matrices for 24 or 72 hours and then fixed for immunostaining for the indicated molecules. L indicates lumen space. Bar equals 20 μm. (B) The 72-hour cultures were stained for acetylated tubulin (green) and F-actin (red); a representative image is shown in the upper right. A 3D reconstruction of these images is shown in the lower right. L indicates lumen space. Bars equal 20 μm. The schematic diagram illustrates major findings in this work including a fundamental role for microtubule assembly and posttranslational modifications in EC lumen and tube formation by supporting the development and maintenance of the apical membrane surface and, thus, controlling cytoskeletal apical–basal polarization of EC-lined tubes.