Fig 1: Myo1e is enriched at the ventral layer of the podosome core. (A) Schematic of axial distribution analysis of podosome core components. Myo1e, F-actin, and various actin-binding proteins in MEF-Src are imaged by confocal microscopy with 200-nm steps along the z-axis. Images from 0 to 2 µm above the adhesion plane are used for axial distribution analysis. (B) MaxZ, a self-defined parameter, represents the axial position with the highest intensity and is used to compare the axial distribution of each podosome component. (C) MaxZ values of Myo1e, F-actin (labeled by UtrCH), ß-actin, and various actin regulators, including filament nucleating factors (Arp3, Cortactin, CARMIL1, Coronin1b, N-WASP, Arpin [inhibitory role]), G-actin–binding proteins (ABP1, CAP1, WIP, Profilin1), depolymerizing factors (Twinfilin1, Cofilin1, Gelsolin), capping proteins (EPS8, CD2AP, CapZ-beta2, CAPG, TMOD3), and cross-linking proteins (Plastin3, VASP, Mena, Alpha-actinin1, FilaminA, Fascin1). Statistical information is in Supplemental Figure S1, B and C. (D) Three-dimensional confocal images of mApple-Myo1e and EBFP2-UtrCH (F-actin marker) in the podosome arc. Inset, the boxed region with the podosome arc (20 × 20 µm2). Myo1e is preferentially distributed at the ventral layer of F-actin. (E) Photoconversion of cytosolic mEOS2-ß-actin reveals the dynamics of actin polymerization at the podosome. After 405-nm spot illumination (cyan circle), newly converted mEOS2-ß-actin (visualized by 561 nm excitation) are first incorporated into the ventral layer of the podosome. (F) Intensity-time plot of photoconverted mEOS2-ß-actin at Z1 (0 µm, ventral layer) and Z9 (1.6 µm above the ventral layer, n = 6). Error bars represent SEM. Scale bars represent 10 µm.