Fig. 3

F-actin is associated with both retraction fibers and migrasomes and tubulin is restricted to migrasomes. A–E Primary human MSCs were cultured on fibronectin-coated glass coverslips for 24 h before being processed for CLSM (A–D) and total internal reflection fluorescence (TIRF) microscopy (E). PFA-fixed cells were saponin-permeabilized prior immunolabeling with anti-α-tubulin antibodies and staining by SiR-Actin (A–C, E) or phalloidin (D) and fluorophore-conjugated WGA, which label F-actin and cellular membrane, respectively. The distribution of F-actin and tubulin in retraction fibers and migrasomes shown in A is highlighted in the insets (a′, a″). A 3D render of a single migrasome highlights the presence of actin and tubulin therein (B). The distribution of cytoskeleton constituents in early-stage and fully formed migrasomes (C, from left to right panels, respectively, see also Additional file 1: Fig. S1) and in cell-free, detached migrasome (D) is shown. The movement of SiR-Actin-stained actin filaments in retraction fibers of living cells is recorded by TIRF video microscopy at a layer height of 250 nm (E). White and yellow arrowheads point the movement of actin filaments within the time frame presented. Elapsed time in minutes is shown on the top-right corner. The images presented in B and E are excerpted from the Additional file 4: Video S3 and Additional file 5: Video S4, respectively. Scale bars, 10 μm (A, E); 5 μm (a′, a″); 1 μm (C, D)