Figure 2. Cortical stress fibers assemble de novo from the actin cortex.

TIRF time-lapse imaging of a migrating human osteosarcoma (U2OS) (A) and mouse embryonic fibroblast (MEF) cell (B) expressing LifeAct-TagGFP2 (gray) and vinculin-mApple (magenta). Selected time-lapse frames from the magnified areas (yellow box) are shown on the right as separate channels and merged frames. These demonstrate the de novo emergence of a cortical stress fiber from the ventral actin cortex. Blue arrows illustrate F-actin bundling and orange arrows point the maturation of the vinculin-positive focal adhesions. See also Videos 1 and 2. Scale bars 10 µm and 2 µm for whole cell images and time-lapse zoom-ins, respectively. Imaging interval 30 s.

Figure 2—figure supplement 1. Cortical stress fibers emerge from the ventral actin cortex through different intermediate assembly states.

(A) Photoactivation of PA-GFP-actin (gray) at both cortical and dorsal stress fibers in human osteosarcoma (U2OS) cells that additionally expressed Lifeact-mKate1.31 (red) and miRFP670-paxillin (magenta). PA-GFP-actin was always activated adjacent to a paxillin positive adhesion. The images of entire cells after photoactivation are shown on the left. Kymographs of PA-GFP-actin dynamics from the selected stress fibers indicated in the whole cell images illustrate that, unlike dorsal stress fibers (4), cortical stress fibers do not elongate through actin polymerization from the adhesion (1,2), and can occasionally even exhibit slow actin flow towards the adhesion (3). All kymographs were oriented in a way that the adhesion is facing to the top. Black arrows on the kymographs indicate the activation time-point, preceded by three time-lapse frames, and the dashed lines movement of activated stress fiber segments. Imaging interval was 10 s, and scale bars are 10 µm in whole cell images. (B) Quantification of the actin polymerization rates from focal adhesions. Actin polymerization rates (µm/min) are shown as half-violin plot, displaying binned individual data points and mean with ±1 SD. p = 5.22 × 10⁻¹⁵ (two-sample t-test). Polymerization rates for individual data points/kymographs were obtained through plot profiles, subtracting the distance of the peak intensities at time points 10 s and 300 s after photoactivation. N (cortical SF) = 13 cells; 33 foci and N (dorsal SF) = 6 cells; seven foci. (C) Cortical stress fiber assembly in a migrating U2OS cell expressing LifeAct-TagGFP2 (F-actin, magenta) and vinculin-mApple (focal adhesions, green) imaged by TIRF microscopy. Blue arrows exemplify the de novo generation of a cortical stress fiber, and the orange arrow highlights a vinculin positive adhesion that was used by several cortical stress fibers over the time. Imaging interval 30 s. See also Video 3. Scale bars 10 μm and 5 μm for the whole cell images and magnified images, respectively.