Fig. 3: Cell cortex imaging.
a Actin cortex imaging of two MK6 macrophages (cell 1 & 2) in TIR-F mode (actin, Lifeact-GFP) at 7 Hz and in TIR-ROCS mode at 100 Hz. In TIR-F, the contact region ROI 1 reveals significant bleaching within frames n = 0, 200, 400. In TIR-ROCS, ROI 2 and ROI 3 reveal actin cytoskeleton-like structures (Supplementary Movie 6). b The magnified snapshot of ROI 2 marks two linescans within < 1 µm distance from cell 1 (yellow) and cell 2 (green). The two kymographs over 600 images show inter-cell communication through the cell protrusions. Two subsequent edge enhanced images of ROI 2 in green and red reveal high dynamics within Δt = 50 ms. c Milliseconds filopodia dynamics of J774 macrophages: 10 ms TIR-ROCS snapshot and activity plot over the complete frequency range (0–100 Hz) with three ROI. Filopodia activity is enhanced by 250% relative to mean cell activity (velocity standard deviation) of 364 nm/s. ROI 1 and ROI 2 reveal strong lateral motions in filopodia tips (green arrows), but no activity in a distance of 1–2 µm away from the tip. Little activity in frequency band Δω = 0–10 Hz, high activity in Δω = 10–50 Hz or Δω = 33–100 Hz. d Latrunculin A injection at time t0 inhibiting F-actin polymerization especially at cell periphery. TIR-ROCS images log(In) at 100 Hz with ROI, 10 ms difference images δIn and high-frequency activity plots (0.5 s STD) reveal high cell dynamics with actin retrograde flow. After 1 s, the cell retracts, loses focal adhesions and retracts filopodia. After 2.5 s activity is minimized. After 45 s actin polymerization restarts, leading to cell extraction and filopodia growth through tip searching. e Magnification of ROI with green and blue line scan. Kymograph of radial linescan 1 shows high actin backflow along a single filopodium with velocities up to v = 5 µm/s. Kymograph of lateral linescan 2 shows fast actin backflow. Blue linescan (length Δy) marks cross-section through the whole cell. Intensity profiles IROCS(t,s0) of single filopodium (in red) and of whole-cell IROCS(t,Δy) (in blue) reveal signal decrease within 0.5 s first in filopodia and 0.4 s later in the cell cortex, followed by a signal recovery within 60 s. Source data are provided as a Source Data file.
