Figure 3
The persistence of individual protrusions is correlated with fiber orientation even when under MII inhibition. HT-1080 cells expressing EGFP-paxillin (green) and Ruby-Lifeact (actin; magenta) were seeded on 2D PCL scaffolds with aligned fibers (blue) in the presence of 20 µM Y-27632 and 10 µM ML-7 and imaged over time. Times indicated in the figure are relative to the time of seeding. All images are maximum projections of z-stacks. Paxillin images have been additionally processed (“flattened”) to reduce background noise (see Materials and Methods). (a) Selected frames from Supplementary Movie 4 representative of seven independent experiments. The cell aligns to the predominant direction of fiber orientation, despite it being highly protrusive in nearly all directions. Adhesions (center column of images) are generally small. (b) The cell forms a protrusion perpendicular to the direction of fiber alignment shortly after 18 min, then turns approximately 90 degrees and protrudes along the aligned fibers, progressively forming small transient adhesions (arrowheads) as it extends. Scale bars: 20 µm (a), 5 µm (b).
