Actin stress fiber dynamics in laterally confined cells

Multiple cellular processes are affected by spatial constraints from the extracellular matrix and neighboring cells. In vitro experiments using defined micro-patterning allow for in-depth analysis and a better understanding of how these constraints impact cellular behavior and functioning. Herein we focused on the analysis of actin cytoskeleton dynamics as a major determinant of mechanotransduction mechanisms in cells. We seeded primary human umbilical vein endothelial cells onto stripe-like cell-adhesive micro-patterns with varying widths and then monitored and quantified the dynamic reorganization of actin stress fibers, including fiber velocities, orientation and density, within these live cells using the cell permeable F-actin marker SiR-actin. Although characteristic parameters describing the overall stress fiber architecture (average orientation and density) were nearly constant throughout the observation time interval of 60 min, we observed permanent transport and turnover of individual actin stress fibers. Stress fibers were more strongly oriented along stripe direction with decreasing stripe width, (5° on 20 μm patterns and 10° on 40 μm patterns), together with an overall narrowing of the distribution of fiber orientation. Fiber dynamics was characterized by a directed movement from the cell edges towards the cell center, where fiber dissolution frequently took place. By kymograph analysis, we found median fiber velocities in the range of 0.2 μm/min with a weak dependence on pattern width. Taken together, these data suggest that cell geometry determines actin fiber orientation, while it also affects actin fiber transport and turnover.