Analysis methods for bulk cytoskeletal flow are distinct from those used to describe speckle motion. (A) A vector-field algorithm identifies the centers of actin features across a whole cell. (B,C) The white square in A is enlarged to clearly show individual vectors in the vector field, and actin features are tracked across multiple frames. New features without tracks are also visible in C. (D) Schematic of the escape time analysis. The time it takes for a feature to travel further than a given escape distance (r) in any direction is its escape time (τ). (E) Escape times are distributed into populations based on each feature's instantaneous position. For example, features within 0.63 μm (10 pixels) of a single pattern boundary and on the opposite side from the cell center are colored dark blue or red, those within 0.63 μm of two boundaries are colored light blue and all other features are colored green. Scale bars: 5 μm.

Actin features slow down in TCR-rich regions. (A,B) Two regions of a cell partially on a grid-patterned supported lipid bilayer equidistant from the cell center were selected (yellow and purple rectangles in A) for analysis of actin escape times (B). (C–E) Representative frames from time lapses show cells interacting with a grid-patterned substrate (C), a cross-patterned substrate (D) or an unpatterned substrate overlaid with the grid used for analysis (E). (F–H) Actin features from the cells in C–E were partitioned into four populations and analyzed for escape times. The inset shows that the green population is composed of all steps occurring further than 0.63 μm (10 pixels) from the outside edge of a grid line, the dark blue and red populations are composed of steps occurring within 0.63 μm of an x or y grid line, respectively, and the light blue population is composed of steps occurring within 0.63 μm of both grid lines. Error bars represent s.e.m. Scale bars: 5 μm.

Increased actin escape times correspond to motion perpendicular to TCR diffusion barriers. (A,D,G) Representative frames from time lapses show cells interacting with a grid-patterned substrate (A), a cross-patterned substrate (D) or an unpatterned substrate overlaid with the grid used for analysis (G). (B,E,H) The escape time analysis from Fig. 3 was repeated for the cells in A,D,G, but the escape time was redefined as the time it takes for a feature to move outside of an area identified by boundaries tangential to the original escape area and parallel to the y grid line (schematic above column). This in effect considers only the x component of motion of the feature. (C,F,I) The escape time analysis from Fig. 3 was repeated for the cells in A,D,G, but the escape time was redefined as the time it takes for a heterogeneity to move outside of an area identified by boundaries tangential to the original escape area and parallel to the x grid line (schematic above column). This in effect considers only the y component of motion of the feature. Error bars represent s.e.m. Scale bars: 5 μm.