JCS092825 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Actin features are identified by a vector field algorithm. (A) A vector field algorithm applied to an EGFP−UtrCH image identifies the centers of actin features (red dots) across a whole cell. (B) The inset square in A is enlarged to clearly show individual vectors in the vector field (blue arrows) leading to local fluorescence intensity maxima in the actin distribution.

• Supplemental Figure S2 -

  Fig. S2. Actin features are tracked across consecutive frames. Nearest neighbor actin features present in consecutive frames of an EGFP−UtrCH image stack are linked to generate actin trajectories. The red dots represent local fluorescence intensity maxima identified by the vector field algorithm in Fig. S1 as actin features. The yellow line shows the trajectory of one particular actin feature across 11 frames (0 to 10 s).

• Supplemental Figure S3 -

  Fig. S3. �Escape time� is determined in two dimensions. The two-dimensional escape time (τ) is defined as the time it takes an actin feature (red dot trailed by a yellow line) to move outside of a circular area (blue circle) with an origin equal to the starting position of the feature and a radius corresponding to the escape distance (r). In this example, the feature moves outside of the boundary at t=4 s. Note that to highlight the trajectory of the actin feature, the escape distance used in this example is larger than the distance used in our analyses.

• Supplemental Figure S4 -

  Fig. S4. �Escape time� is determined in one dimension along the x axis. The one-dimensional escape time (τ) in the x axis is defined as the time it takes an actin feature (red dot trailed by a yellow line) to move outside of an area bounded by lines spaced at the escape distance (r) from the origin of the feature and perpendicular to the x axis (blue lines). This analysis can be thought of as identical to the two-dimensional escape time determination with only the x component of the feature�s motion being taken into account. In this example, the feature moves outside of the boundary at t=4 s. Note that to highlight the trajectory of the actin feature, the escape distance used in this example is larger than the distance used in our analyses. In addition, during our analyses, the x axis was defined relative to the chromium grid lines.

• Supplemental Figure S5 -

  Fig. S5. �Escape time� is determined in one dimension along the y axis. The one-dimensional escape time (τ) in the y axis is defined as the time it takes an actin feature (red dot trailed by a yellow line) to move outside of an area bounded by lines spaced at the escape distance (r) from the origin of the feature and perpendicular to the y axis (blue lines). This analysis can be thought of as identical to the two-dimensional escape time determination with only the y component of the feature�s motion being taken into account. In this example, the feature moves outside of the boundary at t=10 s. Note that to highlight the trajectory of the actin feature, the escape distance used in this example is larger than the distance used in our analyses. In addition, during our analyses, the y axis was defined relative to the chromium grid lines.

• Supplemental Figure S6 -

  Fig. S6. Escape times are not affected by grid opacity or non-triggered TCRs. Full escape times (top row) and x and y directional escape time components (middle and bottom rows, respectively) are displayed for EGFP−UtrCH cells. Cells in the first column were triggered by MCC peptide on a grid substrate (data restated from Figures 3 and 4). Cells in the second column were triggered by MCC peptide on an unpatterned substrate and then superimposed with an 85% opaque grid mask to simulate the appearance of a patterned substrate. Cells in the third column were allowed to interact with a bilayer containing MHC loaded with MCC (T102E) null peptide, which failed to trigger immunological synapse formation. Escape times were taken from actin features of crawling cells.

• Supplemental Figure S7 -

  Fig. S7. Different probes for the actin cytoskeleton generate similar escape time results. Full escape times (top row) and x and y directional escape time components (middle and bottom rows, respectively) are displayed for EGFP−UtrCH cells triggered on a grid substrate (first column), EGFP−β-Actin cells triggered on a grid substrate (second column), and EGFP−β-Actin cells triggered on a cross substrate (control; third column).

• Movie 1 -

  Movie 1. Actin accumulates near grid boundaries. A time-lapse recording of EGFP−β-Actin in a cell triggered by MCC peptide on a grid substrate shows periodic actin accumulations that assemble and dissipate. The labeled time is in the format of mm:ss; the scale bar represents 5 Mm.