Analysis of the distribution of CD3ζ and Lat in native T cell plasma membrane sheets by hsPALM. (a) Three-dimensional probability density plots for CD3ζ-PSCFP2 (top) and Lat-PSCFP2 (bottom) in native plasma membrane sheets from quiescent T cells (on poly-L-lysine (PLL); left) and activated T cells (on I-E^k–MCC plus B7-1 at a ratio of 1:3; right) on immobilized surfaces. Molecules are presented as a normalized Gaussian probability density distribution with a width equal to their positional accuracy. Height and color (key) represent the probability density at that point (x,y), with the highest probability density of all images set as 1. (b) Simulated three-dimensional probability density plot containing the same number of molecules as the plot in a for CD3ζ-PSCFP2 on an activating surface with identical positional accuracies but randomized positions. Key: x and y arrows, 1 μm. (c) Ripley’s K-function analysis of the clustering of CD3ζ-PSCFP2 and Lat-PSCFP2 molecules (left); values for L(r) – r above 1 (where L is (K/π)^0.5 and r is the radius) indicate clustering at distance r. Right, Ripley’s K-function analysis of the clustering of probability density maxima of CD3ζ-PSCFP2 and Lat-PSCFP2; negative values of L(r) – r for small r values in the data curves are due to the dimensions of the peaks themselves. Data are representative of 25–30 experiments (with one sheet per experiment).

TEM analysis of the concatenation of CD3ζ and Lat islands. (a) Localization of CD3ζ and Lat in plasma membrane sheets generated from quiescent T cells (on poly-L-lysine; left) and activated T cells (on I-E^k–MCC plus B7-1 at a ratio of 1:3; right), detected (arrowheads) with 5-nm gold particles (CD3ζ) or 10-nm gold particles (Lat). (b) Ripley’s K-function analysis of the clustering of CD3ζ and Lat molecules (top); values for L(r) – r above 1 indicate clustering of molecules at distance r. Below, bivariate Ripley’s K-function analysis of the coclustering of CD3ζ and Lat molecules (solid blue lines, quiescent T cells; solid red lines, activated T cells). Values above the 99% confidence envelope (blue shaded areas (quiescent) and red shaded areas (activated)) indicate statistically significant association or attraction at distance r; values below the 99% confidence envelope indicate statistically significant repulsion or explicit separation at distance r. Scale bars, 200 nm. Data are representative of 25–30 experiments (with one sheet per experiment).

Kinetics of the correlation between CD3ζ and Lat themselves and each other in T cells on nonactivating and activating bilayers. Above, total entities (N_T) containing CD3ζ, Lat or both (time windows, horizontal axis) on a nonactivating bilayer (~16 ICAM-1 molecules per μm²) or activating bilayer (~30 I-Ek–MCC, ~8 B7-1 molecules and ~16 ICAM-1 molecules per μm²). Below, fraction of correlated movement (F′) between CD3ζ and Lat themselves and each other (same bilayers and time windows as above). Data are representative of two to three experiments with about five measurements each.

Analysis of the distribution of endogenous TCRβ in T cells by hsPALM. (a) Three-dimensional probability density plots for T cells labeled with the monovalent anti-TCRβ-scFv–PSCFP2 probe, on nonactivating bilayers (~16 ICAM-1 molecules per μm²; top left) and activating bilayers (~30 I-E^k –MCC plus ~8 B7-1 plus ~16 ICAM-1 molecules per μm²; top right), as well as nonactivating immobilized surfaces (poly-L-lysine; bottom left) and activating immobilized surfaces (I-E^k–MCC plus B7-1 at a ratio of 1:3; bottom right). Results are presented as described in Figure 1. (b) Simulated three-dimensional probability density plot containing the same number of molecules as the plot in a for TCRβ on an activating surface with identical positional accuracies but randomized positions. Key: x and y arrows, 1 μm. (c) Ripley’s K-function analysis of the clustering of TCRβ (left); values for L(r) – r above 1 indicate clustering of molecules at distance r. Right, Ripley’s K-function analysis of the clustering of probability density maxima of TCRβ; negative values of L(r) – r for small r in the data curves are due to the dimensions of the peaks themselves. Nonact, nonactivating. Data representative of 15–20 experiments (with one T cell per experiment).

Analysis of the correlation of CD3ζ and Lat in live T cells on bilayers by dcFCCS. Cross-correlation of CD3ζ and Lat in T cells expressing all possible combinations of CD3ζ and Lat proteins fused with eGFP or mCherry; T cells were allowed to settle on nonactivating bilayers (~16 ICAM-1 molecules per μm²; top) and activating bilayers (~30 I-E^k–MCC plus ~8 B7-1 plus ~16 ICAM-1 molecules per μm²; bottom), and dcFCCS measurements were obtained within 3 min of adhesion where the plasma membrane contacts the bilayer. Green, eGFP autocorrelation curves; red, mCherry autocorrelation curves; yellow, cross-correlation curves for eGFP and mCherry. Cross-correlation curves with nonzero values for G(τ) indicate correlated movement. Numbers in plots indicate entities containing eGFP alone (Ng), mCherry alone (Nc), or eGFP and mCherry (Ngc). Data are representative of five to seven experiments (with one T cell per experiment).