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. 2020 Sep 2;9:e59035. doi: 10.7554/eLife.59035

Figure 1. Observation of cis-interactions via single-molecule FRET.

(A) Representative heat map of donor and acceptor intensities showing two populations at high and low FRET efficiency indicated by the asterisks. The black line represents the threshold between the two states used to assign each observation to the high or low-FRET state. (B, E, H) Donor and acceptor trajectories for a FRET pair throughout representative trajectories, which are used to determine if the donor E-cad molecule is in a high-FRET or low-FRET state. (C, F, I) X and Y Cartesian coordinates for the donor or acceptor molecule over the length of the trajectory. (D, G, J) Two dimensional trajectory plots of the same trajectories, where the symbol color corresponds to the assigned FRET-state. The background of the trajectory time traces for intensity and position indicate the assigned FRET-state.

Figure 1.

Figure 1—figure supplement 1. Heat maps showing binned acceptor and donor intensities using all molecular observations.

Figure 1—figure supplement 1.

(A–C) Heat maps for the mutant and the intermediate and high concentration wild-type E-cad conditions, respectively. All heat maps show two distinct peaks corresponding to two populations (high-FRET/associated and low-FRET) indicated by the asterisks, where the black line is used to divide the two populations and assign each observation to either the high-FRET or low-FRET state. The low-FRET state is above the dividing line and the high-FRET state is below the dividing line.
Figure 1—figure supplement 2. Overall complementary cumulative distributions of squared displacement calculated using only trajectories from a single movie for all three experimental conditions.

Figure 1—figure supplement 2.

(A) High coverage wild-type, (B) intermediate coverage wild-type, and (C) high coverage mutant CCSDDs. Legends indicate the order of the movie that was used to calculate the CCSDD. No systematic trend in CCSDDs with sample imaging time (i.e. movie number) was observed.
Figure 1—figure supplement 3. Complementary cumulative association time (high-FRET state dwell time) distributions calculated using only trajectories from a single movie for each of the three experimental conditions.

Figure 1—figure supplement 3.

(A) High coverage wild-type, (B) intermediate coverage wild-type, and (C) high coverage mutant association time distributions. Legends indicate the order of the movie that was used to calculate the distributions. No systematic trend in distributions with sample imaging time (i.e. movie number) was observed.
Figure 1—figure supplement 4. Fluorescence recovery after photobleaching (FRAP) analysis indicates the formation of a continuous, fluid supported lipid bilayer.

Figure 1—figure supplement 4.

(A–B) Epifluorescence images showing the photobleached region 0 s and 40 s, respectively, after photobleaching. (C) FRAP recovery curve indicating a mobile fraction of greater than 0.95.
Figure 1—figure supplement 5. All localized and tracked trajectories two frames and longer from the 30 s high surface coverage wild-type movie clip included as Video 1 (left).

Figure 1—figure supplement 5.

Zoomed in view of the region outlined by the dashed black square (right).
Figure 1—figure supplement 6. Representative histogram of positional localization uncertainties for all molecular observations for the high surface coverage wild-type condition indicating a significant number of observations with a large position uncertainty comparable to the size of a pixel (0.43 µm).

Figure 1—figure supplement 6.

Figure 1—figure supplement 7. Trajectory median donor intensity histograms showing the 60th percentile cutoff as a vertical red line used to remove bright contaminants, donor E-cad aggregates, and donor E-cad labeled with multiple fluorophores.

Figure 1—figure supplement 7.

The 60th percentile was selected as a consistent cutoff to remove trajectories corresponding to the tail of these distributions, as indicated by the figures. (A–C) Histograms for the mutant, high surface coverage wild-type, and intermediate surface coverage wild-type conditions, respectively.