(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 …
(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 …
(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 …
(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 …
(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.
Zoomed in view of the region outlined by the dashed black square (right).
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 …
(A–C) Complementary cumulative squared displacement distributions in the high-FRET and low-FRET states for the mutant and two wild-type E-cad conditions, along with the respective Gaussian mixture …
Gaussian mixture model fits are shown as the black curve. Error bars represent the standard deviation of 100 bootstrapped samples.
(A) Complementary cumulative squared displacement distributions for the three FRET conditions and a lipid in the bilayer. Solid black curves correspond to a two-term Gaussian mixture model fit. …
Error bars were estimated as the square root of the Cramèr-Rao lower bound.
Notably, the low coverage control shows dramatically shorter dwell times than all other conditions.
Qualitatively comparing the two wild-type high-FRET distributions indicates the probability of a long surface residence time increases with increasing surface coverage due to stronger lateral …
(A) E-cad extracellular domains (orange), nonspecific and specific cis-interactions. Cis-donor sites are labeled in purple, and cis-acceptor sites are labeled in red. A structural model of the E-cad …
(A–B) Representative experimental cluster size probability distribution functions for wild-type and mutant E-cad at low, intermediate, and high surface coverages. Error bars correspond to the …
The 1st, 2nd and 3rd columns correspond to on rate 2 × 106 s−1, 2 × 105 s−1, and 2 × 104 s−1, respectively. The 1st, 2nd and 3rd rows correspond to off rate 104 s−1, 103 s−1, and 102 s−1, …
The columns correspond to on rate 108 s−1, 107 s−1, and 106 s−1, respectively. The rows correspond to off rate 103 s−1, 102 s−1, and 10 s−1, respectively. Nonspecific interaction on and off rates, …
(A) Ensemble-time-averaged MSDs for mutant E-cad at high, intermediate and low surface coverage and a lipid in the bilayer. Linear fits are shown as the dot-dashed line. (B) Ensemble-time-averaged …
The peak at a friction factor of ~0.5 s/µm2 corresponds to monomeric E-cad and was used to normalize all cluster size calculations.
The data are fit to a single exponential.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (human) | HEK293T | ATCC, Dr. Keith Johnson, University of Nebraska, Lincoln | CRL-3216 (RRID:CVCL_0063) | authenticated using STR-PCR and tested negative for mycoplasma |
Transfected construct (human) | CEP 4.2 plasmid | Dr. Lawrence Shapiro, Columbia University | Encoding hexahistidine-tagged wild-type E-cad and L175D mutant | |
Commercial assay or kit | Alexa Fluor 555 NHS-ester antibody labelling kit | Invitrogen | A20187 | Labeling E-cad |
Commercial assay or kit | Alexa Fluor 647 NHS-ester antibody labelling kit | Invitrogen | A20186 | Labeling E-cad |
Chemical compound, drug | DOPC | Sigma-Aldrich | P6354 | |
Chemical compound, drug | DGS-NTA(Ni) | Avanti Polar Lipids | 790404 | |
Chemical compound, drug | DOPE-LR | Avanti Polar Lipids | 810150 | |
Software, algorithm | Custom Matlab-based software | 10.1021/acsmacrolett.8b00004; 10.1021/acsnano.8b02956; 10.1021/acs.jpclett.9b00004 | Image analysis | |
Software, algorithm | simjFRAP | 10.1038/srep11655 | Image analysis |
Additional tables showing experimental parameters, simulation parameters, and sample size values.