Figure 8.
TfR undergoes hop diffusion within the FA region but with a smaller median compartment size of 74 nm (vs. 110 nm in the bulk basal PM) and a longer dwell lifetime of 36 ms (vs. 24 ms in the bulk basal PM). (A) Typical ultrafast single fluorescent-molecule trajectories (0.167-ms resolution; 6-kHz frame rate) of TMR-Halo-TfR diffusing outside the FA (top), entering the FA from the bulk PM (middle), and diffusing inside the FA (bottom). In the middle figure, the background of the trajectory is the mGFP-paxillin image, and the yellow line shows the boundary of the FA region determined by binarization, using the minimum cross entropy thresholding. (B) Distributions of RDs (relative deviation from ideal Brownian diffusion; for the definition and explanation, see Fig. 4 B in the companion paper) for the trajectories of TfR diffusing inside (top) and outside (bottom) the FA region in the basal PM (shaded histograms; 54 and 57 molecules, respectively). The open histogram (the same for both inside and outside the FA) represents the RD distribution for simple-Brownian trajectories generated by Monte-Carlo simulations (5,000 trajectories), with the red and blue vertical lines indicating the 2.5 percentiles from both ends of the distribution (RDmin and RDMAX, respectively). The trajectories exhibiting RD values below RDmin are categorized into the suppressed diffusion mode. For details, see Fig. 4 B of the companion paper. The data for “Outside FA” shown here in B–D are reproduced from Fig. 7, B–D (top), in the companion paper for the direct comparison with the “Inside FA” data. (C) Distributions of the compartment sizes determined by the hop-diffusion fitting of the MSD-∆t plot of each TfR trajectory (see Fig. 4 A, Supplemental theory 2, and related main text in the companion paper), inside (top) and outside (bottom) the FA region in the basal PM. Arrowheads indicate the median values (statistically significant difference with P = 1.7 × 10−7, using the Brunner–Munzel test). (D) Distributions of the TfR residency times within a compartment, obtained for each molecule in each compartment determined by the TILD analysis (see Fig. S5 and its legend in the companion paper), with the best-fit exponential curves (providing the dwell lifetimes), inside (top) and outside (bottom) the FA region. A statistically significant difference exists between before and after stimulation with P = 4.9 × 10−4, using the log-rank test. For the derivation of the single exponential dependence of the dwell lifetime distribution, see the subsection “Expected distribution of the residency times: development of the hop diffusion theory” in the legend to Fig. S5 in the companion paper. (E) Schematic model of our proposed FA architecture, based on the observations made in the present study. The FA-protein islands with various molecular stoichiometric compositions (24–32 nm in the mean diameter in MEFs; green hexagons) form loose clusters with a diameter of ≈320 nm in the compartmentalized fluid (actin meshwork schematically shown by the green lattice). Recruitment of paxillin to these islands might not occur randomly at individual islands but instead occur synchronously at islands within the same loose cluster. The fluid membrane in the inter-island channels in the FA is partitioned into 74-nm compartments (110 nm in the bulk PM). These compartment boundaries are probably composed of the actin-based membrane-skeleton mesh, which might be bound and stabilized by various FA proteins as monomers, oligomers, and islands. (F) Distributions of DMACRO for TfR determined by the hop-diffusion fitting of the MSD-∆t plot for each TfR trajectory obtained at 6 kHz. Arrowheads indicate the median values (statistically significant difference with P = 9.3 × 10−9, using the Brunner–Munzel test).

TfR undergoes hop diffusion within the FA region but with a smaller median compartment size of 74 nm (vs. 110 nm in the bulk basal PM) and a longer dwell lifetime of 36 ms (vs. 24 ms in the bulk basal PM). (A) Typical ultrafast single fluorescent-molecule trajectories (0.167-ms resolution; 6-kHz frame rate) of TMR-Halo-TfR diffusing outside the FA (top), entering the FA from the bulk PM (middle), and diffusing inside the FA (bottom). In the middle figure, the background of the trajectory is the mGFP-paxillin image, and the yellow line shows the boundary of the FA region determined by binarization, using the minimum cross entropy thresholding. (B) Distributions of RDs (relative deviation from ideal Brownian diffusion; for the definition and explanation, see Fig. 4 B in the companion paper) for the trajectories of TfR diffusing inside (top) and outside (bottom) the FA region in the basal PM (shaded histograms; 54 and 57 molecules, respectively). The open histogram (the same for both inside and outside the FA) represents the RD distribution for simple-Brownian trajectories generated by Monte-Carlo simulations (5,000 trajectories), with the red and blue vertical lines indicating the 2.5 percentiles from both ends of the distribution (RDmin and RDMAX, respectively). The trajectories exhibiting RD values below RDmin are categorized into the suppressed diffusion mode. For details, see Fig. 4 B of the companion paper. The data for “Outside FA” shown here in B–D are reproduced from Fig. 7, B–D (top), in the companion paper for the direct comparison with the “Inside FA” data. (C) Distributions of the compartment sizes determined by the hop-diffusion fitting of the MSD-∆t plot of each TfR trajectory (see Fig. 4 A, Supplemental theory 2, and related main text in the companion paper), inside (top) and outside (bottom) the FA region in the basal PM. Arrowheads indicate the median values (statistically significant difference with P = 1.7 × 10−7, using the Brunner–Munzel test). (D) Distributions of the TfR residency times within a compartment, obtained for each molecule in each compartment determined by the TILD analysis (see Fig. S5 and its legend in the companion paper), with the best-fit exponential curves (providing the dwell lifetimes), inside (top) and outside (bottom) the FA region. A statistically significant difference exists between before and after stimulation with P = 4.9 × 10−4, using the log-rank test. For the derivation of the single exponential dependence of the dwell lifetime distribution, see the subsection “Expected distribution of the residency times: development of the hop diffusion theory” in the legend to Fig. S5 in the companion paper. (E) Schematic model of our proposed FA architecture, based on the observations made in the present study. The FA-protein islands with various molecular stoichiometric compositions (24–32 nm in the mean diameter in MEFs; green hexagons) form loose clusters with a diameter of ≈320 nm in the compartmentalized fluid (actin meshwork schematically shown by the green lattice). Recruitment of paxillin to these islands might not occur randomly at individual islands but instead occur synchronously at islands within the same loose cluster. The fluid membrane in the inter-island channels in the FA is partitioned into 74-nm compartments (110 nm in the bulk PM). These compartment boundaries are probably composed of the actin-based membrane-skeleton mesh, which might be bound and stabilized by various FA proteins as monomers, oligomers, and islands. (F) Distributions of DMACRO for TfR determined by the hop-diffusion fitting of the MSD-∆t plot for each TfR trajectory obtained at 6 kHz. Arrowheads indicate the median values (statistically significant difference with P = 9.3 × 10−9, using the Brunner–Munzel test).

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