Figure S5.
TfR molecules diffuse within the FA, but in certain subregions, TfR is excluded or has limited diffusibility. Typical trajectories of single TMR-labeled Halo-TfR obtained at a frame rate of 60 Hz (durations of 1.0–5.5 s) are overlaid on the simultaneously recorded TIRF image of the mGFP-paxillin (not a super-resolution image). Trajectories are colored to aid discernment where the trajectories are crowded (the color was not changed in a single trajectory). The contours of the FA regions (yellow lines) were determined by binarization, using the minimum cross entropy thresholding. Previously, our observations at slower frame rates (30–250 Hz) revealed that non-FA protein TfR molecules entered the FA region and diffused more or less freely in the fluid membrane region inside the FA (Shibata et al., 2012; Shibata et al., 2013; Tsunoyama et al., 2018). However, the presence of loose clusters of FA-protein islands shown in Figs. 6 and 7 raises the possibility that TfR molecules might not enter these island cluster domains, or if they do enter, they may not undergo free diffusion there. To address this question, we examined the movements of TMR-labeled Halo-TfR molecules located in and near the FAs at a frame rate of 60 Hz, to determine the long-range, long-time movements, such as those occurring over a time frame of 1.0–5.5 s (Fig. S5). The typical trajectories overlaid on the fluorescent paxillin images (not super-resolution) suggest that these trajectories probably represent the smeared-out (indistinct) hop diffusion of TfR, due to the slow rate of observation (60 Hz; see Fig. 8). The superimposed image in Fig. S5 implies that TfR is somewhat excluded from the areas with elevated paxillin concentrations, and when TfR molecules occasionally entered these areas, their diffusion is slowed and confined, although a quantitative analysis was beyond the scope of this study. These results indicate that the molecular diffusion within the FA is spatially quite heterogeneous, which aligns with the presence of loose FA-protein island clusters. Meanwhile, by applying the hop-diffusion fitting to 250-ms-long TfR trajectories recorded at 6 kHz in the FA (Fig. 8, A–D), we obtained the distribution of the macroscopic diffusion coefficients of individual molecules (representing the diffusion rate over several compartments rather than that within a compartment; DMACRO; Fig. 8 F). It shows that ≈19% of TfR molecules are almost immobile in the FA, with DMACRO values <0.0063 µm2/s (Fig. 8 F). This result provides clear evidence for the existence of FA subdomains where TfR diffusion is suppressed. This is likely to occur in the FA subdomains where the FA-protein islands, which act as diffusion obstacles, exist at higher number densities, such as the loose clusters of FA-protein islands.

TfR molecules diffuse within the FA, but in certain subregions, TfR is excluded or has limited diffusibility. Typical trajectories of single TMR-labeled Halo-TfR obtained at a frame rate of 60 Hz (durations of 1.0–5.5 s) are overlaid on the simultaneously recorded TIRF image of the mGFP-paxillin (not a super-resolution image). Trajectories are colored to aid discernment where the trajectories are crowded (the color was not changed in a single trajectory). The contours of the FA regions (yellow lines) were determined by binarization, using the minimum cross entropy thresholding. Previously, our observations at slower frame rates (30–250 Hz) revealed that non-FA protein TfR molecules entered the FA region and diffused more or less freely in the fluid membrane region inside the FA (Shibata et al., 2012; Shibata et al., 2013; Tsunoyama et al., 2018). However, the presence of loose clusters of FA-protein islands shown in Figs. 6 and 7 raises the possibility that TfR molecules might not enter these island cluster domains, or if they do enter, they may not undergo free diffusion there. To address this question, we examined the movements of TMR-labeled Halo-TfR molecules located in and near the FAs at a frame rate of 60 Hz, to determine the long-range, long-time movements, such as those occurring over a time frame of 1.0–5.5 s (Fig. S5). The typical trajectories overlaid on the fluorescent paxillin images (not super-resolution) suggest that these trajectories probably represent the smeared-out (indistinct) hop diffusion of TfR, due to the slow rate of observation (60 Hz; see Fig. 8). The superimposed image in Fig. S5 implies that TfR is somewhat excluded from the areas with elevated paxillin concentrations, and when TfR molecules occasionally entered these areas, their diffusion is slowed and confined, although a quantitative analysis was beyond the scope of this study. These results indicate that the molecular diffusion within the FA is spatially quite heterogeneous, which aligns with the presence of loose FA-protein island clusters. Meanwhile, by applying the hop-diffusion fitting to 250-ms-long TfR trajectories recorded at 6 kHz in the FA (Fig. 8, A–D), we obtained the distribution of the macroscopic diffusion coefficients of individual molecules (representing the diffusion rate over several compartments rather than that within a compartment; DMACRO; Fig. 8 F). It shows that ≈19% of TfR molecules are almost immobile in the FA, with DMACRO values <0.0063 µm2/s (Fig. 8 F). This result provides clear evidence for the existence of FA subdomains where TfR diffusion is suppressed. This is likely to occur in the FA subdomains where the FA-protein islands, which act as diffusion obstacles, exist at higher number densities, such as the loose clusters of FA-protein islands.

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