Figure 6.
Ultrafast SFMI of TfR and Cy3-DOPE revealed that the basal PM outside the FAs (bulk basal PM) is compartmentalized like the apical PM and that the dwell lifetimes of TfR and Cy3-DOPE within a compartment in the bulk basal PM are the same as those in the apical PM. The data about the apical PM are reproduced in figures B–E here from Fig. 4, B and D; and Fig. 5 A for the ready comparison. (A) Typical ultrafast single fluorescent-molecule trajectories of TfR (6 kHz) and Cy3-DOPE (10 kHz), diffusing in the bulk basal PM and in the apical PM. The order of the compartments visited by the molecules (parenthesized integers) and their respective dwell lifetimes there, as determined by the TILD analysis (Fig. S5), are shown in the figure. TMR was used to label the Halo-tag protein fused to the cytoplasmic domain of TfR (N-terminus). Since Cy3 (and Alexa555), which exhibited superior throughput among all the dyes tested (Figs. S2 and S3), is membrane impermeable, the availability of a membrane-permeable dye, TMR, for ultrafast observations is very useful. (B) Motional mode classification based on RD, performed as described in Fig. 4 B. Shaded and open bars show the RD distributions, obtained by experiments and simulation for simple-Brownian particles, respectively. The percentages of molecules categorized into the suppressed and simple-Brownian diffusion modes are indicated. (C) Distributions of the compartment sizes determined from the hop-diffusion fitting of the MSD-∆t plot for each TfR and Cy3-DOPE molecule. The MSD-∆t plot data that could not be fit (due to large noise and the closeness of Dmicro and DMACRO) were not included. Arrowheads indicate the median values. (D) Distributions of the TfR and Cy3-DOPE residency times within a compartment determined by the TILD analysis, with the best-fit single exponential functions. The decay time constants of these curves provide the dwell lifetimes (see the caption to Fig. 5 A). (E) Distribution of DMACRO determined by the hop-diffusion fitting of the MSD-∆t plot for each TfR and Cy3-DOPE molecule (shaded histogram, bulk basal PM; open histogram, apical PM). Arrowheads indicate the median values. The statistical test methods, parameters (number of experiments), and P values are summarized in Table 3.

Ultrafast SFMI of TfR and Cy3-DOPE revealed that the basal PM outside the FAs (bulk basal PM) is compartmentalized like the apical PM and that the dwell lifetimes of TfR and Cy3-DOPE within a compartment in the bulk basal PM are the same as those in the apical PM. The data about the apical PM are reproduced in figures B–E here from Fig. 4, B and D; and Fig. 5 A for the ready comparison. (A) Typical ultrafast single fluorescent-molecule trajectories of TfR (6 kHz) and Cy3-DOPE (10 kHz), diffusing in the bulk basal PM and in the apical PM. The order of the compartments visited by the molecules (parenthesized integers) and their respective dwell lifetimes there, as determined by the TILD analysis (Fig. S5), are shown in the figure. TMR was used to label the Halo-tag protein fused to the cytoplasmic domain of TfR (N-terminus). Since Cy3 (and Alexa555), which exhibited superior throughput among all the dyes tested (Figs. S2 and S3), is membrane impermeable, the availability of a membrane-permeable dye, TMR, for ultrafast observations is very useful. (B) Motional mode classification based on RD, performed as described in Fig. 4 B. Shaded and open bars show the RD distributions, obtained by experiments and simulation for simple-Brownian particles, respectively. The percentages of molecules categorized into the suppressed and simple-Brownian diffusion modes are indicated. (C) Distributions of the compartment sizes determined from the hop-diffusion fitting of the MSD-∆t plot for each TfR and Cy3-DOPE molecule. The MSD-∆t plot data that could not be fit (due to large noise and the closeness of Dmicro and DMACRO) were not included. Arrowheads indicate the median values. (D) Distributions of the TfR and Cy3-DOPE residency times within a compartment determined by the TILD analysis, with the best-fit single exponential functions. The decay time constants of these curves provide the dwell lifetimes (see the caption to Fig. 5 A). (E) Distribution of DMACRO determined by the hop-diffusion fitting of the MSD-∆t plot for each TfR and Cy3-DOPE molecule (shaded histogram, bulk basal PM; open histogram, apical PM). Arrowheads indicate the median values. The statistical test methods, parameters (number of experiments), and P values are summarized in Table 3.

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