Figure 4.
Ultrafast SFMI can detect hop diffusion of Cy3-DOPE and TfR in the apical PM, previously found by ultrafast single gold-particle tracking. (A a–c) Typical MSD-∆t plot for a single molecule (a), and the ensemble-averaged MSD-∆t plots with two different y-axis scales (b and c: 20× of b) in the intact apical PM (black) and the blebbed apical PM (purple). The MSD-∆t plots of the top and middle boxes in (b) are those after subtracting 4σxy2, shown in Fig. S4 (σxy = single-molecule localization precision). Only in the typical MSD-∆t plot for a single 5xCy3-Tf molecule bound to TfR obtained at 45 kHz (a, bottom), 4σxy2 was not subtracted due to large errors in its estimation. Green curves are the best-fit functions for the hop-diffusion fitting (top and middle rows) and confined-diffusion fitting (bottom row for 45 kHz observations of 5xCy3-Tf). (B) Approximately 80% of Cy3-DOPE and TfR undergo suppressed diffusion in the intact apical PM, whereas >90% of them undergo simple-Brownian diffusion in the actin-depleted blebbed PM (shaded histograms in b), as revealed by the method for the statistical classification of each single-molecule trajectory into a suppressed-, simple-Brownian-, or directed-diffusion mode. (B a) The basic idea for the classification of trajectories into suppressed, simple-Brownian, and directed diffusion: the parameter RD (relative deviation) describes the extent to which the observed diffusion deviates from simple-Brownian diffusion at a time sufficiently later from time 0; i.e., the actual MSD divided by the calculated MSD from the short-term diffusion coefficient (D2-4) assuming simple-Brownian diffusion. See Materials and methods. The RD value is <<, ≈, or >> 1, when the molecules are undergoing suppressed, simple-Brownian, or directed diffusion, respectively (Fujiwara et al., 2002; Kusumi et al., 1993; Murase et al., 2004; Suzuki et al., 2005). The suppressed-diffusion mode includes both the confined-diffusion and hop-diffusion modes. (B b) Classification of individual trajectories based on the RD histograms for simulated simple-Brownian particles (open bars; n = 5,000). The RD values giving the 2.5 percentiles of the particles from both ends of the distribution, referred to as RDmin and RDMAX, were obtained (red and blue vertical lines, respectively). Each experimental single-molecule trajectory was classified into suppressed (confined and hop), simple-Brownian, or directed diffusion when its RD value was smaller than RDmin, between RDmin and RDMAX, and greater than RDMAX (no trajectory fell in this category), respectively. The distributions of RDs for the Cy3-DOPE and TfR trajectories are shown by shaded histograms (n = 50 and 20 for the intact and actin-depleted blebbed PM, respectively). (C a and b) In the blebbed apical PM, Cy3-DOPE (a) and Cy3-Tf bound to TfR (b) exhibited single diffusion coefficients that are ≈20× greater than DMACRO in the intact apical PM. The distributions of Dmicro (=D2–4) are underestimated due to the insufficient time resolution for measuring Dmicro within a compartment, even at a 0.1-ms resolution. Arrowheads indicate the median values (summarized in Table 2). These diffusion coefficients in the blebbed PM are slightly smaller than those obtained with gold probes (Fujiwara et al., 2002; Fujiwara et al., 2016). This is probably due to the residual actin filaments in the T24 cells employed here, since the membrane-bound actin filament meshes are much denser in T24 cells than the NRK cells used previously. (D) Cy3-DOPE and TfR exhibited similar compartment size distributions, suggesting that the underlying mechanism for the compartmentalization would be the same for the phospholipid and the transmembrane protein. Arrowheads indicate the median values. The statistical test methods, parameters (number of experiments), and P values are summarized in Table 2.

Ultrafast SFMI can detect hop diffusion of Cy3-DOPE and TfR in the apical PM, previously found by ultrafast single gold-particle tracking. (A a–c) Typical MSD-∆t plot for a single molecule (a), and the ensemble-averaged MSD-∆t plots with two different y-axis scales (b and c: 20× of b) in the intact apical PM (black) and the blebbed apical PM (purple). The MSD-∆t plots of the top and middle boxes in (b) are those after subtracting 4σxy2, shown in Fig. S4 (σxy = single-molecule localization precision). Only in the typical MSD-∆t plot for a single 5xCy3-Tf molecule bound to TfR obtained at 45 kHz (a, bottom), 4σxy2 was not subtracted due to large errors in its estimation. Green curves are the best-fit functions for the hop-diffusion fitting (top and middle rows) and confined-diffusion fitting (bottom row for 45 kHz observations of 5xCy3-Tf). (B) Approximately 80% of Cy3-DOPE and TfR undergo suppressed diffusion in the intact apical PM, whereas >90% of them undergo simple-Brownian diffusion in the actin-depleted blebbed PM (shaded histograms in b), as revealed by the method for the statistical classification of each single-molecule trajectory into a suppressed-, simple-Brownian-, or directed-diffusion mode. (B a) The basic idea for the classification of trajectories into suppressed, simple-Brownian, and directed diffusion: the parameter RD (relative deviation) describes the extent to which the observed diffusion deviates from simple-Brownian diffusion at a time sufficiently later from time 0; i.e., the actual MSD divided by the calculated MSD from the short-term diffusion coefficient (D2-4) assuming simple-Brownian diffusion. See Materials and methods. The RD value is <<, ≈, or >> 1, when the molecules are undergoing suppressed, simple-Brownian, or directed diffusion, respectively (Fujiwara et al., 2002; Kusumi et al., 1993; Murase et al., 2004; Suzuki et al., 2005). The suppressed-diffusion mode includes both the confined-diffusion and hop-diffusion modes. (B b) Classification of individual trajectories based on the RD histograms for simulated simple-Brownian particles (open bars; n = 5,000). The RD values giving the 2.5 percentiles of the particles from both ends of the distribution, referred to as RDmin and RDMAX, were obtained (red and blue vertical lines, respectively). Each experimental single-molecule trajectory was classified into suppressed (confined and hop), simple-Brownian, or directed diffusion when its RD value was smaller than RDmin, between RDmin and RDMAX, and greater than RDMAX (no trajectory fell in this category), respectively. The distributions of RDs for the Cy3-DOPE and TfR trajectories are shown by shaded histograms (n = 50 and 20 for the intact and actin-depleted blebbed PM, respectively). (C a and b) In the blebbed apical PM, Cy3-DOPE (a) and Cy3-Tf bound to TfR (b) exhibited single diffusion coefficients that are ≈20× greater than DMACRO in the intact apical PM. The distributions of Dmicro (=D2–4) are underestimated due to the insufficient time resolution for measuring Dmicro within a compartment, even at a 0.1-ms resolution. Arrowheads indicate the median values (summarized in Table 2). These diffusion coefficients in the blebbed PM are slightly smaller than those obtained with gold probes (Fujiwara et al., 2002; Fujiwara et al., 2016). This is probably due to the residual actin filaments in the T24 cells employed here, since the membrane-bound actin filament meshes are much denser in T24 cells than the NRK cells used previously. (D) Cy3-DOPE and TfR exhibited similar compartment size distributions, suggesting that the underlying mechanism for the compartmentalization would be the same for the phospholipid and the transmembrane protein. Arrowheads indicate the median values. The statistical test methods, parameters (number of experiments), and P values are summarized in Table 2.

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