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Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

Fujiwara TK, Iwasawa K, Kalay Z, Tsunoyama TA, Watanabe Y, Umemura YM, Murakoshi H, Suzuki KG, Nemoto YL, Morone N, Kusumi A - Mol. Biol. Cell (2016)

Bottom Line: Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion.The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion.These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

View Article: PubMed Central - PubMed

Affiliation: Center for Meso-Bio Single-Molecule Imaging, Institute for Integrated Cell-Material Sciences, Kyoto 606-8501, Japan.

No MeSH data available.


Related in: MedlinePlus

Hop diffusion becomes visible only with enhanced frame rates (improved time resolution). (A) Representative trajectories of gold-TfR (left) and DOPE (right) in the PtK2-cell PM obtained at systematically varied frame times of 33, 2, 0.22, and 0.025 ms. The trajectories obtained at 0.22- and 0.025-ms resolution are enlarged (see scales). Color coding in the 0.025-ms-resolution trajectories represents plausible compartments detected by a computer program (Fujiwara et al., 2002). The residency time within each compartment is shown. The overlaps of trajectories in adjacent compartments occur due to noise (limited single-molecule localization precision of 19.3 nm for both the horizontal and vertical directions of the camera at 0.025-ms resolution; see Materials and Methods). (B) Distributions of RD(n steps, nδt) (δt = time resolution) for gold-TfR and DOPE in the PtK2-cell PM. For the data obtained at time resolution of 33, 2, and 0.025 ms, the values of the (N, n) pair used here were (100, 30), (500, 30), and (2500, 60), respectively, in terms of the number of steps and (3.3 s, 1 s), (2 s, 60 ms), and (62.5 ms, 1.5 ms), respectively, in terms of time. The (N, n) pair of (100, 30) for the 33-ms resolution data was used, for consistency with the data for Cy3-TfR and Cy3-DOPE (Figure 2C). For the analysis of the data obtained at 2- and 0.025-ms resolution, n values were selected so that the analysis time scale of nδt would be useful to detect the non–simple-Brownian nature of the trajectories (Murase et al., 2004). The shapes of the RD distributions for simulated simple-Brownian particles at different time resolutions shown here seem to be quite different because we used the same x-axis scale for all of the RD distributions obtained at different time resolutions, whereas the ratios n/N, which strongly affect the appearance of the RD histograms, used here were quite different for the data obtained on different time scales. To show the shapes of the RD distributions obtained at different time resolutions more clearly, histograms with different x-scales for the same data sets are shown in Supplemental Figure S3.
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Figure 3: Hop diffusion becomes visible only with enhanced frame rates (improved time resolution). (A) Representative trajectories of gold-TfR (left) and DOPE (right) in the PtK2-cell PM obtained at systematically varied frame times of 33, 2, 0.22, and 0.025 ms. The trajectories obtained at 0.22- and 0.025-ms resolution are enlarged (see scales). Color coding in the 0.025-ms-resolution trajectories represents plausible compartments detected by a computer program (Fujiwara et al., 2002). The residency time within each compartment is shown. The overlaps of trajectories in adjacent compartments occur due to noise (limited single-molecule localization precision of 19.3 nm for both the horizontal and vertical directions of the camera at 0.025-ms resolution; see Materials and Methods). (B) Distributions of RD(n steps, nδt) (δt = time resolution) for gold-TfR and DOPE in the PtK2-cell PM. For the data obtained at time resolution of 33, 2, and 0.025 ms, the values of the (N, n) pair used here were (100, 30), (500, 30), and (2500, 60), respectively, in terms of the number of steps and (3.3 s, 1 s), (2 s, 60 ms), and (62.5 ms, 1.5 ms), respectively, in terms of time. The (N, n) pair of (100, 30) for the 33-ms resolution data was used, for consistency with the data for Cy3-TfR and Cy3-DOPE (Figure 2C). For the analysis of the data obtained at 2- and 0.025-ms resolution, n values were selected so that the analysis time scale of nδt would be useful to detect the non–simple-Brownian nature of the trajectories (Murase et al., 2004). The shapes of the RD distributions for simulated simple-Brownian particles at different time resolutions shown here seem to be quite different because we used the same x-axis scale for all of the RD distributions obtained at different time resolutions, whereas the ratios n/N, which strongly affect the appearance of the RD histograms, used here were quite different for the data obtained on different time scales. To show the shapes of the RD distributions obtained at different time resolutions more clearly, histograms with different x-scales for the same data sets are shown in Supplemental Figure S3.

Mentions: Second, we calculated the parameter RD(N, n) = MSD(nδt)/4D1–3nδt for each trajectory, where n is the number of steps used for the analysis in the trajectory of N steps (1 ≤ n ≤ N), δt is the camera frame time (thus the actual time for n steps is nδt; Figure 2B, right), and D1–3 is the initial slope of the MSD–∆t plot divided by 4 (see Materials and Methods and Figure 2B; as a macroscopic diffusion coefficient obtained from data recorded at video rate, D2–4 was used for consistency with the previous results). Here RD(N, n) describes the long-term (for a period of nδt) relative deviation of MSD(nδt) from the simple-Brownian model (see Materials and Methods). Because nδt (Figure 2B, right) is the key time scale used for evaluating the deviation from the ideal simple-Brownian diffusion mode, in this article, RD(N, n) will be expressed in the form of RD(n, nδt) to clearly indicate the time scale of the classification of each trajectory (see the x-axes of Figures 2, B right, and C, and 3B). The average value of RD(N, n) (or RD(n, nδt)) for the ensemble of molecules undergoing simple-Brownian diffusion will be 1, whereas those for the ensemble of molecules undergoing directed or suppressed diffusion will be >1 or <1, respectively. However, note that the RD(N, n) value for each individual trajectory would vary greatly from trajectory to trajectory.


Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

Fujiwara TK, Iwasawa K, Kalay Z, Tsunoyama TA, Watanabe Y, Umemura YM, Murakoshi H, Suzuki KG, Nemoto YL, Morone N, Kusumi A - Mol. Biol. Cell (2016)

Hop diffusion becomes visible only with enhanced frame rates (improved time resolution). (A) Representative trajectories of gold-TfR (left) and DOPE (right) in the PtK2-cell PM obtained at systematically varied frame times of 33, 2, 0.22, and 0.025 ms. The trajectories obtained at 0.22- and 0.025-ms resolution are enlarged (see scales). Color coding in the 0.025-ms-resolution trajectories represents plausible compartments detected by a computer program (Fujiwara et al., 2002). The residency time within each compartment is shown. The overlaps of trajectories in adjacent compartments occur due to noise (limited single-molecule localization precision of 19.3 nm for both the horizontal and vertical directions of the camera at 0.025-ms resolution; see Materials and Methods). (B) Distributions of RD(n steps, nδt) (δt = time resolution) for gold-TfR and DOPE in the PtK2-cell PM. For the data obtained at time resolution of 33, 2, and 0.025 ms, the values of the (N, n) pair used here were (100, 30), (500, 30), and (2500, 60), respectively, in terms of the number of steps and (3.3 s, 1 s), (2 s, 60 ms), and (62.5 ms, 1.5 ms), respectively, in terms of time. The (N, n) pair of (100, 30) for the 33-ms resolution data was used, for consistency with the data for Cy3-TfR and Cy3-DOPE (Figure 2C). For the analysis of the data obtained at 2- and 0.025-ms resolution, n values were selected so that the analysis time scale of nδt would be useful to detect the non–simple-Brownian nature of the trajectories (Murase et al., 2004). The shapes of the RD distributions for simulated simple-Brownian particles at different time resolutions shown here seem to be quite different because we used the same x-axis scale for all of the RD distributions obtained at different time resolutions, whereas the ratios n/N, which strongly affect the appearance of the RD histograms, used here were quite different for the data obtained on different time scales. To show the shapes of the RD distributions obtained at different time resolutions more clearly, histograms with different x-scales for the same data sets are shown in Supplemental Figure S3.
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Related In: Results  -  Collection

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Figure 3: Hop diffusion becomes visible only with enhanced frame rates (improved time resolution). (A) Representative trajectories of gold-TfR (left) and DOPE (right) in the PtK2-cell PM obtained at systematically varied frame times of 33, 2, 0.22, and 0.025 ms. The trajectories obtained at 0.22- and 0.025-ms resolution are enlarged (see scales). Color coding in the 0.025-ms-resolution trajectories represents plausible compartments detected by a computer program (Fujiwara et al., 2002). The residency time within each compartment is shown. The overlaps of trajectories in adjacent compartments occur due to noise (limited single-molecule localization precision of 19.3 nm for both the horizontal and vertical directions of the camera at 0.025-ms resolution; see Materials and Methods). (B) Distributions of RD(n steps, nδt) (δt = time resolution) for gold-TfR and DOPE in the PtK2-cell PM. For the data obtained at time resolution of 33, 2, and 0.025 ms, the values of the (N, n) pair used here were (100, 30), (500, 30), and (2500, 60), respectively, in terms of the number of steps and (3.3 s, 1 s), (2 s, 60 ms), and (62.5 ms, 1.5 ms), respectively, in terms of time. The (N, n) pair of (100, 30) for the 33-ms resolution data was used, for consistency with the data for Cy3-TfR and Cy3-DOPE (Figure 2C). For the analysis of the data obtained at 2- and 0.025-ms resolution, n values were selected so that the analysis time scale of nδt would be useful to detect the non–simple-Brownian nature of the trajectories (Murase et al., 2004). The shapes of the RD distributions for simulated simple-Brownian particles at different time resolutions shown here seem to be quite different because we used the same x-axis scale for all of the RD distributions obtained at different time resolutions, whereas the ratios n/N, which strongly affect the appearance of the RD histograms, used here were quite different for the data obtained on different time scales. To show the shapes of the RD distributions obtained at different time resolutions more clearly, histograms with different x-scales for the same data sets are shown in Supplemental Figure S3.
Mentions: Second, we calculated the parameter RD(N, n) = MSD(nδt)/4D1–3nδt for each trajectory, where n is the number of steps used for the analysis in the trajectory of N steps (1 ≤ n ≤ N), δt is the camera frame time (thus the actual time for n steps is nδt; Figure 2B, right), and D1–3 is the initial slope of the MSD–∆t plot divided by 4 (see Materials and Methods and Figure 2B; as a macroscopic diffusion coefficient obtained from data recorded at video rate, D2–4 was used for consistency with the previous results). Here RD(N, n) describes the long-term (for a period of nδt) relative deviation of MSD(nδt) from the simple-Brownian model (see Materials and Methods). Because nδt (Figure 2B, right) is the key time scale used for evaluating the deviation from the ideal simple-Brownian diffusion mode, in this article, RD(N, n) will be expressed in the form of RD(n, nδt) to clearly indicate the time scale of the classification of each trajectory (see the x-axes of Figures 2, B right, and C, and 3B). The average value of RD(N, n) (or RD(n, nδt)) for the ensemble of molecules undergoing simple-Brownian diffusion will be 1, whereas those for the ensemble of molecules undergoing directed or suppressed diffusion will be >1 or <1, respectively. However, note that the RD(N, n) value for each individual trajectory would vary greatly from trajectory to trajectory.

Bottom Line: Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion.The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion.These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

View Article: PubMed Central - PubMed

Affiliation: Center for Meso-Bio Single-Molecule Imaging, Institute for Integrated Cell-Material Sciences, Kyoto 606-8501, Japan.

No MeSH data available.


Related in: MedlinePlus