Limits...
Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton.

Tomishige M, Sako Y, Kusumi A - J. Cell Biol. (1998)

Bottom Line: When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton.Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral.These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

ABSTRACT
Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

Show MeSH
(a and b) Typical plots of MSD against the time interval  for a particle–band 3 complex. The MSD-Δt plot in a shows a  steep rise and fast leveling off, indicating confined diffusion of  this particle in a time window of 10 ms. Intercompartmental hops  took place in a time scale of 1,000 ms as they appear as the positive slope in b (the slope gives 4DMACRO). (c) MSD-Δt plots for intact and cleaved band 3 in a time window of 2 s, averaged over all  of the respective particles studied in this work (in c, ensemble averaging over all of the particles was taken, whereas a running average over a single particle's trajectory was calculated in a and b).  Cleaved band 3 shows a distinctly greater slope in the time window of 2 s. Inset, the ensemble-averaged MSDs for intact and  cleaved band 3 in a time window of 20 ms. In this time scale, the  difference between intact and cleaved band 3 was slight, suggesting that their diffusion characteristics within a compartment are  similar to each other.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132872&req=5

Figure 6: (a and b) Typical plots of MSD against the time interval for a particle–band 3 complex. The MSD-Δt plot in a shows a steep rise and fast leveling off, indicating confined diffusion of this particle in a time window of 10 ms. Intercompartmental hops took place in a time scale of 1,000 ms as they appear as the positive slope in b (the slope gives 4DMACRO). (c) MSD-Δt plots for intact and cleaved band 3 in a time window of 2 s, averaged over all of the respective particles studied in this work (in c, ensemble averaging over all of the particles was taken, whereas a running average over a single particle's trajectory was calculated in a and b). Cleaved band 3 shows a distinctly greater slope in the time window of 2 s. Inset, the ensemble-averaged MSDs for intact and cleaved band 3 in a time window of 20 ms. In this time scale, the difference between intact and cleaved band 3 was slight, suggesting that their diffusion characteristics within a compartment are similar to each other.

Mentions: For a quantitative analysis of the movement of mobile band 3 molecules, the MSD of the particle was plotted against the time interval (Δt) (Fig. 6, a and b). Almost all of the MSDs showed a rapid rise and leveling off in a time window of ∼10 ms (Fig. 6 a). Statistical analysis according to the method by Kusumi et al. (1993) indeed showed that all of the mobile band 3 molecules undergo confined diffusion in a time window of 20 ms. The rapid rise near Δt = 0 reflects fast diffusion within a compartment (the microscopic diffusion coefficient Dmicro). In a longer time window, the MSD-Δt plot asymptotically approached a straight line with a constant positive slope (Fig. 6 b). Since the absence of hops would result in a horizontal line in a longer time regime (Saxton, 1989; Kusumi et al., 1993), the positive slope represents hop diffusion over different compartments (the macroscopic diffusion coefficient DMACRO). Therefore, the MSD-Δt plots in Fig. 6, a and b indicate that intercompartmental hops take place on a time scale of 1,000 ms, which can be detected even in a time window of 50 ms (Fig. 6 a). These results are consistent with the impression of the trajectories shown in Fig. 5, b–d, and suggest that the macroscopic diffusion takes place as a result of a series of intercompartmental hops.


Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton.

Tomishige M, Sako Y, Kusumi A - J. Cell Biol. (1998)

(a and b) Typical plots of MSD against the time interval  for a particle–band 3 complex. The MSD-Δt plot in a shows a  steep rise and fast leveling off, indicating confined diffusion of  this particle in a time window of 10 ms. Intercompartmental hops  took place in a time scale of 1,000 ms as they appear as the positive slope in b (the slope gives 4DMACRO). (c) MSD-Δt plots for intact and cleaved band 3 in a time window of 2 s, averaged over all  of the respective particles studied in this work (in c, ensemble averaging over all of the particles was taken, whereas a running average over a single particle's trajectory was calculated in a and b).  Cleaved band 3 shows a distinctly greater slope in the time window of 2 s. Inset, the ensemble-averaged MSDs for intact and  cleaved band 3 in a time window of 20 ms. In this time scale, the  difference between intact and cleaved band 3 was slight, suggesting that their diffusion characteristics within a compartment are  similar to each other.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132872&req=5

Figure 6: (a and b) Typical plots of MSD against the time interval for a particle–band 3 complex. The MSD-Δt plot in a shows a steep rise and fast leveling off, indicating confined diffusion of this particle in a time window of 10 ms. Intercompartmental hops took place in a time scale of 1,000 ms as they appear as the positive slope in b (the slope gives 4DMACRO). (c) MSD-Δt plots for intact and cleaved band 3 in a time window of 2 s, averaged over all of the respective particles studied in this work (in c, ensemble averaging over all of the particles was taken, whereas a running average over a single particle's trajectory was calculated in a and b). Cleaved band 3 shows a distinctly greater slope in the time window of 2 s. Inset, the ensemble-averaged MSDs for intact and cleaved band 3 in a time window of 20 ms. In this time scale, the difference between intact and cleaved band 3 was slight, suggesting that their diffusion characteristics within a compartment are similar to each other.
Mentions: For a quantitative analysis of the movement of mobile band 3 molecules, the MSD of the particle was plotted against the time interval (Δt) (Fig. 6, a and b). Almost all of the MSDs showed a rapid rise and leveling off in a time window of ∼10 ms (Fig. 6 a). Statistical analysis according to the method by Kusumi et al. (1993) indeed showed that all of the mobile band 3 molecules undergo confined diffusion in a time window of 20 ms. The rapid rise near Δt = 0 reflects fast diffusion within a compartment (the microscopic diffusion coefficient Dmicro). In a longer time window, the MSD-Δt plot asymptotically approached a straight line with a constant positive slope (Fig. 6 b). Since the absence of hops would result in a horizontal line in a longer time regime (Saxton, 1989; Kusumi et al., 1993), the positive slope represents hop diffusion over different compartments (the macroscopic diffusion coefficient DMACRO). Therefore, the MSD-Δt plots in Fig. 6, a and b indicate that intercompartmental hops take place on a time scale of 1,000 ms, which can be detected even in a time window of 50 ms (Fig. 6 a). These results are consistent with the impression of the trajectories shown in Fig. 5, b–d, and suggest that the macroscopic diffusion takes place as a result of a series of intercompartmental hops.

Bottom Line: When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton.Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral.These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

ABSTRACT
Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

Show MeSH