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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.

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Typical trajectories of band 3 that is (or is not) undergoing macroscopic diffusion, spectrin, and Fl-PE (artificially incorporated lipid) in erythrocyte membranes with time resolutions  of 33 and 0.22 ms (total observation times of 6.7 s and 67 ms), respectively. The macroscopically mobile band 3 was undergoing  apparent simple Brownian diffusion at a time resolution of 33 ms.  However, the diffusion rate was slow compared with that of  Fl-PE in the same time scale. Macroscopically immobile band 3  showed oscillatory movements at both time scales, which were  similar to that of spectrin. Note that the magnification for Fl-PE  with a 33 ms resolution is reduced by a factor of two. Bars, 500 nm.
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Figure 3: Typical trajectories of band 3 that is (or is not) undergoing macroscopic diffusion, spectrin, and Fl-PE (artificially incorporated lipid) in erythrocyte membranes with time resolutions of 33 and 0.22 ms (total observation times of 6.7 s and 67 ms), respectively. The macroscopically mobile band 3 was undergoing apparent simple Brownian diffusion at a time resolution of 33 ms. However, the diffusion rate was slow compared with that of Fl-PE in the same time scale. Macroscopically immobile band 3 showed oscillatory movements at both time scales, which were similar to that of spectrin. Note that the magnification for Fl-PE with a 33 ms resolution is reduced by a factor of two. Bars, 500 nm.

Mentions: In SPT observations, one-third of band 3 molecules did not exhibit long-range diffusion at 37°C. The percentage of such particles was close to the immobile fractions in translational (FRAP) and rotational (anisotropy decay) diffusion measurements (Tsuji and Ohnishi, 1986; Tsuji et al., 1988), suggesting that the immobile fractions in these experiments represent band 3 molecules bound to the membrane skeleton. The band 3 molecules that do not undergo long-range diffusion in SPT showed oscillatory movements (Fig. 2 b), which was observed even at a time resolution of 0.22 ms (Fig. 3). When such particles were dragged along the membrane by optical tweezers at a maximum trapping force of 0.25 pN, they could be dragged ∼300 nm until they escaped from the optical trap and returned to the initial position (Fig. 4, a and c). Such movement and responses to being dragged are very similar to those of gold particles attached to spectrin (Figs. 3 and 4). Taken together, these results indicate that band 3 molecules that are not undergoing long-range diffusion are bound to the spectrin network, and that the oscillatory restricted motion that these band 3 molecules exhibit would represent thermal conformational fluctuations of the membrane skeletal network.


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)

Typical trajectories of band 3 that is (or is not) undergoing macroscopic diffusion, spectrin, and Fl-PE (artificially incorporated lipid) in erythrocyte membranes with time resolutions  of 33 and 0.22 ms (total observation times of 6.7 s and 67 ms), respectively. The macroscopically mobile band 3 was undergoing  apparent simple Brownian diffusion at a time resolution of 33 ms.  However, the diffusion rate was slow compared with that of  Fl-PE in the same time scale. Macroscopically immobile band 3  showed oscillatory movements at both time scales, which were  similar to that of spectrin. Note that the magnification for Fl-PE  with a 33 ms resolution is reduced by a factor of two. Bars, 500 nm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132872&req=5

Figure 3: Typical trajectories of band 3 that is (or is not) undergoing macroscopic diffusion, spectrin, and Fl-PE (artificially incorporated lipid) in erythrocyte membranes with time resolutions of 33 and 0.22 ms (total observation times of 6.7 s and 67 ms), respectively. The macroscopically mobile band 3 was undergoing apparent simple Brownian diffusion at a time resolution of 33 ms. However, the diffusion rate was slow compared with that of Fl-PE in the same time scale. Macroscopically immobile band 3 showed oscillatory movements at both time scales, which were similar to that of spectrin. Note that the magnification for Fl-PE with a 33 ms resolution is reduced by a factor of two. Bars, 500 nm.
Mentions: In SPT observations, one-third of band 3 molecules did not exhibit long-range diffusion at 37°C. The percentage of such particles was close to the immobile fractions in translational (FRAP) and rotational (anisotropy decay) diffusion measurements (Tsuji and Ohnishi, 1986; Tsuji et al., 1988), suggesting that the immobile fractions in these experiments represent band 3 molecules bound to the membrane skeleton. The band 3 molecules that do not undergo long-range diffusion in SPT showed oscillatory movements (Fig. 2 b), which was observed even at a time resolution of 0.22 ms (Fig. 3). When such particles were dragged along the membrane by optical tweezers at a maximum trapping force of 0.25 pN, they could be dragged ∼300 nm until they escaped from the optical trap and returned to the initial position (Fig. 4, a and c). Such movement and responses to being dragged are very similar to those of gold particles attached to spectrin (Figs. 3 and 4). Taken together, these results indicate that band 3 molecules that are not undergoing long-range diffusion are bound to the spectrin network, and that the oscillatory restricted motion that these band 3 molecules exhibit would represent thermal conformational fluctuations of the membrane skeletal network.

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