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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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(a and b) Representative trajectories of gold particles  attached to band 3 that does not exhibit macroscopic diffusion (a)  and spectrin (b) when they were dragged using optical tweezers  at a velocity of 0.6 μm/s (maximum trapping force of 0.25 pN).  Gold particles attached to macroscopically immobile band 3 molecules could be dragged ∼300 nm (red line) until they escaped  from the trap and returned to the initial position (blue line). Gold  particles attached to spectrin showed similar behavior to that of  immobile band 3. (c and d) Histograms showing distributions of  the dragged distance of immobile band 3 and spectrin. Dragged  distance is defined as the distance from the initial position to the  farthest point reached by the particle (Sako and Kusumi, 1995;  Sako et al., 1998). Arrowheads, median values for band 3 and  spectrin, which were 290 nm and 330 nm, respectively. Bar, 100 nm.
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Figure 4: (a and b) Representative trajectories of gold particles attached to band 3 that does not exhibit macroscopic diffusion (a) and spectrin (b) when they were dragged using optical tweezers at a velocity of 0.6 μm/s (maximum trapping force of 0.25 pN). Gold particles attached to macroscopically immobile band 3 molecules could be dragged ∼300 nm (red line) until they escaped from the trap and returned to the initial position (blue line). Gold particles attached to spectrin showed similar behavior to that of immobile band 3. (c and d) Histograms showing distributions of the dragged distance of immobile band 3 and spectrin. Dragged distance is defined as the distance from the initial position to the farthest point reached by the particle (Sako and Kusumi, 1995; Sako et al., 1998). Arrowheads, median values for band 3 and spectrin, which were 290 nm and 330 nm, respectively. Bar, 100 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)

(a and b) Representative trajectories of gold particles  attached to band 3 that does not exhibit macroscopic diffusion (a)  and spectrin (b) when they were dragged using optical tweezers  at a velocity of 0.6 μm/s (maximum trapping force of 0.25 pN).  Gold particles attached to macroscopically immobile band 3 molecules could be dragged ∼300 nm (red line) until they escaped  from the trap and returned to the initial position (blue line). Gold  particles attached to spectrin showed similar behavior to that of  immobile band 3. (c and d) Histograms showing distributions of  the dragged distance of immobile band 3 and spectrin. Dragged  distance is defined as the distance from the initial position to the  farthest point reached by the particle (Sako and Kusumi, 1995;  Sako et al., 1998). Arrowheads, median values for band 3 and  spectrin, which were 290 nm and 330 nm, respectively. Bar, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132872&req=5

Figure 4: (a and b) Representative trajectories of gold particles attached to band 3 that does not exhibit macroscopic diffusion (a) and spectrin (b) when they were dragged using optical tweezers at a velocity of 0.6 μm/s (maximum trapping force of 0.25 pN). Gold particles attached to macroscopically immobile band 3 molecules could be dragged ∼300 nm (red line) until they escaped from the trap and returned to the initial position (blue line). Gold particles attached to spectrin showed similar behavior to that of immobile band 3. (c and d) Histograms showing distributions of the dragged distance of immobile band 3 and spectrin. Dragged distance is defined as the distance from the initial position to the farthest point reached by the particle (Sako and Kusumi, 1995; Sako et al., 1998). Arrowheads, median values for band 3 and spectrin, which were 290 nm and 330 nm, respectively. Bar, 100 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
Related in: MedlinePlus