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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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Examination of the viscous drag induced by the membrane skeleton dragging. (a and b) Latex bead attached to the  membrane skeleton (Bead) was dragged using optical tweezers at  a velocity of 0.15 μm/s (a) and 1.8 μm/s (b) for a distance of 2.5  μm as shown in the blue lines. Gold particles bound to band 3  that was undergoing macroscopic diffusion (Gold) followed the  bead even at a slow rate of 0.15 μm/s. The movement of band 3  was like a superposition of hop diffusion and directed motion of  the membrane skeletal network. (c) Effect of dragging the membrane skeleton on the lateral diffusion of lipid. When the latex  bead attached to the membrane skeleton (Bead) was dragged at  a velocity of 0.6 μm/s, gold particles attached to Fl-PE (Gold) did  not exhibit any forced displacement (blue and green lines). Bar,  1 μm.
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Figure 9: Examination of the viscous drag induced by the membrane skeleton dragging. (a and b) Latex bead attached to the membrane skeleton (Bead) was dragged using optical tweezers at a velocity of 0.15 μm/s (a) and 1.8 μm/s (b) for a distance of 2.5 μm as shown in the blue lines. Gold particles bound to band 3 that was undergoing macroscopic diffusion (Gold) followed the bead even at a slow rate of 0.15 μm/s. The movement of band 3 was like a superposition of hop diffusion and directed motion of the membrane skeletal network. (c) Effect of dragging the membrane skeleton on the lateral diffusion of lipid. When the latex bead attached to the membrane skeleton (Bead) was dragged at a velocity of 0.6 μm/s, gold particles attached to Fl-PE (Gold) did not exhibit any forced displacement (blue and green lines). Bar, 1 μm.

Mentions: Dragging of the membrane skeleton moves skeleton-bound membrane proteins and lipids, which would induce a viscous drag in the fluid membrane. To examine the extent to which such viscous drag affects the movements of free band 3 and lipids, the following two experiments were carried out. First, the scan rate of the optical trap to drag the membrane skeleton was decreased by a factor of 12 to 0.15 μm/s (Fig. 9 a). Gold particles attached to band 3 that are undergoing hop diffusion followed just as they did at a dragging rate of 1.8 μm/s (Fig. 9 b). The deviation of the trajectories of band 3 perpendicular to the direction of dragging increased as the dragging rate was decreased. This can be explained by the presence of hop diffusion of band 3, which was superimposed on the unidirectional movement due to dragging of the membrane skeleton.


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)

Examination of the viscous drag induced by the membrane skeleton dragging. (a and b) Latex bead attached to the  membrane skeleton (Bead) was dragged using optical tweezers at  a velocity of 0.15 μm/s (a) and 1.8 μm/s (b) for a distance of 2.5  μm as shown in the blue lines. Gold particles bound to band 3  that was undergoing macroscopic diffusion (Gold) followed the  bead even at a slow rate of 0.15 μm/s. The movement of band 3  was like a superposition of hop diffusion and directed motion of  the membrane skeletal network. (c) Effect of dragging the membrane skeleton on the lateral diffusion of lipid. When the latex  bead attached to the membrane skeleton (Bead) was dragged at  a velocity of 0.6 μm/s, gold particles attached to Fl-PE (Gold) did  not exhibit any forced displacement (blue and green lines). Bar,  1 μm.
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Related In: Results  -  Collection

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Figure 9: Examination of the viscous drag induced by the membrane skeleton dragging. (a and b) Latex bead attached to the membrane skeleton (Bead) was dragged using optical tweezers at a velocity of 0.15 μm/s (a) and 1.8 μm/s (b) for a distance of 2.5 μm as shown in the blue lines. Gold particles bound to band 3 that was undergoing macroscopic diffusion (Gold) followed the bead even at a slow rate of 0.15 μm/s. The movement of band 3 was like a superposition of hop diffusion and directed motion of the membrane skeletal network. (c) Effect of dragging the membrane skeleton on the lateral diffusion of lipid. When the latex bead attached to the membrane skeleton (Bead) was dragged at a velocity of 0.6 μm/s, gold particles attached to Fl-PE (Gold) did not exhibit any forced displacement (blue and green lines). Bar, 1 μm.
Mentions: Dragging of the membrane skeleton moves skeleton-bound membrane proteins and lipids, which would induce a viscous drag in the fluid membrane. To examine the extent to which such viscous drag affects the movements of free band 3 and lipids, the following two experiments were carried out. First, the scan rate of the optical trap to drag the membrane skeleton was decreased by a factor of 12 to 0.15 μm/s (Fig. 9 a). Gold particles attached to band 3 that are undergoing hop diffusion followed just as they did at a dragging rate of 1.8 μm/s (Fig. 9 b). The deviation of the trajectories of band 3 perpendicular to the direction of dragging increased as the dragging rate was decreased. This can be explained by the presence of hop diffusion of band 3, which was superimposed on the unidirectional movement due to dragging of the membrane skeleton.

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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