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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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Movements of band 3 during deformation of the membrane skeleton. (a) An experimental design to test the occurrence  of collisions between the cytoplasmic portion of band 3 and the  membrane skeleton. Using optical tweezers, the membrane–skeletal network was deformed by dragging a 1-μm-φ latex bead that  had been attached to the network, and the movement of band 3  molecules that were undergoing hop diffusion on the same cell  was monitored. If band 3 collides with the membrane skeleton, it  will be moved along with the bead. (b) Sequential images obtained in a membrane-skeleton dragging experiment. When the  1-μm-φ latex bead attached to the membrane skeleton was  moved by the optical trap at a velocity of 1.8 μm/s (carets), gold  particles attached to band 3 that were undergoing hop diffusion  (arrows) were displaced toward the same direction as the bead.  Note that the change in the contour of the cell was almost negligible. (c) Trajectories of the latex bead (Bead) and gold particles  (Gold) shown in b (refer to text for details). The time resolution  was 33 ms. Bars: (b) 2 μm; (c) 1 μm.
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Figure 8: Movements of band 3 during deformation of the membrane skeleton. (a) An experimental design to test the occurrence of collisions between the cytoplasmic portion of band 3 and the membrane skeleton. Using optical tweezers, the membrane–skeletal network was deformed by dragging a 1-μm-φ latex bead that had been attached to the network, and the movement of band 3 molecules that were undergoing hop diffusion on the same cell was monitored. If band 3 collides with the membrane skeleton, it will be moved along with the bead. (b) Sequential images obtained in a membrane-skeleton dragging experiment. When the 1-μm-φ latex bead attached to the membrane skeleton was moved by the optical trap at a velocity of 1.8 μm/s (carets), gold particles attached to band 3 that were undergoing hop diffusion (arrows) were displaced toward the same direction as the bead. Note that the change in the contour of the cell was almost negligible. (c) Trajectories of the latex bead (Bead) and gold particles (Gold) shown in b (refer to text for details). The time resolution was 33 ms. Bars: (b) 2 μm; (c) 1 μm.

Mentions: To examine whether or not the cytoplasmic domain of band 3 actually collides with the membrane skeleton, we displaced the mesh of the membrane skeleton by dragging and deforming the skeletal network with optical tweezers, and investigated whether the displacement of the network causes forced movements of band 3 molecules that are not bound to the membrane skeleton (Fig. 8 a). If band 3 is confined by collision with the spectrin skeleton, even band 3 molecules that are not bound to the skeleton should be displaced by moving the membrane skeleton 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)

Movements of band 3 during deformation of the membrane skeleton. (a) An experimental design to test the occurrence  of collisions between the cytoplasmic portion of band 3 and the  membrane skeleton. Using optical tweezers, the membrane–skeletal network was deformed by dragging a 1-μm-φ latex bead that  had been attached to the network, and the movement of band 3  molecules that were undergoing hop diffusion on the same cell  was monitored. If band 3 collides with the membrane skeleton, it  will be moved along with the bead. (b) Sequential images obtained in a membrane-skeleton dragging experiment. When the  1-μm-φ latex bead attached to the membrane skeleton was  moved by the optical trap at a velocity of 1.8 μm/s (carets), gold  particles attached to band 3 that were undergoing hop diffusion  (arrows) were displaced toward the same direction as the bead.  Note that the change in the contour of the cell was almost negligible. (c) Trajectories of the latex bead (Bead) and gold particles  (Gold) shown in b (refer to text for details). The time resolution  was 33 ms. Bars: (b) 2 μm; (c) 1 μm.
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Related In: Results  -  Collection

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Figure 8: Movements of band 3 during deformation of the membrane skeleton. (a) An experimental design to test the occurrence of collisions between the cytoplasmic portion of band 3 and the membrane skeleton. Using optical tweezers, the membrane–skeletal network was deformed by dragging a 1-μm-φ latex bead that had been attached to the network, and the movement of band 3 molecules that were undergoing hop diffusion on the same cell was monitored. If band 3 collides with the membrane skeleton, it will be moved along with the bead. (b) Sequential images obtained in a membrane-skeleton dragging experiment. When the 1-μm-φ latex bead attached to the membrane skeleton was moved by the optical trap at a velocity of 1.8 μm/s (carets), gold particles attached to band 3 that were undergoing hop diffusion (arrows) were displaced toward the same direction as the bead. Note that the change in the contour of the cell was almost negligible. (c) Trajectories of the latex bead (Bead) and gold particles (Gold) shown in b (refer to text for details). The time resolution was 33 ms. Bars: (b) 2 μm; (c) 1 μm.
Mentions: To examine whether or not the cytoplasmic domain of band 3 actually collides with the membrane skeleton, we displaced the mesh of the membrane skeleton by dragging and deforming the skeletal network with optical tweezers, and investigated whether the displacement of the network causes forced movements of band 3 molecules that are not bound to the membrane skeleton (Fig. 8 a). If band 3 is confined by collision with the spectrin skeleton, even band 3 molecules that are not bound to the skeleton should be displaced by moving the membrane skeleton 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