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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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Distributions of the microscopic diffusion coefficient  Dmicro (a), the macroscopic diffusion coefficient DMACRO (b), and  the size of the confinement domain L (c). The median values are  indicated by arrowheads. Open bars, intact band 3; hatched bars,  trypsin-cleaved band 3.
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Figure 7: Distributions of the microscopic diffusion coefficient Dmicro (a), the macroscopic diffusion coefficient DMACRO (b), and the size of the confinement domain L (c). The median values are indicated by arrowheads. Open bars, intact band 3; hatched bars, trypsin-cleaved band 3.

Mentions: By analyzing the MSD-Δt plots in the time window of 10 ms using the method described in Kusumi et al. (1993), Dmicro and the size of the confinement domain L were obtained. Distribution of Dmicro is shown in Fig. 7 a. The median value is 5.3 × 10−9 cm2/s (Table I), which is close to a value one might expect for freely diffusing membrane proteins in the plasma membrane (Poo and Cone, 1974; Golan et al., 1984; Berk and Hochmuth, 1992; Cole et al., 1996), suggesting that band 3 undergoes free diffusion within a compartment. Dmicro is also similar to the diffusion rate of band 3 in spectrin-deficient mouse erythrocyte ghosts (Sheetz et al., 1980) and in spectrin-deficient erythrocytes from patients with hereditary spherocytosis (Corbett et al., 1994) as measured by FRAP (Table I), which again suggests that band 3 undergoes uninhibited diffusion within each compartment.


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)

Distributions of the microscopic diffusion coefficient  Dmicro (a), the macroscopic diffusion coefficient DMACRO (b), and  the size of the confinement domain L (c). The median values are  indicated by arrowheads. Open bars, intact band 3; hatched bars,  trypsin-cleaved band 3.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Distributions of the microscopic diffusion coefficient Dmicro (a), the macroscopic diffusion coefficient DMACRO (b), and the size of the confinement domain L (c). The median values are indicated by arrowheads. Open bars, intact band 3; hatched bars, trypsin-cleaved band 3.
Mentions: By analyzing the MSD-Δt plots in the time window of 10 ms using the method described in Kusumi et al. (1993), Dmicro and the size of the confinement domain L were obtained. Distribution of Dmicro is shown in Fig. 7 a. The median value is 5.3 × 10−9 cm2/s (Table I), which is close to a value one might expect for freely diffusing membrane proteins in the plasma membrane (Poo and Cone, 1974; Golan et al., 1984; Berk and Hochmuth, 1992; Cole et al., 1996), suggesting that band 3 undergoes free diffusion within a compartment. Dmicro is also similar to the diffusion rate of band 3 in spectrin-deficient mouse erythrocyte ghosts (Sheetz et al., 1980) and in spectrin-deficient erythrocytes from patients with hereditary spherocytosis (Corbett et al., 1994) as measured by FRAP (Table I), which again suggests that band 3 undergoes uninhibited diffusion within each compartment.

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