Limits...
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.

Show MeSH
Representative trajectories of band 3 in the  erythrocyte membrane observed using a high-speed  video system. (a) A representative image of a colloidal  gold-labeled ghost (2-ms  frame time). (b and c) Typical trajectories of intact  band 3 in a ghost with time  resolutions of 2 and 0.22 ms  (total observation times of  700 and 120 ms), respectively. In b, by following the  trajectories closely by eye,  the portions of the trajectories that were presumably  within individual compartments and of intercompartmental hops were identified and are shown in different colors (red, orange, and magenta for compartments;  blue and green for hops). In c, the color is changed simply to indicate the passage of time, and these trajectories suggest that the particle  stayed in the same compartment during this observation period (120 ms). (d) A typical trajectory of intact band 3 observed with a time  resolution of 2 ms, showing many returns to the same domains it passed through previously with various residency times (180, 250, 210,  50, 210, 190, and 240 ms for each domains). Individual compartments and intercompartmental hops are shown in different colors (blue,  hops). Individual compartments are numbered sequentially. Note that domains 1, 5, and 7 (also domains 2 and 6) match quite well with  regard to position and shape. (e and f) Typical trajectories of trypsin-cleaved band 3 observed with time resolutions of 2 and 0.22 ms (total observation times of 700 and 120 ms), respectively. In e, the color change merely indicates the passage of time. Hop diffusion becomes apparent only at a higher time resolution of 0.22 ms (f). These results show that rapid intercompartmental hop diffusion of  cleaved band 3 can only be detected when the temporal resolution is enhanced. Bars: (a) 2 μm; (b–f) 100 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132872&req=5

Figure 5: Representative trajectories of band 3 in the erythrocyte membrane observed using a high-speed video system. (a) A representative image of a colloidal gold-labeled ghost (2-ms frame time). (b and c) Typical trajectories of intact band 3 in a ghost with time resolutions of 2 and 0.22 ms (total observation times of 700 and 120 ms), respectively. In b, by following the trajectories closely by eye, the portions of the trajectories that were presumably within individual compartments and of intercompartmental hops were identified and are shown in different colors (red, orange, and magenta for compartments; blue and green for hops). In c, the color is changed simply to indicate the passage of time, and these trajectories suggest that the particle stayed in the same compartment during this observation period (120 ms). (d) A typical trajectory of intact band 3 observed with a time resolution of 2 ms, showing many returns to the same domains it passed through previously with various residency times (180, 250, 210, 50, 210, 190, and 240 ms for each domains). Individual compartments and intercompartmental hops are shown in different colors (blue, hops). Individual compartments are numbered sequentially. Note that domains 1, 5, and 7 (also domains 2 and 6) match quite well with regard to position and shape. (e and f) Typical trajectories of trypsin-cleaved band 3 observed with time resolutions of 2 and 0.22 ms (total observation times of 700 and 120 ms), respectively. In e, the color change merely indicates the passage of time. Hop diffusion becomes apparent only at a higher time resolution of 0.22 ms (f). These results show that rapid intercompartmental hop diffusion of cleaved band 3 can only be detected when the temporal resolution is enhanced. Bars: (a) 2 μm; (b–f) 100 nm.

Mentions: To characterize the movement of mobile band 3 molecules, short-term movements of mobile band 3 in the ghost membrane were observed using a high-speed video system (Fig. 5 a). Fig. 5, b and c show typical trajectories of band 3 recorded with time resolutions of 2 and 0.22 ms, respectively. These recording rates are greater than those of normal video by factors of 17 and 150, respectively. By following the trajectory in Fig. 5 b closely by eye, plausible compartments (domains) were found, as shown in different colors. Occasions of hops between compartments can be clearly identified as shown in the blue and green lines in Fig. 5, b and d. The adjacent compartments are closely apposed to each other, and the particles did not return to the previous compartment before the next hop occurs. When a particle did return within several seconds to the domain that it had passed through previously, the domain looked similar with regard to position and shape (Fig. 5 d). Therefore, these trajectories suggest that these band 3 molecules are temporarily confined within domains and occasionally hop to adjacent domains.


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)

Representative trajectories of band 3 in the  erythrocyte membrane observed using a high-speed  video system. (a) A representative image of a colloidal  gold-labeled ghost (2-ms  frame time). (b and c) Typical trajectories of intact  band 3 in a ghost with time  resolutions of 2 and 0.22 ms  (total observation times of  700 and 120 ms), respectively. In b, by following the  trajectories closely by eye,  the portions of the trajectories that were presumably  within individual compartments and of intercompartmental hops were identified and are shown in different colors (red, orange, and magenta for compartments;  blue and green for hops). In c, the color is changed simply to indicate the passage of time, and these trajectories suggest that the particle  stayed in the same compartment during this observation period (120 ms). (d) A typical trajectory of intact band 3 observed with a time  resolution of 2 ms, showing many returns to the same domains it passed through previously with various residency times (180, 250, 210,  50, 210, 190, and 240 ms for each domains). Individual compartments and intercompartmental hops are shown in different colors (blue,  hops). Individual compartments are numbered sequentially. Note that domains 1, 5, and 7 (also domains 2 and 6) match quite well with  regard to position and shape. (e and f) Typical trajectories of trypsin-cleaved band 3 observed with time resolutions of 2 and 0.22 ms (total observation times of 700 and 120 ms), respectively. In e, the color change merely indicates the passage of time. Hop diffusion becomes apparent only at a higher time resolution of 0.22 ms (f). These results show that rapid intercompartmental hop diffusion of  cleaved band 3 can only be detected when the temporal resolution is enhanced. Bars: (a) 2 μm; (b–f) 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Representative trajectories of band 3 in the erythrocyte membrane observed using a high-speed video system. (a) A representative image of a colloidal gold-labeled ghost (2-ms frame time). (b and c) Typical trajectories of intact band 3 in a ghost with time resolutions of 2 and 0.22 ms (total observation times of 700 and 120 ms), respectively. In b, by following the trajectories closely by eye, the portions of the trajectories that were presumably within individual compartments and of intercompartmental hops were identified and are shown in different colors (red, orange, and magenta for compartments; blue and green for hops). In c, the color is changed simply to indicate the passage of time, and these trajectories suggest that the particle stayed in the same compartment during this observation period (120 ms). (d) A typical trajectory of intact band 3 observed with a time resolution of 2 ms, showing many returns to the same domains it passed through previously with various residency times (180, 250, 210, 50, 210, 190, and 240 ms for each domains). Individual compartments and intercompartmental hops are shown in different colors (blue, hops). Individual compartments are numbered sequentially. Note that domains 1, 5, and 7 (also domains 2 and 6) match quite well with regard to position and shape. (e and f) Typical trajectories of trypsin-cleaved band 3 observed with time resolutions of 2 and 0.22 ms (total observation times of 700 and 120 ms), respectively. In e, the color change merely indicates the passage of time. Hop diffusion becomes apparent only at a higher time resolution of 0.22 ms (f). These results show that rapid intercompartmental hop diffusion of cleaved band 3 can only be detected when the temporal resolution is enhanced. Bars: (a) 2 μm; (b–f) 100 nm.
Mentions: To characterize the movement of mobile band 3 molecules, short-term movements of mobile band 3 in the ghost membrane were observed using a high-speed video system (Fig. 5 a). Fig. 5, b and c show typical trajectories of band 3 recorded with time resolutions of 2 and 0.22 ms, respectively. These recording rates are greater than those of normal video by factors of 17 and 150, respectively. By following the trajectory in Fig. 5 b closely by eye, plausible compartments (domains) were found, as shown in different colors. Occasions of hops between compartments can be clearly identified as shown in the blue and green lines in Fig. 5, b and d. The adjacent compartments are closely apposed to each other, and the particles did not return to the previous compartment before the next hop occurs. When a particle did return within several seconds to the domain that it had passed through previously, the domain looked similar with regard to position and shape (Fig. 5 d). Therefore, these trajectories suggest that these band 3 molecules are temporarily confined within domains and occasionally hop to adjacent domains.

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