© The Rockefeller University Press,
0021-9525/1998//989 $5.00
The Journal of Cell Biology, Volume 142, Number 4,
, 1998 989-1000
Regulation Mechanism of the Lateral Diffusion of Band 3 in Erythrocyte Membranes by the Membrane Skeleton
Michio Tomishige*,
,
Yasushi Sako*, and
Akihiro Kusumi*
* Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan and Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-0041, Japan
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.
Key Words: lateral diffusion • erythrocyte membrane • membrane skeleton • single particle tracking • optical tweezers
Abbreviations used in this paper: 
, diameter; D, lateral diffusion coefficient; DMACRO, macroscopic diffusion coefficient; Dmicro, microscopic diffusion coefficient; Fl-PE, fluorescein-phosphatidylethanolamine; FRAP, fluorescence redistribution after photobleaching; MSD, mean square displacement; pA-PMSF, (p-amidinophenyl) methanesulfonyl fluoride hydrochloride; SPT, single particle tracking.
Address all correspondence to: Akihiro Kusumi, Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. Tel.: (81) 52-789-2969. Fax: (81) 52-789-2968. E-mail: akusumi{at}bio.nagoya-u.ac.jp
Y. Sako's present address is First Department of Physiology, Medical School of Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan.
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