| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1994: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1993: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Research Abstract |
Movements of transferrin and alpha2-macroglobulin receptor molecules in the plasma membrane of cultured normal rat kidney (NRK) fibroblastic cells were investigated by video-enhanced optical microscopy with a nanometer-level spatial precision and a temporal resolution up to 0.2ms by labeling the receptors with the ligand-coated nanometer-sized colloidal gold particles. For both receptor species, approximately 90% of the movement trajectories are of the confined diffusion type, within domains of <approximately equal> 0.25mum^2 (500-700nm in diagonal length). Movement within the domains is randon with a microscopic diffusion coefficient (D_<micro>) <approximately equal> 10^<-9>cm^2/s, which is consistent with a value expected for freely diffusing proteins in the plasma membrane. The receptor molecules move from one domain to one of the adjacent domains every 25 s on average, indicating that the plasma membrane is compartmentalized for diffusion of membrane receptors and that long-range d
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iffusion occurs as a result of successive intercompartmental hops. The macroscopic diffusion coefficients for these two receptor molecules are <approximately equal> 3x10^<-11>cm^2/s, which is smaller than D_<micro> by a factor of 30. The above results indicate that the macroscopic diffusion is slowed due to confinement by the boundaries, and not due to the intrinsically slow rate of diffusion. Partial destruction of the cytoskeleton and partial deletion of the cytoplasmic domains of many membrane receptors strongly influenced their diffusion properties, indicating that the boundaries between compartments are made of the membrane-associated part of the cytoskeleton or the membrane skeleton (membrane-skeleton fence model).
The mechanical properties of intercompartmental boundaries were then studied by tagging transferrin receptor (TR) with either 210nm-phi latex or 40nm-phi colloidal gold particles, and by dragging the particle-TR complexes laterally along the plasma membrane using laser tweezers. Approximately 90% of the TR-particle complexes, which showed confined-type diffusion with D_<micro> of <approximately equal> 10^<-9>cm^2/s, could be dragged past the intercompartmental boundaries in their path by laser tweezers at a trapping force of 0.35-0.8pN.At the dragging forces between 0.05 and 0.1pN,particle-TR complexes tended to escape from the laser trap at the boundaries, and such escape occurred in both the forward and backward directions of dragging. The boundaries are elastic with an effective elastic constant of 1-10pN/mum. These results are consistent with the proposal that the compartment boundaries consist of membrane skeleton. Approximately 10% of TR exhibited slower diffusion (D_<micro> <approximately equal> 10^<-10>-10^<-11>cm^2/s) and binding to elastic structures. Less
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