Abstract
Movements of transferrin and α2-macroglobulin receptor molecules in the plasma membrane of cultured normal rat kidney (NRK) fibroblastic cells were investigated by video-enhanced optical microscopy with 1.8 nm spatial precision and 33 ms temporal resolution 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=0.25 μm2 (500-700 nm in diagonal length). Movement within the domains is random with a microscopic diffusion coeffcient (Dmicro) =10-9cm2/s, which is consistent with that expected for free Brownian diffusion of proteins in the plasma membrane. The receptor molecules move from one domain to one of the adjacent domains at an average frequency of 0.034s-1 (the residence time within a domain=29s), indicating that the plasma membrane is compartmentalized for diffusion of membrane receptors and that longrange diffusion is the result of successive intercompartmental jumps. The macroscopic diffusion coefficients for these two receptor molecules calculated on the basis of the compartment size and the intercompartmental jump rate are=2.4 × 10-11cm2/s, which is consistent with those determimed determined by averaging the long-term movements of many particles. Partial destruction of the cytoskeleton decreased the confined diffusion mode, increased the simpple diffusion mode, and induced the directed diffusion (transport) mode, Those resules suggest that the boundaries between compartments are made of dynamically fluctuating membrane skeletons (membrane-skeletion fence model).
The mechanical properties of intercompartmental boundaries were then studied by tagging transferrin receptor (TR) with either 210 nm-φ latex or 40nm-φ 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 that showed confined-type diffusion with Dmicro of=10-9cm2/s could be dragged past the intercompartmental boundaries in their path by laser tweezers at a trapping force of 0.35-0.8 pN. At the dragging forces between 0.05 and 0.1 pN, 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 average distance dragged was half of the confined distance of TR, which further indicates that particle-TR complexes escape at the compartment boundaries. The boundaries are likely present in the cytoplasmic domain, and are elastic. These results are consistent with the proposal that the compartment boundaries consist of membrane skeleton or a membrane-associated part of the cytoskeleton. Approximately 10% or TR exhibited slower diffusion (Dmicro=10-10-10-11cm2/s) and binding to elastic structures.
