Diffusion of low density lipoprotein-receptor complex on human fibroblasts

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Abstract
Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts was measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovery and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10.degree. C, < 20% of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor-internalization-defective J. D. cells GM-2408A. At 21.degree. and 28.degree. C, the diffusion coefficients of the LDL-receptor complex were 1.4 and 4.5 .times. 10-11 cm2/s with diffusible fractions of .apprx. 75 and 60%, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5 .times. 10-8 cm2/s at 22.degree. C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to .apprx. 10-9 cm2/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD-phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on lateral diffusion observed on blebs. The internalization defect of J. D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least 2 orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.