Early appearance of dispersed low density lipoprotein receptors on the fibroblast surface during recycling.

We have examined the distributions of recycling low density lipoprotein receptors (LDL-Rs) as they emerge onto and cluster on the surfaces of cultured cells. Surface LDL-Rs were labeled with colloidal gold-LDL conjugates (AuLDL) and cells viewed as whole-mounts in the transmission electron microscope. The steady-state distribution of LDL-Rs on the cell surface, labeled with AuLDL at 4 degrees C, comprised ring-shaped clusters of receptors with dispersed receptors scattered amongst them. After 12 min of incubation at 37 degrees C, virtually all AuLDL probes were internalized. Electron microscopy of thin sections revealed clustered receptors in coated pits and the progressive accumulation of AuLDL in endosomes, multivesicular bodies and lysosomes. By initially blocking all surface LDL-Rs, either with unconjugated LDL or AuLDL of one size, the clustering behavior of newly emerged receptors which recycled to the cell surface was selectively visualized with an AuLDL probe of a second size over a defined time-course. Release of the blocking ligand during the time-course was found to be negligible. Newly appearing dispersed LDL-Rs were detected as early as 2 min and these were often concentrated at the cell margins. The newly labeled and preblocked LDL-Rs did not cocluster before 6 min. By 8 to 12 min, ring-shaped clusters of newly emerged receptors had formed and these were often seen associated with pre-blocked LDL-Rs. The clustering of LDL-Rs on the cell surface was independent of the presence of ligand, AuLDL. Our results indicate that LDL-Rs recycle to the cell surface where they form a dispersed population which gives rise to the ring-shaped clusters of cell surface LDL-Rs associated with coated pits.