Experiments are proposed to study the subcellular events in lipoprotein processing by monocyte-derived macrophages cultured from White Carneau pigeons. Our objective is to determine if differences in metabolism of lipoproteins are related to differential intracellular sorting by pigeon macrophages. The internalization routes and intracellular sorting of lipoproteins in pigeon monocyte-derived macrophages will be studied ultrastructurally for four lipoproteins; beta migrating very low density lipoprotein (BVLDL), acetylated low density lipoprotein (AcLDL), low density lipoprotein (LDL), and aggregated LDL (LDL). Two approaches will be used to compare the lipoproteins. First, lipoproteins will be compared from the perspective of internalization route; clathrin-coated (LDL and small BVLDL), non-coated (AcLDL) and large BVLDL) membrane receptor- mediated endocytosis, or receptor-mediated phagocytosis (LDL). Secondly, lipoprotein subcellular processing will be compared with their ability to stimulate acyl-C0A:cholesterol acyltransferase (ACAT) and cholesterol ester accumulation. Initially, the surface distribution for each lipoprotein and receptor will be determined by TEM, video-enhanced light microscopy and SEM. Next, the ultrastructural pathway of internalization for each lipoprotein will be determined by thin section TEM and video-microscopy (AVEC-DIC, nanovid and fluorescence). These ultrastructural pathways will be compared to standards for 1) fluid-phase endocytosis (horseradish peroxidase and lucifer yellow), 2) receptor-mediated endocytosis (alpha2macroglobulin) and 3) phagocytosis (latex beads). The relationships-among differential lipoprotein uptake, pre-lysosomal sorting, lysosomal activity and cytoplasmic lipid accumulation will be determined by co-incubation of pairs of lipoproteins (BVLDL subfractions, LDL, AcLDL, and LDL). Lipoproteins will be differentially labeled by using gold colloids with differing diameters or different fluorescent labeling. The subcellular localization and spatial (3-D) organization of bound lipoproteins or endocytosed particles will be established through computer reconstructions, and 3-D (stereo pair) IVEM thick section of whole mounts. All studies which will be carried out in the atherosclerosis research center, capitalizing on the presence of two unique technologies, a resource of intermediate microscopy and an institutional facility for video-enhanced light microscopy.
Showing the most recent 10 out of 15 publications
