The basic cell biology of transport of lipids and proteins in cells will be studied using quantitative fluorescence microscopy coupled with biochemical and ultrastructural tools. The proposed studies build directly on previous work establishing the basic methods to be used. The proposed work Is divided in two main themes relating to membrane protein transport (Aims l-lll) and cholesterol and lipid transport (Aims IV and V). These two themes interact synergistically because proteins are responsible for lipid transport, and membrane lipids strongly Influence the transport and distribution of membrane proteins. In Aim I the role of specific proteins will be studied using rapid and selective protein inactivation by chemical crosslinking or other methods. The effect bf inactivating target proteins (e.g, clathrin, GGAs, or Rab proteins) will be studied by following the transport of specific marker proteins (e.g., labeled Tac-Furin, Tac-TGN38, CI-MPR) within minutes after inactivation. In Aim II the role of Clc-7 and Ostml In regulating lysosomal pH will be studied. Delivery of these proteins to late endosomes and lysosomes is essential for proper acidification of these organelles In microglial cells. The role of these proteins in organelle acidification in other cell types, Including dendritic cells, will be examined. The transcriptional regulation of Clc-7 and Ostml In microglia and other cell types will also be examined. In Aim III the ability of macrophages and other cell types to create functional extracellular lysosomes will be examined. These lysosomal synapses play an important role In degradation of extracellular digestion of lipoprotein deposits, and their role in other types of extracellular degradation will be examined. In Aim IV intracellular sterol transport and distribution will be characterized using fluorescent sterols and biochemical analyses. Although proteins such as ABCA1 and ABCG4 are essential for export of cholesterol to HDLs, the precise role of these proteins remains unclear. The effect of expression of these proteins on the transbilayer distribution of sterol at the plasma membrane will be determined using previously developed fluorescence quenching assays. Similarly, the effect of NPC1 and NPC2 on the transbilayer distribution of sterol in late endosomes will be examined. The mechanisms and function of high levels of nonvesicular sterol transport within cells will also be'examined. In Aim V the Interplay between sterols and membrane traffic will be explored. In particular, the function of tubules that emanate from late endosomes In response to transient sterol loading will be studied. RELEVANCE fSee instructions): We are studying the basic mechanisms by which proteins and lipids move within cells. Findings from these studies are directly related to diseases associated with disorders in lipid and cholesterol transport, including diabetes, atherosclerosis, and lysosomal storage disorders. Our studies of protein degradation may also be related to diseases associated with abnormal protein deposits, including Alzheimer's disease and other amyloid-associated diseases.