Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.

Acyl coenzyme A:cholesterol O-acyltransferase (ACAT) is the enzyme responsible for cholesterol esterification in macrophages leading to foam cell formation. The determination of its localization is a critical step in understanding its regulation by cholesterol. Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers of the endoplasmic reticulum, but in addition, ACAT was found in an unidentified paranuclear site. In the present study, we further define the localization of paranuclear ACAT. First, we found that ACAT does not colocalize with sorting endosomes or late endosomes labeled with fluorescent alpha(2)-macroglobulin. The paranuclear ACAT is close to the endocytic recycling compartment labeled with fluorescent transferrin. We also show that the paranuclear structure containing ACAT is very close to TGN38, a membrane protein of the trans-Golgi network (TGN), but farther from Gos28, a marker of cis, medial, and trans Golgi. After treatment with nocodazole, the central localization of ACAT did not colocalize with markers of the TGN. These data indicate that a significant fraction of ACAT resides in membranes that may be a subcompartment of the endoplasmic reticulum in proximity to the TGN and the endocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma membrane, esterification of cholesterol in these membranes may play an important role in macrophage foam cell formation during atherogenesis.

Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer's amyloid beta-peptide by microglial cells.

Microglia are phagocytic cells that are the main inflammatory response cells of the central nervous system. In Alzheimer's disease brain, activated microglia are concentrated in regions of compact amyloid deposits that contain the 39-43-amino acid Abeta peptide. We examined the uptake, degradation, and release of small aggregates of fibrillar Abeta (fAbeta) or soluble Abeta (sAbeta) by microglia. We found that although some degradation of fAbeta was observed over 3 days, no further degradation was observed over the next 9 days. Instead, there was a slow release of intact Abeta. The poor degradation was not due to inhibition of lysosomal function, since the rate of alpha2-macroglobulin degradation was not affected by the presence of fAbeta in the late endosomes/lysosomes. In contrast to fAbeta, internalization of sAbeta was not saturable. After internalization, sAbeta was released rapidly from microglia, and very little was degraded. These data show that fAbeta and sAbeta interact differently with microglia but that after internalization a large fraction of both are released without degradation.

Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails.

To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1'- didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in their alkyl tail length or unsaturation; these differences are expected to affect their distribution in membrane domains of varying fluidity or curvature. All three DiIs initially enter sorting endosomes containing endocytosed transferrin. DiIC16(3), with two long 16-carbon saturated tails is then delivered to late endosomes, whereas FAST DiI, with two cis double bonds in each tail, and DiIC12(3), with saturated but shorter (12-carbon) tails, are mainly found in the endocytic recycling compartment. We also find that DiOC16(3) (3,3'- dihexadecyloxacarbocyanine perchlorate) and FAST DiO (3, 3'-dilinoleyloxacarbocyanine perchlorate) behave similarly to their DiI counterparts. Furthermore, whereas a phosphatidylcholine analogue with a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore attached at the end of a 5-carbon acyl chain is delivered efficiently to the endocytic recycling compartment, a significant fraction of another derivative with BODIPY attached to a 12-carbon acyl chain entered late endosomes. Our results thus suggest that endocytic organelles can sort membrane components efficiently based on their preference for association with domains of varying characteristics.

An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.

To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.