Studying phagocytosis by live-cell scintillation proximity assay.

Phagocytosis of microorganisms, senescent cells, apoptotic bodies, and effete tissue material is an important process in host defense and tissue homeostasis. A method is described to measure, in living macrophages, the kinetics of particle engulfment and lysosome/phagosome targeting. Plasma membranes or lysosomes are labeled with tritiated lipids, followed by exposure of cells to scintillant microbeads.

A cell-free scintillation proximity assay for studies on lysosome-to-phagosome targeting.

Phagocytes, such as macrophages, neutrophils, and dendritic cells, play important roles in the innate immune system through their ability to engulf, kill, and digest invading microbes. In cooperation with the humoral adaptive immune system, coating of substrates with immunoglobulin G (IgG) antibodies enhances several aspects of phagocytosis, including the recognition of substrates by cell surface IgG (Fcgamma) receptors, particle internalization, generation of microbicidal oxygen species, and targeting of lysosomes to phagosomes. We describe a cell-free scintillation proximity assay developed to study the mechanisms of lysosome targeting to phagosomes and the regulation of this process by IgG.

Immunoglobulin G signaling activates lysosome/phagosome docking.

An important role of IgG antibodies in the defense against microbial infections is to promote the ingestion and killing of microbes by phagocytes. Here, we developed in vivo and in vitro approaches to ask whether opsonization of particles with IgG enhances intracellular targeting of lysosomes to phagosomes. To eliminate the effect of IgG on the ingestion process, cells were exposed to latex beads at 15-20 degrees C, which allows engulfment of both IgG-coated and uncoated beads but prevents the fusion of lysosomes with phagosomes.

Differential requirements for actin polymerization, calmodulin, and Ca2+ define distinct stages of lysosome/phagosome targeting.

Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies, and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process, we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. Incubation of tritium-labeled lysosomes with phagosomes containing scintillant latex beads led to light emission in a reaction requiring cytosol, ATP, and low Ca(2+) concentrations.

Transcriptional regulation of phagocytosis-induced membrane biogenesis by sterol regulatory element binding proteins.

In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate approximately 100 lipids at appropriate concentrations. To study the regulation of bilayer assembly in a cell cycle-independent manner, we have exploited the fact that phagocytes replenish membranes expended during particle engulfment in a rapid phase of lipid synthesis. In response to phagocytosis of latex beads, human embryonic kidney 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles.

Modulation of endosomal cholesteryl ester metabolism by membrane cholesterol.

Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood.

Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay.

A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells. Mouse macrophages were cultured in the presence of tritiated cholesterol and scintillant microspheres. Microspheres were taken up by phagocytosis and stored in phagolysosomes.

The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor.

Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to specific membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane trafficking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein.