Translate this page to:
In JoVE (1)
Other Publications (10)
- Science (New York, N.Y.)
- Journal of Lipid Research
- The Journal of Biological Chemistry
- Proceedings of the National Academy of Sciences of the United States of America
- Molecular Biology of the Cell
- Proceedings of the National Academy of Sciences of the United States of America
- Science's STKE : Signal Transduction Knowledge Environment
- Methods in Molecular Biology (Clifton, N.J.)
- PloS One
- Annual Review of Cell and Developmental Biology
Articles by Axel Nohturfft in JoVE
Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay
Shaochong Zhang1, Axel Nohturfft1,2
1Department of Molecular and Cell Biology, Harvard, 2Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St. George's University of London
Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.
Other articles by Axel Nohturfft on PubMed
Journal of Lipid Research. Nov, 2004 | Pubmed ID: 15314094
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. Absorption of tritium beta particles by the scintillant produces light signals that can be measured in standard scintillation counters. Because of the short range of tritium beta particles and for geometric reasons, scintillant microspheres detect only that fraction of tritiated cholesterol localized inside phagolysosomes or within a distance of approximately 600 nm. By incubating cultures in a temperature-controlled microplate reader, the kinetics of phagocytosis and cholesterol transport could be analyzed in near-real time. Scintillation signals were significantly increased in response to inhibitors of lysosomal cholesterol export. This method should prove a useful new tool for the study of endosomal trafficking of lipids and other molecules.
The Journal of Biological Chemistry. Mar, 2005 | Pubmed ID: 15657032
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. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.
Transcriptional Regulation of Phagocytosis-induced Membrane Biogenesis by Sterol Regulatory Element Binding Proteins
Proceedings of the National Academy of Sciences of the United States of America. Sep, 2005 | Pubmed ID: 16141315
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. Lipid synthesis was accompanied by increased transcription of several lipogenic proteins, including the low-density lipoprotein receptor, enzymes required for cholesterol synthesis (3-hydroxy-3-methylglutaryl CoA synthase, 3-hydroxy-3-methylglutaryl CoA reductase), and fatty acid synthase. Phagocytosis triggered the proteolytic activation of two lipogenic transcription factors, sterol regulatory element binding protein-1a (SREBP-1a) and SREBP-2. Proteolysis of SREBPs coincided with the appearance of their transcriptionally active N termini in the nucleus and 3-fold activation of an SREBP-specific reporter gene. In previous studies with cultured cells, proteolytic activation of SREBP-1a and SREBP-2 has been observed in response to selective starvation of cells for cholesterol and unsaturated fatty acids. However, under the current conditions, SREBP-1a and SREBP-2 are induced without lipid deprivation. SREBP activation is inhibited by high levels of the SREBP-interacting proteins Insig1 or the cytosolic domain of SREBP cleavage-activating protein. Upon overexpression of these proteins, phagocytosis-induced transcription and lipid synthesis were blocked. These results identify SREBPs as essential regulators of membrane biogenesis and provide a useful system for further studies on membrane homeostasis.
Differential Requirements for Actin Polymerization, Calmodulin, and Ca2+ Define Distinct Stages of Lysosome/phagosome Targeting
Molecular Biology of the Cell. Apr, 2006 | Pubmed ID: 16452628
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. The nascent complex was sensitive to disruption by alkaline carbonate, indicating that the organelles had "docked" but not fused. Through inhibitor studies and fluorescence microscopy we show that docking is preceded by a tethering step that requires actin polymerization and calmodulin. In the docked state ongoing actin polymerization and calmodulin are no longer necessary. The tethering/docking activity was purified to near homogeneity from rat liver cytosol. Major proteins in the active fractions included actin, calmodulin and IQGAP2. IQGAPs are known to bind calmodulin and cross-link F-actin, suggesting a key coordinating role during lysosome/phagosome attachment. The current results support the conclusion that lysosome/phagosome interactions proceed through distinct stages and provide a useful new approach for further experimental dissection.
Proceedings of the National Academy of Sciences of the United States of America. Nov, 2006 | Pubmed ID: 17110435
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. Upon shifting the temperature to 37 degrees C, phagosomes containing IgG beads matured significantly faster into phagolysosomes as judged by colocalization with lysosomal markers. The IgG effect was independent of other particle-associated antigens or serum factors. Lysosome/phagosome attachment was also quantified biochemically with a cytosol-dependent scintillation proximity assay. Interactions were enhanced significantly in reactions containing cytosol from mouse macrophages that had been exposed to IgG-coated beads, indicating that IgG signaling modulates the cytosolic-targeting machinery. Similar results were obtained with cytosol from primary human monocytes, human U-937 histiocytic lymphoma cells and from Chinese hamster ovary (CHO) cells transfected with a human IgG (Fcgamma) receptor. IgG-induced activation is shown to affect the actin-dependent tethering/docking stage of the targeting process and to proceed through a pathway involving protein kinase C. These results provide a rare example of an extracellular signal controlling membrane targeting on the level of tethering and docking. We propose that this pathway contributes to the role of antibodies in the protection against microbial infections.
Science's STKE : Signal Transduction Knowledge Environment. Jul, 2007 | Pubmed ID: 17595222
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. The approach involves the use of isolated phagosomes containing scintillant latex beads and lysosomes labeled with a tritiated marker. Scintillation results only when lysosomes and phagosomes come into immediate contact and requires supplementation of reactions with adenosine triphosphate and cytosol; addition of cytosol from IgG-conditioned cells enhances this signal. The method is useful for investigating the biochemistry and regulation of the early tethering and docking steps of lysosome and phagosome interactions.
Methods in Molecular Biology (Clifton, N.J.). 2008 | Pubmed ID: 18369943
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. Because of the short range of tritium beta-particles, geometric factors, and the confinement of lipids to membranes, scintillation can only be elicited by tracer molecules in membranes immediately vicinal to the scintillant. When the plasma membrane.is labeled with [(3)H]cholesterol, a signal is produced on bead-cell contact and engulfment and then reaches steady state within 45 min. When lysosomes are labeled with nonhydrolyzable [(3)H]cholesterol oleyl ether, scintillation requires intracellular lysosome/phagosome attachment or fusion, and steady state is attained only after several hours. The live-cell scintillation proximity approach is useful for examining the effects of pharmacological and genetic manipulations on particle uptake and on lysosome/phagosome targeting.
PloS One. 2009 | Pubmed ID: 19381295
Lipid metabolism in mammals is orchestrated by a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control the expression of genes required for the uptake and synthesis of cholesterol, fatty acids, and triglycerides. SREBPs are thus essential for insulin-induced lipogenesis and for cellular membrane homeostasis and biogenesis. Although multiple players have been identified that control the expression and activation of SREBPs, gaps remain in our understanding of how SREBPs are coordinated with other physiological pathways.
Annual Review of Cell and Developmental Biology. 2009 | Pubmed ID: 19575637
Bilayer synthesis during membrane biogenesis involves the concerted assembly of multiple lipid species, requiring coordination of the level of lipid synthesis, uptake, turnover, and subcellular distribution. In this review, we discuss some of the salient conclusions regarding the coordination of lipid synthesis that have emerged from work in mammalian and yeast cells. The principal instruments of global control are a small number of transcription factors that target a wide range of genes encoding enzymes that operate in a given metabolic pathway. Critical in mammalian cells are sterol regulatory element binding proteins (SREBPs) that stimulate expression of genes for the uptake and synthesis of cholesterol and fatty acids. From work with Saccharomyces cerevisiae, much has been learned about glycerophospholipid and ergosterol regulation through Ino2p/Ino4p and Upc2p transcription factors, respectively. Lipid supply is fine-tuned through a multitude of negative feedback circuits initiated by both end products and intermediates of lipid synthesis pathways. Moreover, there is evidence that the diversity of membrane lipids is maintained through cross-regulatory effects, whereby classes of lipids activate the activity of enzymes operating in another metabolic branch.