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

Stockinger, Walter; Castoreno, Adam; Wang, Yan; Pagnon, Joanne; Nohturfft, Axel

doi:10.1194/jlr.d400011-jlr200

Cited by 12 publications

(12 citation statements)

References 32 publications

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“…To test whether expression of Insig and C-terminal SCAP were interfering with particle uptake, the kinetics of phagocytosis were analyzed with a live-cell scintillation proximity assay. In this assay, photons are emitted as scintillant microspheres are enveloped with [ 3 H]cholesterolloaded plasma membrane (28). No significant differences were seen between samples expressing the three viruses (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Castoreno

Wang

Stockinger

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate Ϸ100 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.

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Section: Resultsmentioning

confidence: 99%

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

Castoreno

Wang

Stockinger

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The key components of this assay are phagosomes containing scintillant beads and lysosomes labeled with tritiated cholesteryl ether. Because of the short range of tritiumderived ␤ particles and as a consequence of geometric constraints, scintillation requires immediate contact between lysosomes and phagosomes (15)(16)(17). Using this assay, we previously showed that lysosomes and phagosomes will attach when reactions are supplemented with ATP and rat liver cytosol (15).…”

Section: Resultsmentioning

confidence: 99%

“…ARPE-19, CHO-K1, J774, and RAW 264.7 cells were grown in medium C. Human embryonic kidney 293 cells were grown in DMEM supplemented with antibiotics and 10% FBS. Primary mouse peritoneal macrophages were obtained from BALB/c mice as described (16). Human monocytes were obtained from the blood (20 ml) of a healthy volunteer by using Ficoll-Paque as described in ref.…”

Section: Methodsmentioning

confidence: 99%

Immunoglobulin G signaling activates lysosome/phagosome docking

Trivedi

Zhang

Castoreno

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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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°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°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 (Fc␥) 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. membrane fusion ͉ protein kinase C ͉ scintillation proximity assay ͉ Fc receptor ͉ macrophages

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“…HRP-tagging of ER-or Golgi-resident proteins would allow researchers to determine cholesterol trafficking along the secretory pathway in the same manner [31]. While this method is still subject to some concerns regarding cholesterol redistribution during membrane isolation and centrifugation steps, another new method based on a scintillation proximity assay allows for determining cholesterol transport with a time resolution of a few minutes [56]. Nohturfft and colleagues used phagocytosis of scintillation beads in 3 Hcholesterol labelled murine macrophages to measure absorption of tritium radiation in immediate proximity of counting beads (i.e., radiance within a radius of about 600 nm) in order to track cholesterol transport out of the phagolysosome [56].…”

Section: Biochemical Assays For Characteriza-tion Of Inter-compartmenmentioning

confidence: 98%

Following Intracellular Cholesterol Transport by Linear and Non-Linear Optical Microscopy of Intrinsically Fluorescent Sterols

Wüstner

2012

CPB

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Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow the cellular movement of this essential lipid molecule. In this article, a survey of the various methods being used for analysis of sterol trafficking is given. Various classical biochemical methods are presented and their suitability for analysis of sterol trafficking is assessed. Special emphasis is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented.

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

Cited by 12 publications

References 32 publications

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

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

Immunoglobulin G signaling activates lysosome/phagosome docking

Following Intracellular Cholesterol Transport by Linear and Non-Linear Optical Microscopy of Intrinsically Fluorescent Sterols

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