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| Regulation of Phagosome Maturation by Signals from Toll-Like Receptors |
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By J. Magarian Blander and Ruslan Medzhitov in Science Magazine 304:1014-1018 (2004).
The authors studied the mechanism of phagocytosis in higher metazoans. They found, that for the phagocytosis of bacterial but not for apoptotic cells a Toll-like receptor signaling pathway is activated.
For their work the authors used a Carl Zeiss Axioplan 2 imaging e microscope and an AxioCam camera. Images were taken and processed using AxioVision with the modules Multichannel Fluorescence, Z-Stack, 3D-Deconvolution and Extended Focus.
Abstract and Figure reprinted with permission from Science 304:1014-1018 (2004). Copyright 2004 AAAS. Authors address: Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. Figure S7 and technical information presented with the authors kind permission.
Abstract:
"In higher metazoans, phagocytosis is essential in host defense against microbial pathogens and in clearance of apoptotic cells. Both microbial and apoptotic cells are delivered on a common route from phagosomes to lysosomes for degradation. Here, we found that activation of the Toll-like receptor (TLR) signaling pathway by bacteria, but not apoptotic cells, regulated phagocytosis at multiple steps including internalization and phagosome maturation. Phagocytosis of bacteria was impaired in the absence of TLR signaling. Two modes of phagosome maturation were observed, constitutive and inducible; their differential engagement depended on the ability of the cargo to trigger TLR signaling."
Figure 4B shows the effect of two inhibitors (lower two panels) on the ability of macrophages (nuclei stained blue with DAPI) to target phagocytosed GFP labeled E.coli bacteria (green) to lysosomes (stained red) for degradation. In the positive control (upper panel) the bacteria (labelled green) are internalized into the cytoplasm of the macrophages and colocalize with lysosomes resulting in a yellow color in the overlay. An inactive compound does not influence this colocalization (second panel from top). Two inhibitory compounds allow phagocytosis of bacteria but prevent loading of lysosomes which results in a lack of yellow colocalization signal.
Technical Details:
Cells were fixed, stained and observed on an Axioplan 2 imaging microscope with a 63x/1.4 Oil Plan Apochromate objective. 4-channel Z-Stacks were acquired (DAPI, GFP, LysoTracker-Red, DIC) using the Carl Zeiss AxioCam and AxioVision. For presentation Z-Stacks were collapsed to produce 2D images using the module Extended Focus.
Colocalization of LysoTracker and LAMP-2 in macrophages:
This figure shows that LysoTracker preferentially labels lysosomes as the main acidic compartments in macrophages. Lysosomal membranes were counterstained with antibodies specific for LAMP-2, a lysosomal membrane glycoprotein, followed by AlexaFluor 488 secondary anti-rat Antibody.
Note that all LysoTracker positive vesicles are LAMP-2 positive. Whereas LAMP-2 labels the membrane of lysosomes, LysoTracker labels their lumen (see enlarged insets).
Technical Details:
Cells were fixed, stained and observed on an Axioplan 2 imaging e microscope with a 63x/1.4 Oil Plan Apochromate objective. 3-channel Z-Stacks were acquired (Alexa 488, LysoTracker-Red, DIC) using the Carl Zeiss AxioCam and AxioVision. Fluorescent channels were deconvolved using the AxioVision module 3D-Deconvolution. For presentation Z-Stacks were combined to produce 2D images using the module Extended Focus. | |
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