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Abstract

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Materials

References

Immunology and Infection

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Published: August 26th, 2016

DOI:

10.3791/54470

1Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation.

We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

Phagocytosis is a major cell function that starts with the recognition and binding of material to surface receptors, which then leads to the internalization and degradation of the ingested material. While single-celled eukaryotes such as the mold Dictyostelium discoideum and amoebae use phagocytosis for feeding on bacteria, higher organisms have evolved with professional cells. Macrophages or dendritic cells are the first line of defense against pathogens in various tissues and organs, and are crucial to activate the adaptive immune system through antigen presentation and cytokine production 1-4. Under certain circumstances phagocytosis can be perf....

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Note: The plasmid used Lifeact-mCherry is a kind gift of Dr. Guillaume Montagnac, Institut Curie, Paris, generated after 17.

1. Cells and Transfection

Note: RAW264.7 macrophages are grown to sub confluency in complete medium (RPMI (Roswell Park Memorial Institute) 1640 medium, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 2 mM L-Glutamine and 10% FCS (Fetal Calf Serum)) in a 100 mm plate. They are transfected with plasmids encoding fluorescently tagged proteins by electropor.......

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The experimental system described in this manuscript is schematically represented in Figure 1. Transfected RAW264.7 macrophages expressing the proteins of interest fused to a fluorescent tag are placed into contact with IgG-opsonized sheep red blood cells (SRBCs) that were non-covalently fixed on the coverslip. The macrophages can detach the SRBC from the coverslip to engulf it. The TIRF microscope used allows concomitant acquisition of signals from the TIRF area correspo.......

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The experimental protocol described here proposes an unprecedented method to follow in real time and in living cells, with high-resolution, the formation of a phagosome and in particular its closure. Several technical aspects have to be discussed. Firstly, the assay is very sensitive to temperature. It is very important to check that the heating chamber is at 37 °C and that all media, devices or cells are kept within the chamber to avoid temperature changes that could impair the efficiency of phagocytosis. We notice.......

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We thank Dr. Alexandre Benmerah (Institut Imagine Necker, Paris, France) for initial discussions on the experimental approach and Dr. Jamil Jubrail for reading the manuscript. Nadège Kambou and Susanna Borreill are acknowledged for performing experiments with the method in our laboratory. This work was supported by grants from CNRS (ATIP Program), Ville de Paris and Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche Médicale (FRM INE20041102865, FRM DEQ20130326518 including a doctoral fellowship for FMA) to FN, and Agence Nationale de Recherche sur le SIDA et les hépatites virales" (ANRS) including a doctoral fellowship for....

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Name Company Catalog Number Comments
Anti-sheep red blood cells IgG MP Biomedicals 55806
Bovine Serum Albumin heat shock fraction, pH 7, ≥98%  Sigma A7906
Cell lifter Corning 3008
Cuvettes 4mm Cell project EP104
DPBS, no calcium, no magnesium Thermo Fischer Scientific 14190-094 Room temperature
Electrobuffer kit Cell project EB110
100mm TC-Treated Cell Culture Dish Corning 353003
Gene X-cell pulser Biorad 165-2661
Gentamicin solution Sigma G1397
Glass Bottom Dishes 35 mm uncoated 1.5 MatTek corporation P35G-1.5-14-C Case
iMIC  TILL Photonics  Oil-immersion objective (N 100x, NA1.49.),  heating chamber with CO2, a camera single photon detection EMCCD ( Electron Multiplying Charge Coupled Device) and a 1.5X lens
Poly-L-Lysine Solution 0.1% Sigma P8920-100ml Dilution at 0.01% in water 
RPMI 1640 medium GLUTAMAX  Supplement Life technologies 61870-010
RPMI 1640 medium, no phenol red (10x500 ml) Life technologies 11835-105 Warm in 37°C water bath before use
Sheep red blood cells (SRBCs) Eurobio DSGMTN00-0Q Conserved in Alsever buffer at 4°C before use

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