Microscopic versus Biochemical Assays

Two alternatives to observe organelle fusion in a cell-free system are microscopical or biochemical detection.

For microscopic assays each of the fusion partners is tagged with a microscopically detectable label and organelle fusion presents itself as colocalization of both labels. All published microscopic in vitro phagosome-endosome fusion assays are based on electron microscopy. Endocytic compartments are labeled with electron-dense gold nanoparticles via endocytic uptake, while phagosomes can be readily discriminated in transmission electron microscopy without a specific label because of the size and unique structure of the enclosed particle. Fusion of the organelles results in a membrane-surrounded structure containing both the phagocytosed particle and endosomal gold (Figure 6.1d). To determine fusion efficiency, a sufficiently high number of phagosomes is analyzed and frequency of colocalization of phagocytosed particles and endosomal gold is quantified [9, 11, 12]. Biochemical assays for organelle fusion, on the other hand, are based on the formation of a specific fusion product that can be quantified enzymatically or fluorometrically. Some examples are listed in detail below.

Both microscopic and biochemical assays have certain advantages over the other. Microscopic assays are normally more sensitive than biochemical ones. Theroretically, a content of few hundred phagosomes in one microscopical sample is sufficient to calculate fusion efficiency, a number usually too low to overcome the detection limit in a biochemical assay containing many thousands of phagosomes. On the other hand, enzymatic or fluorometric quantification is typically much less laborious than the preparation and examination ofelectron microscopical samples and so allows a higher throughput. Furthermore, different aspects of membrane fusion are quantified in the two types of assay. While microscopic assays indicate how many phagosomes in a sample have turned into fusion products (e.g., phagolysosomes), biochemical assays integrate over the whole phagosome population and can state how much material has been transferred from endocytic to phagocytic organelles.

Our lab has recently established a fluorescence microscopy-based in vitro assay for fusion of lysosomes with bacteria-containing phagosomes (Becken and Haas, unpublished, similar to an assay described by Brandhorst et al. [13] for homotypic early endosome fusion. In our assay, bacteria are covalently surface-labeled with a green fluorescent dye before phagocytosis and lysosomes are preloaded with a red fluorophor. After vesicle fusion both dyes are present in one compartment. This can be visualized as a spatial overlap of both signals using conventional fluorescence microscopy (Figure 6.1e). Using this method there is little danger of false-positive signals caused by fluorescent dye leaking from ruptured lysosomes. If that did happen, it would be strongly diluted in the reaction buffer and would not be able to

Rhodamine Assay Facs
Figure 6.1 Assays reconstituting fusion or attachment of phagosomes and endocytic compartments in vitro. HRP, horseradish peroxidase; N-Rh-PE, lissamine rhodamine phosphatidylethanolamine; NBD-PE,

benzoxadiazole phosphatidylethanolamine; BSA, bovine serum albumin; GFP, green fluorescent protein; PE, phycoerythrin; FACS, fluorescence-activated cell sorting. See text for details.

label further compartments. This means there is no need for special precautions to avoid a fusion-independent positive readout.

This assay system can be further validated by choosing suitable fluorophores with an overlap between the emission spectrum of the one and the excitation spectrum of the second fluorophore. Excitation of the first dye thus leads to the fluorescence emission of the second one in fused organelles (fluorescence resonance energy transfer, FRET). This interaction requires very close contact between the fluorophores (10 nm or less [14]), which can only be established by organelle fusion but not by an attachment of vesicles. Therefore, using FRET to quantify phagosome-lysosome fusion is an appropriate and even more stringent tool than colocalization studies.

Software tools to automate the taking and analyzing of fluorescence microscopic pictures have already been used in cell-free endosome-endosome fusion systems [13]. Use of such systems will hopefully also allow a time-efficient interpretation of microscopic data deriving from the described in vitro phagosome-lysosome fusion assay and so help to combine the high sensitivity of microscopy with the high capacity of biochemical approaches in one assay.

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