Endosome recycling assay

To investigate vesicular trafficking in a cell-free transport assay (e.g., from endosomal membranes to recycling endosomes) four prerequisites are essential first, a physiologically relevant marker by which the vesicular transport step can be followed and quantified second, defined donor membranes third, defined acceptor membranes and fourth, appropriate transport conditions. 

Fig. 1. Principle of the in vitro fusion assay. After labeling the endocytic pathway with acridinium-transferrin (Ac-Tfn) labeled endosomes (donor) are enriched by density centrifugation. Recycling endosomes (acceptor) are purilied from unlabeled cells first by density centrifugation followed by immunoisolation using anti-Rabll coated magnetic beads. Transport of Ac-Tfn from the donor endosomes to acceptor recycling endosomes is performed at 3T and in the presence of cytosol and ATP. After washing the beads-bound membranes, transferred Ac-Tfn is measured by detecting acridinium cleavage in a luminometer.

The combined methods of preparing an endosome-enriched fraction labeled with acridinium-transferrin and specific immunoadsorption of unlabeled Rabll-positive recycling endosomes provide the basis of our in vitro fusion assay. Using this assay, transport of the physiological marker transferrin from labeled donor endosomes to immunoisolated acceptor recycling endosomes can be followed by measuring arrival of acridinium-transferrin to beads-bound endosomes as detected by lashlight-luminescence in a luminometer. 

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