Lysosome fusion in cultured mammalian cells.

Abstract

In mammalian cells, lysosomes fuse with late endosomes to form endolysosomes from which lysosomes are reformed. Lysosomal fusion events were initially inferred from light and electron microscopy studies, demonstrated in cell-free content mixing assays and, more recently, shown directly with live cell microscopy. Currently, there is a focus on studying lysosome fusion in cultured cells using various forms of microscopy, especially under conditions in which the use of overexpression of dominant-negative protein constructs or the use of RNA interference to deplete individual proteins allows the investigation of the molecular machinery of fusion. Here, we review a variety of fluorescence, live cell, and electron microscopy techniques with which to study lysosome fusion in cultured mammalian cells. We address the merits and limitations of different techniques when choosing an assay system and provide a series of protocols with which to study endocytic delivery to lysosomes and fusion events between lysosomes and endosomes.