Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.

This chapter describes two imaging-based approaches for examining the localization of bridge-like lipid transfer proteins at membrane contact sites during native biological processes. These approaches use multi-color fluorescence imaging, enabling high spatial and temporal resolution and overcoming the limitations of biochemical methods. The first approach involves immunofluorescence in fixed cells, while the second utilizes time-lapse imaging in live cells. These methods are showcased through the example of ATG2, an essential autophagy-related protein, and demonstrate the ability to overcome technical difficulties such as large protein size, lack of high-quality antibodies, and imaging highly dynamic subcellular structures. These described methods provide a powerful tool for understanding protein function and biological processes and can be widely applied to various research questions in cell biology.