Fluorescence Imaging of Autophagy-Mediated ER-to-Vacuole Trafficking in Plants

Macroautophagy (hereafter referred to as autophagy) is a conserved mechanism in eukaryotic cells that delivers unneeded cellular components for degradation in the lytic organelle. In plants, as in other eukaryotes, autophagy begins in the formation of cup-shaped double membranes that engulf cytosolic material. The double membrane closes to form autophagosomes that are then transported to the vacuole for degradation. Autophagy can function as a bulk nonselective process or as a selective process targeting specific proteins, protein aggregates, organelles, or other cellular components for degradation. The endoplasmic reticulum (ER) is linked to autophagy-related processes in multiple ways. The ER was suggested as a possible site for the nucleation of autophagosomes, and as a source for autophagosomal membranes. Furthermore, autophagy has an important role in ER homeostasis, and the ER is a target for a selective type of autophagy, ER-phagy, in response to ER stress. However, the detailed molecular mechanisms, especially in plants, are only now starting to be revealed.In this chapter, we describe the use of confocal imaging to follow the delivery of fluorescently tagged ER-associated proteins to the vacuole. We also describe the utilization of fluorescent protein fusions to look at the co-localization of a protein of interest with the autophagosome marker protein ATG8, a core autophagy machinery protein that is essential for selective autophagy processes.