Protocol for 4D live fluorescence microscopy to image rapid cellular responses in yeast

Micro-autophagy is thought to occur through cargo engulfment by the lysosomal membrane based on electron microscopy of fixed cells. We present a protocol that follows micro-ER (endoplasmic reticulum)-phagy in live yeast cells that can be used for following other rapid cellular processes. We describe steps for inducing the accumulation of misfolded proteins and their selective shuttling for degradation during nutritional stress. We then detail procedures for live-cell time-lapse confocal microscopy and 3D reconstruction showing individual events of cargo engulfment and internalization into the lysosome that occur within seconds. For complete details on the use and execution of this protocol, please refer to Gyurkovska et al.¹.