Table 3.

The summaries of biochemical technique/assays used to study properties of membrane in autophagy

Membrane Properties to study	Technique/assay to use		Principle and observable(s)	References
Lipid composition	Lipidomics of purified subcellular fraction by MS (Mass spectrometry)		MS of lipids from purified organelles	(Takamori et al., 2006; de la Ballina et al., 2020)
	IF (immunofluorescence) of specific PI probes		IF of specific PI targeting proteins	(Di Paolo and De Camilli, 2006)
Membrane tethering	DLS		Vesicle size change upon tethering or clustering	(Diao et al., 2015; Liu et al., 2016)
	EM		Morphology change of vesicles upon tethering or clustering.	(Diao et al., 2015)
	TIRF-based Single vesicle tethering assay		Overlap of fluorescence labeled vesicles	(Diao et al., 2015)
Lipid transfer	FRET based lipid transfer assay		FRET signal change upon lipid transfer	(Maeda et al., 2019)
Membrane fusion	Ensemble average assay (Bulk assay)	Lipid mixing assay: NBD/Rhod or DiI/DiD	FRET signal changes of FRET pairs upon lipid mixing	(Weber et al., 1998)
		Content mixing assay: SrB	Recover of self-quench high concentration fluorescence signal upon fusion	(Ma et al., 2013; Diao et al., 2015)
		Simultaneous lipid mixing and content mixing NBD/Marine blue and PhycoE and Cy5	FRET signal changes of two FRET pairs. One pair on the membrane, the other pair inside the vesicles.	(Zucchi and Zick, 2011; Liu et al., 2016; Liu et al., 2017)
	TIRF based Single vesicle fusion assay	Lipid mixing DiI/DiD	TIRF fluorescence signal changes	(Kyoung et al., 2011; Diao et al., 2012; Kyoung et al., 2013)
		Content mixing SrB or Cy5/Cy3
Membrane scission	High resolution TEM		Visualize the closure under EM	(Zhen et al., 2019)
	Optogenetic closure assay Photo active release assay		Photo active to dissociation and association LOVTRAP tag reversibly to monitor the closure state of mitophagosome	(Zhen et al., 2019)
	HaloTag based IF assay		Utilizing membrane permeable and impermeable Halo tag fluorescent substrate, visualize closed autophagosome and unclosed phagophore	(Takahashi et al., 2018; Takahashi et al., 2019)
	Optical tweezer-based force test		Giant unilamellar vesicles pulled by optical tweezer and measure the force changing upon ATP release	(Schoneberg et al., 2018)
	Atomic Force Microscope based visualization of in vitro ESCRT-III contain membrane system		In vitro assemble ESCRT-III on lipid bilayers, and visualize it by Atomic Force Microscope	(Chiaruttini et al., 2015)