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Furthermore, selective autophagy can be clarified by labelling corresponding substrates and autophagosomes or lysosomes simultaneously. With the help of two-photon microscopy, the process of autophagy in live animals has been uncovered. Here, we summarize the methods for observing autophagy by optical microscopy and the selection of fluorescent markers.Autophagy, a highly conserved metabolic process in eukaryotes, is a widespread degradation/recycling system. However, there are significant differences (as well as similarities) between autophagy in animals, plants, and microorganisms such as yeast. While the overall process of autophagy is similar between different organisms, the molecular mechanisms and the pathways regulating autophagy are different, which is manifested in the diversity and specificity of the genes involved. In general, the autophagy system is much more complicated in mammals than in yeast. In addition, there are some differences in the types of autophagy present in animals, plants, and microorganisms. For example, there is a unique type of selective autophagy called the cytoplasm-to-vacuole targeting (Cvt) pathway in yeast, and a special kind of autophagy, chloroplast autophagy, exists in plants. In conclusion, although autophagy is highly conserved in eukaryotes, there are still many differences between autophagy of animals, plants, and microorganisms.Autophagy is a lysosome-dependent degradation process. During autophagy, cytoplasmic components are sequestered and catabolized to supply nutrition and energy under starvation conditions. Recent work has demonstrated that many cargos can be specifically recognized and then eliminated via the core mechanism of autophagy which is termed as selective autophagy. The cargo recognition program provides the basis for the specific degradation of selective autophagy; thus, the exploration of the interaction between the cargo and the receptor is the key for revealing the underlying mechanism. Also, receptor protein complexes are required in various selective autophagy subtypes which process and guide the cargo to the core mechanism. Ubiquitination and phosphorylation are the main methods to modulate the affinity of the receptor toward cargo. Although many key processes of selective autophagy subtypes have been discovered and intensively studied, the precise ways in which the mechanisms of cargo recognition function remain mostly elusive. A fuller mechanistic understanding of selective autophagy will be important for efforts to promote disease treatment and drug development.Autophagy is a major intracellular degradation/recycling system that ubiquitously exists in eukaryotic cells. Autophagy contributes to the turnover of cellular components through engulfing portions of the cytoplasm or organelles and delivering them to the lysosomes/vacuole to be degraded. The trafficking of autophagosomes and their fusion with lysosomes are important steps that complete their maturation and degradation. In cells such as neuron, autophagosomes traffic long distances along the axon, while in other specialized cells such as cardiomyocytes, it is unclear how and even whether autophagosomes are transported. Therefore, it is important to learn more about the processes and mechanisms of autophagosome trafficking to lysosomes/vacuole during autophagy. The mechanisms of autophagosome trafficking are similar to those of other organelles trafficking within cells. The machinery mainly includes cytoskeletal systems such as actin and microtubules, motor proteins such as myosins and the dynein-dynactin complex, and other proteins like LC3 on the membrane of autophagosomes. Factors regulating autophagosome trafficking have not been widely studied. To date the main reagents identified for disrupting autophagosome trafficking include 1. Microtubule polymerization reagents, which disrupt microtubules by interfering with microtubule dynamics, thus directly influence microtubule-dependent autophagosome trafficking 2. F-actin-depolymerizing drugs, which inhibit autophagosome formation, and also subsequently inhibit autophagosome trafficking 3. Motor protein regulators, which directly affect autophagosome trafficking.The autophagosome delivers engulfed substrates to the lysosome for degradation via membrane fusion between the autophagosome and the lysosome. The process of membrane fusion is highly conserved in evolution. It is widely accepted that membrane fusion in general is driven by the zippering of the SNARE complex to form a four-helix bundle. Besides SNAREs, other proteins are required to complete fusion efficiently, including tethering proteins, Rab GTPases, and SM proteins (Sec1/SM family proteins). This chapter will summarize the current knowledge of the key machinery involved in autophagosome-lysosome fusion, including autophagic SNAREs, involved ATG proteins, the HOPS complex, Rab GTPase, and other relevant aspects.Phagophore closure is a critical step during macroautophagy. However, the proteins and mechanisms to regulate this step have been elusive for a long time. In 2017, Rab5 was affirmed to play a role in phagophore closure in yeast. TAK-243 molecular weight Furthermore, in mammalian cells, ESCRT III was reported to have roles in phagophore closure and mitophagosome closure in vivo in 2018 and 2019, respectively. The role of ESCRT in phagophore closure was confirmed in yeast, both in vivo and in vitro, in 2019. Most importantly, the latter paper found that Atg17 recruited the ESCRT III subunit Snf7 to the phagophore to close it under the control of Rab5. To determine the closure characteristics of autophagosome-like membrane structures in ESCRT mutants, a traditional protease protection assay with immunoblotting was used, accompanied by new techniques that were developed, including immunofluorescence assays, autophagosome completion assays, and the optogenetic closure assay. This study delivered our current understanding of phagophore closure and provided more reference methods to detect membrane closure.Autophagosome formation is a regulated membrane remodeling process, which involves the generation of autophagosomal membrane precursors (vesicles), the assembly of the autophagosomal membrane precursors to form the phagophore, and phagophore elongation to complete the autophagosome. The sources of the autophagosomal membrane precursors are endomembrane compartments, such as the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), ER-exit sites (ERES), and endosomes. In response to stress, these structures are remodeled, to generate the early autophagosomal membrane precursors. The phagophore assembly site (PAS), which mainly localizes on the ER, harbors the site for autophagosomal membrane assembly, elongation, and completion. ATG proteins, membrane remodeling factors, and autophagic membranes follow a precise choreography to complete the overall process. In this chapter, we briefly discuss our current knowledge on the membrane origins of the autophagosome, as well as autophagosomal precursor generation, assembly, and expansion.