Reconstruction of destruction – in vitro reconstitution methods in autophagy research

Abstract

Autophagy is one of the most elaborative membrane remodeling systems in eukaryotic cells. Its major function is to recycle cytoplasmic material by delivering it to lysosomes for degradation. To achieve this, a membrane cisterna is formed that gradually captures cargo such as organelles or protein aggregates. The diversity of cargo requires autophagy to be highly versatile to adapt the shape of the phagophore to its substrate. Upon closure of the phagophore, a doublemembrane-surrounded autophagosome is formed that eventually fuses with lysosomes. In response to environmental cues such as cytotoxicity or starvation, bulk cytoplasm can be captured and delivered to lysosomes. Autophagy thus supports cellular survival under adverse conditions. During the past decades, groundbreaking genetic and cell biological studies have identified the core machinery involved in the process. In this Review, we are focusing on in vitro reconstitution approaches to decipher the details and spatiotemporal control of autophagy, and how such studies contributed to our current understanding of the pathways in yeast and mammals. We highlight studies that revealed the function of the autophagy machinery at a molecular level with respect to its capacity to remodel membranes.

Active Particle Based Selective Transport and Release of Cell Organelles and Mechanical Probing of a Single Nucleus

“…[ 51 ] Moreover, the trapping and transport of organelles on the particle provide a new ex vivo platform for organelle manipulation and analysis, such as study of the elastic properties of a single nucleus; fusion, fission, and dysfunction of mitochondrion; [ 52 ] and autophagosome–lysosome fusion. [ 53 ] As a conclusion, this study constitutes a potentially important experimental tool for single‐cell analysis and manipulation.…”

A Yeast Chronological Lifespan Assay to Assess Activity of Proteasome Stimulators

“…DMC did not appear to impact clearance of α‐synuclein. Although some autophagic processes can be recapitulated in cell‐free systems, [31] the complete autophagic process may not be functioning sufficiently to degrade α‐synuclein. We also investigated the levels of G6PDH enzyme as a control protein and found that G6PDH was not appreciably affected by UA treatment (Supporting Information Figure S10).…”

Cross‐talk between autophagy and sporulation in Saccharomyces cerevisiae

“… The protein machinery involved in autophagy.It consists mainly of ATG proteins. The details about the molecular players involved in autophagy are discussed elsewhere (Chinchwadkar et al, 2017; Moparthi & Wollert, 2018). Briefly, during the autophagy process in yeast, the Atg1 complex (Atg1‐Atg13‐Atg17‐Atg31‐Atg29) forms the initiation complex and leads to the pre‐autophagosomal structure (PAS) formation near the vacuole, where the autophagosomes emerge (Figure 1a) (Kamada et al, 2010).…”