Autophagy Pathway Mapping to Elucidate the Function of Novel Autophagy Regulators Identified by High-Throughput Screening

Methods Mol Biol. 2019:1880:375-387. doi: 10.1007/978-1-4939-8873-0_25.

Abstract

Autophagy is an evolutionarily conserved degradative pathway, and the core autophagy machinery acts in a highly regulated, hierarchical manner to engulf cytoplasmic material in a double-membrane-bound organelle and deliver it to the lysosome. High-throughput screening approaches lead to the identification of novel autophagy regulators, and we describe an autophagy pathway mapping strategy to determine the stage of the autophagy pathway at which these novel candidate proteins function.

Keywords: ATG9; Autophagic flux; Autophagosome maturation; Autophagy pathway mapping; LC3; ULK complex; WIPI2; mTORC1.

MeSH terms

  • Autophagosomes / metabolism
  • Autophagy / physiology*
  • Autophagy-Related Proteins / metabolism
  • HEK293 Cells
  • High-Throughput Screening Assays / instrumentation
  • High-Throughput Screening Assays / methods*
  • Humans
  • Intracellular Membranes / metabolism
  • Lysosomes / metabolism
  • Membrane Proteins / metabolism
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Microtubule-Associated Proteins / metabolism
  • Protein Interaction Mapping / instrumentation
  • Protein Interaction Mapping / methods*
  • Signal Transduction / physiology*

Substances

  • Autophagy-Related Proteins
  • Membrane Proteins
  • Microtubule-Associated Proteins