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Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery.

ABSTRACT: Macroautophagy (also known as autophagy) is a major pathway for selective degradation of misfolded/aggregated proteins and damaged organelles and non-selective degradation of cytoplasmic constituents for the generation of power during nutrient deprivation. The multi-step degradation process, from sequestering cytoplasmic cargo into the double-membrane vesicle termed autophagosome to the delivery of the autophagosome to the lysosome or lytic vacuole for breakdown, is mediated by the core autophagy machinery composed of multiple Atg proteins, as well as the divergent sequence family of selective autophagy receptors. Single-particle electron microscopy (EM) is a molecular imaging approach that has become an increasingly important tool in the structural characterization of proteins and macromolecular complexes. This article summarizes the contributions single-particle EM have made in advancing our understanding of the core autophagy machinery and selective autophagy receptors. We also discuss current technical challenges and roadblocks, as well as look into the future of single-particle EM in autophagy research.

SUBMITTER: Cheung YWS

PROVIDER: S-EPMC7663694 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery.

Cheung Yiu Wing Sunny YWS Nam Sung-Eun SE Yip Calvin K CK

International journal of molecular sciences 20201028 21

Macroautophagy (also known as autophagy) is a major pathway for selective degradation of misfolded/aggregated proteins and damaged organelles and non-selective degradation of cytoplasmic constituents for the generation of power during nutrient deprivation. The multi-step degradation process, from sequestering cytoplasmic cargo into the double-membrane vesicle termed autophagosome to the delivery of the autophagosome to the lysosome or lytic vacuole for breakdown, is mediated by the core autophag ...[more]

PMID: 33126766

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Project description:The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior, and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, the AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin-dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule-dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses.IMPORTANCE Baculoviruses are used widely as environmentally benign pesticides, protein expression systems, and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrated that the alphabaculovirus AcMNPV exhibited actin- and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubules impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single-particle level, which advances our understanding of the early steps of baculovirus entry.

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Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery.

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Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery.

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