bims-nocaut 2023-01-01 papers

Issue of 2023‒01‒01
two papers selected by
Quentin Frenger
University of Strasbourg

Sci Rep. 2022 Dec 27. 12(1): 22452

FLIP-based autophagy-detecting technique reveals closed autophagic compartments.

Hajime Tajima Sakurai, Satoko Arakawa, Saori Noguchi, Shigeomi Shimizu.

Autophagy results in the degradation of cytosolic components via two major membrane deformations. First, the isolation membrane sequesters components from the cytosol and forms autophagosomes, by which open structures become closed compartments. Second, the outer membrane of the autophagosomes fuses with lysosomes to degrade the inner membrane and its contents. The efficiency of the latter degradation process, namely autophagic flux, can be easily evaluated using lysosomal inhibitors, whereas the dynamics of the former process is difficult to analyze because of the challenges in identifying closed compartments of autophagy (autophagosomes and autolysosomes). To resolve this problem, we here developed a method to detect closed autophagic compartments by applying the FLIP technique, and named it FLIP-based Autophagy Detection (FLAD). This technique visualizes closed autophagic compartments and enables differentiation of open autophagic structures and closed autophagic compartments in live cells. In addition, FLAD analysis detects not only starvation-induced canonical autophagy but also genotoxic stress-induced alternative autophagy. By the combinational use of FLAD and LC3, we were able to distinguish the structures of canonical autophagy from those of alternative autophagy in a single cell.

DOI: https://doi.org/10.1038/s41598-022-26430-5

Nat Commun. 2022 12 24. 13(1): 7929

A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis.

Jianting Shi, Xun Wu, Ziyi Wang, Fang Li, Yujiao Meng, Rebecca M Moore, Jian Cui, Chenyi Xue, Katherine R Croce, Arif Yurdagul, John G Doench, Wei Li, Konstantinos S Zarbalis, Ira Tabas, Ai Yamamoto, Hanrui Zhang.

Phagocytic clearance of dying cells, termed efferocytosis, is essential for maintaining tissue homeostasis, yet our understanding of efferocytosis regulation remains incomplete. Here we perform a FACS-based, genome-wide CRISPR knockout screen in primary mouse macrophages to search for novel regulators of efferocytosis. The results show that Wdfy3 knockout in macrophages specifically impairs uptake, but not binding, of apoptotic cells due to defective actin disassembly. Additionally, WDFY3 interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Mechanistically, while the C-terminus of WDFY3 is sufficient to rescue the impaired degradation induced by Wdfy3 knockout, full-length WDFY3 is required to reconstitute the uptake of apoptotic cells. Finally, WDFY3 is also required for efficient efferocytosis in vivo in mice and in vitro in primary human macrophages. This work thus expands our knowledge of the mechanisms of macrophage efferocytosis, as well as supports genome-wide CRISPR screen as a platform for interrogating complex functional phenotypes in primary macrophages.

DOI: https://doi.org/10.1038/s41467-022-35604-8