Biology subjects

Saito, C.

Publications and source records attributed to Saito, C..

3 recordsLinked to original sources

bioRxiv2022

Experimental determination and mathematical modeling of standard shapes of forming autophagosomes

The formation of autophagosomes involves dynamic morphological changes of a phagophore from a disk-shaped membrane cisterna into a cup-shaped intermediate and a spherical autophagosome. However, the physical mechanism behind these morphological changes remains elusive. Here, we determined the average shapes of phagophores by statistically investigating three-dimensional electron micrographs of more than 100 phagophores. The results showed that the cup-shaped structures adopted a characteristic morphology; they were longitudinally elongated, and the rim was catenoidal with an outwardly recurved shape. To understand these characteristic shapes, we established a theoretical model of the shape of entire phagophores. The model quantitatively reproduced the average morphology and revealed that the characteristic shape of phagophores (i.e., an elongated shape with a catenoidal rim) was primarily determined by the relative size of the open rim to the total surface area. These results suggest that autophagosomal membranes are highly flexible and that the morphological changes during autophagosome formation follow a stable path determined by elastic bending energy minimization. SummaryThe formation of autophagosomes involves dynamic morphological changes of membrane cisternae. Sakai et al. determined the average shapes of forming autophagosomes by statistically investigating three-dimensional electron micrographs and established a theoretical model that quantitatively reproduces the phagophore shapes.

cell biology↗

bioRxiv2022

Cis-membrane association of human ATG8 proteins N-terminus mediates autophagy

Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. The cis-membrane association of the N-terminus is critical to maintain membrane expansion and the size of autophagosomes in cells, consequently, mediating the efficient degradation of p62. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.

cell biology↗

bioRxiv2022

NCOA4 drives ferritin phase separation to facilitate macroferritinophagy and endosomal microferritinophagy

A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, the exact function of NCOA4 remains elusive. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).

cell biology↗