Biology subjects

Saito, C.

Publications and source records attributed to Saito, C..

5 recordsLinked to original sources

bioRxiv2023

The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate lysosomal stress responses

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinsons disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

molecular biology↗

bioRxiv2023

Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of β-propeller associated neurodegeneration

{beta}-propellers that bind polyphosphoinositides (PROPPINs) are an autophagy-related protein family conserved throughout eukaryotes. The PROPPIN family includes Atg18, Atg21, and Hsv2 in yeast and WD-repeat protein interacting with phosphoinositides (WIPI)1-4 in mammals. Mutations in the WIPI genes are associated with human neuronal diseases, including {beta}-propeller associated neurodegeneration (BPAN) caused by mutations in WDR45 (encoding WIPI4). In contrast to yeast PROPPINs, the functions of mammalian WIPI1-WIPI4 have not been systematically investigated. Although the involvement of WIPI2 in autophagy has been clearly shown, the functions of WIPI1, WIPI3, and WIPI4 in autophagy remain poorly understood. In this study, we comprehensively analyzed the roles of WIPI proteins by using WIPI-knockout (single, double, and quadruple knockout) HEK293T cells and recently developed HaloTag-based reporters, which enable us to monitor autophagic flux sensitively and quantitatively. We found that WIPI2 was nearly essential for autophagy and partially redundant with WIPI1. Autophagic flux was unaffected or only slightly reduced by single deletion of WIPI3 (encoded by WDR45B) or WIPI4 but was profoundly reduced by double deletion of WIPI3 and WIPI4. Furthermore, we revealed variable effects of BPAN-related missense mutations on the autophagic activity of WIPI4. BPAN is characterized by neurodevelopmental and neurodegenerative abnormalities, and we found a possible association between the magnitude of the defect of the autophagic activity of WIPI4 mutants and the severity of neurodevelopmental symptoms. However, some of the BPAN-related missense mutations, which produce neurodegenerative signs, showed almost normal autophagic activity, suggesting that non-autophagic functions of WIPI4 may be related to neurodegeneration in BPAN.

cell biology↗

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↗