Prefused lysosomes cluster on autophagosomes regulated by VAMP8

Qixin Chen,Xintian Shao,Jun-Lin Guan,Axel T Brunger,Linsen Li,Mingang Hao,Lei Wang,Zhiqi Tian,Qing Zhong,Yang Chen,Richard A Pfuetzner,Jiajie Diao

doi:10.1038/s41419-021-04243-0

Abstract

Lysosome–autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes—vesicle-associated membrane protein 8 (VAMP8)—plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

Highlights

Lysosomes are acidic organelles ranging from 0.025 to 0.8 μm in diameter that are the degradation centers of cellular materials in eukaryotic cells [1, 2]
To associated membrane protein 8 (VAMP8), the sensitive factor attachment protein receptor (SNARE) protein on further confirm the reliability of DAPG dyes as autophagosome lysosomes, regulates the lysosome–autophagosome association. markers, we examined the colocalization of the DAPRed dyes, phosphorylation of VAMP8 reduces fusion by preventing which is similar to DAPG except emitting red fluorescence, with full SNARE complex assembly, suggesting a new regulatory LC3B-GFP puncta
The precise mechanism of membrane fusion involved in autophagosome maturation remains unknown

Summary

INTRODUCTION

Lysosomes are acidic organelles ranging from 0.025 to 0.8 μm in diameter that are the degradation centers of cellular materials in eukaryotic cells [1, 2]. To confirm the VAMP8KD on autophagosome-lysosome fusion, we used negative potential interaction of lysosome clusters and autophagosomes, stain EM (Fig. 3i) [34] and an autolysosome dye to stain cells for we stained lysosomes and autophagosomes with LTR and DAPG SIM experiments (Fig. 3j) in order to investigate the formation of dyes, respectively (Fig. 2a), and observed a substantial overlap of autolysosomes. We monitored the formation of autolysosomes using SIM of DAPG-stained samples (Fig. 4i, j) and found that their formation increased inVAMP8Ala cells but decreased in VAMP8Glu cells We confirmed this observation with another autophagy indicator, LC3B with CCCP-treatment (Supplementary Fig. S8) or in RMtreated HeLa cells stably expressing GFP-LC3B (Supplementary Fig. S9). We observed that both autophagosome-lysosome fusion and the formation of lysosome clusters that are associated with autolysosomes increased in PKC inhibitor-treated HeLa cells (Supplementary Fig. S10). These results suggest that VAMP8 phosphorylation plays an important role in TMZ resistance by influencing autophagic flux

CONCLUSION

MATERIALS AND METHODS

Findings

ETHICS STATEMENT