DRAM1 requires PI(3,5)P₂generation by PIKfyve to deliver vesicles and their cargo to endolysosomes

Abstract

Endolysosomal vesicle trafficking and autophagy are crucial degradative pathways in maintenance of cellular homeostasis. The transmembrane protein DRAM1 is a potential therapeutic target that primarily localises to endolysosomal vesicles and promotes autophagy and vesicle fusion with lysosomes. However, the molecular mechanisms underlying DRAM1-mediated vesicle fusion events remain unclear. Using high-resolution confocal microscopy in the zebrafish model, we show that mCherry-Dram1 labelled vesicles interact and fuse with early endosomes marked by PI(3)P. Following these fusion events, early endosomes mature into late endosomes in a process dependent on the conversion of PI(3)P into PI(3,5)P2 by the lipid kinase PIKfyve. Chemical inhibition of PIKfyve reduces the targeting of Dram1 to acidic endolysosomal vesicles, arresting Dram1 in multivesicular bodies, early endosomes, or non-acidified vesicles halted in their fusion with early endosomes. In conclusion, Dram1-mediated vesicle fusion requires the formation of PI(3,5)P2 to deliver vesicles and their cargo to the degradative environment of the lysosome.

Stimulating the autophagic-lysosomal axis enhances host defense against fungal infection in a zebrafish model of invasive Aspergillosis

“…Finally, using an mCherry-Dram1 construct [ 38 ], we demonstrated that Dram1 colocalizes with A. fumigatus conidia in phagocytes ( Figure 5(g )), supporting the conclusion that this autophagy-stimulating factor contributes to the response of phagocytes to A. fumigatus .…”

“…It has recently been reported that microbial-targeted autophagy (xenophagy) and its related process LAP contribute to efficient intracellular fungal clearance by innate immune cells in vitro [ 22–24 ]. To visualize the in vivo autophagy activation dynamics in innate immune cells after A. fumigatus phagocytosis, we imaged the fluorescent map1lc3b /Lc3b reporter zebrafish line Tg(CMV:EGFP -map1lc3b ) zf155 [ 37 ] or Tg( bactin : mCherry- map1lc3b ) [ 38 ] – further referred to as GFP-Lc3 and mCherry-Lc3, respectively – by confocal microscopy shortly after infection. These transgenic lines allow the study of autophagy-related dynamics in real time by analyzing the accumulation of fluorescently labeled Lc3b in specific vesicles ( Figure 2 , Movie S2).…”

“…Zebrafish were handled in compliance with the local animal welfare directives of Leiden University (License number 10,612) and following standard zebrafish rearing protocols ( https://zfin.org ), which adhere to the international guidelines from the EU Animal Protection Direction 2010/63/EU. In this work we used the lines ABTL (wild-type), Tg(CMV:EGFP -map1lc3b ) zf155 , Tg( bactin :mCherry- map1lc3b ) [ 38 ], Tg(CMV:EGFP -map1lc3b zf155 ; mpeg1.1 :mCherryF ump2 ), Tg( mpeg1.1 :EGFP gl22 , mpx :GAL4-VP16 sh267 /UAS-E1B:NTR-mCherry i149 ), and Tg (mpx :GAL4-VP16 sh267 /UAS-E1B:NTR-mCherry i149 ). All experiments were performed on zebrafish embryos or larvae up to 5 days post fertilization, which have not yet reached the free-feeding stage.…”