A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body
biorxiv(2021)
摘要
For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: “HA Frankenbody”. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.
### Competing Interest Statement
The authors have declared no competing interest.
* APF
: after puparium formation
ATG
: autophagy-related
mCh
: monomeric Cherry
DIOM
: dorsal internal oblique muscle
GOI
: gene of interest
LBWM
: larval body wall muscle
LEC
: larval epidermal cell
tAL
: tubular autolysosome
TF
: GFP-mCh tandem fluorescent
POI
: protein of interest
V-ATPase
: vacuolar H+ ATPase
3IL
: third instar larvae
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