Gene expression profiling identifies FKBP39 as an inhibitor of autophagy in larval Drosophila fat body

Abstract

In Drosophila, the fat body undergoes a massive burst of autophagy at the end of larval development in preparation for the pupal transition. To identify genes involved in this process, we carried out a microarray analysis. We found that mRNA levels of the homologs of Atg8, the coat protein of early autophagic structures, and lysosomal hydrolases were upregulated, consistent with previous results. Genes encoding mitochondrial proteins and many chaperones were downregulated, including the inhibitor of eIF2alpha kinases and the peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39). Genetic manipulation of FKBP39 expression had a significant effect on autophagy, potentially through modulation of the transcription factor Foxo. Accordingly, we found that Foxo mutants cannot properly undergo autophagy in response to starvation, and that overexpression of Foxo induces autophagy.

Figure 1

Overexpression of FKBP39 inhibits developmental and starvation-induced autophagy, whereas loss of FKBP39 function leads to higher than wild-type induction of autophagy. a-c: Lysotracker staining of fat bodies. No Lysotracker staining is seen in feeding early third instar larvae (a), whereas Lysotracker-positive granules (shown in red) accumulate in fat body cells of wandering late third instars (b). Overexpression of FKBP39 in the fat body inhibits the formation of Lysotracker-positive granules (c), and leads to an inhibition of cell growth. This effect is best seen by cortical actin staining (red) of fat bodies clonally overexpressing FKBP39, marked by co-overexpression of nuclear GFP (green) (d, top panel). Lower panel shows DNA staining. Note that the nuclei of FKBP39-overexpressing cells (green arrows) are of the same size but contain less DNA than surrounding wild-type cells (blue arrows). e-f: electron microscopic images show fat body cells of late third instar larvae. Developmental autophagy results in the accumulation of large autolysosomes in wild-type larvae (arrow in e), whereas only small autolysosomes are seen in FKBP39-overexpressing fat body cells of the same age (arrows in f). Note the greatly enlarged nucleolus in f (asterisk), also caused by the overexpression of FKBP39. g shows quantitation of Lysotracker data on developmental autophagy. h-j: Lysotracker staining of fat bodies. Autophagy in fat body cells of early third instar larvae, induced by a 4-hour starvation (h, compare to a). Fat body-specific overexpression of FKBP39 also blocks this starvation response (i). Co-overexpression of PTEN restores autophagy inhibited by FKBP39 (j). k shows quantitation of Lysotracker data. l-n: Lysotracker staining of fat bodies. Short (80-minute) starvation induces higher levels of autophagy in fat bodies of FKBP39 mutant larvae than in wild-type controls (m, n, compare to l). o shows quantification of the results. Panels a-d, h-j and l-n are of the same magnification (200x). Panels e-f are of the same magnification and the bar equals 1 um. N marks the nucleus in electron microscopical images. Error bars represent standard deviation in panels g, k and o. Genotypes: UASFKBP39/+ (a, b, e, h), cgGal4/UASFKBP39 (c, f, i), hsFLP; UASFKBP39/+; Act>CD2>Gal4, UASGFPnls/+ (d), cgGal4/UASFKBP39, UASPTEN (j), w¹¹¹⁸ (l), w¹¹¹⁸; 5-HA-2590/Df(3R)Exel6194 (m), w¹¹¹⁸; 5-HA-2440/Df(3R)Exel6194 (n).

Figure 2

The autophagy-inhibiting effect of FKBP39 overexpression is potentially mediated by inhibition of Foxo. a: the membrane localization of a probe used as an indicator of PI3K activity (GFP-PH, green) is greatly reduced compared to the cytoplasmic signal in fat body cells of feeding larvae that clonally overexpress FKBP39 (marked by punctate myrRFP expression, red). a‘ shows the green channel separately. b: Foxo staining (red) in fat bodies of wild-type mid-third instar larvae shows both nuclear and cytoplasmic labeling. In cells overexpressing FKBP39 (and GFP, green), the nuclear pool of Foxo is completely abolished, and only cytoplasmic signal is seen. b‘ shows the red channel separately. c: null mutation of Foxo (Foxo²¹/Foxo²⁵) strongly decreases starvation-induced autophagy in the larval fat body, as shown by Lysotracker staining (red) (compare to Figure 1h). d: overexpression of FKBP39 in a Foxo null mutant background results in a reduction similar to that seen in case of Foxo mutation or FKBP39 overexpression alone, also shown by quantitation of Lysotracker data in panel e. f: overexpression of an activated form of Foxo, Foxo^™ strongly induces autophagy in fat body cells of feeding larvae. Inset shows the red channel (Lysotracker staining) in the overexpressing cells (delineated by a yellow line). Magnification is 300x for panel a and 200x for panels b-d and f. Error bars represent standard deviation in panel e. Genotypes: hsFLP; UASFKBP39/UASmyrRFP; Act>CD2>Gal4, tGPH/+ (a), hsFLP; UASFKBP39/+; Act>CD2>Gal4, UASGFPnls/+ (b), Foxo²¹/Foxo²⁵ (c), cgGal4/UASFKBP39; Foxo²¹/Foxo²⁵ (d), hsFLP; UASFoxo^™/+; Act>CD2>Gal4, UASGFPnls/+ (f).