Loss of the starvation-induced gene Rack1 leads to glycogen deficiency and impaired autophagic responses in Drosophila

Abstract

Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. We compared genome-wide transcriptional profiles of fed and starved control, autophagy-deficient Atg7 and Atg1 null mutant Drosophila larvae to search for novel regulators of autophagy. Genes involved in catabolic processes including autophagy were transcriptionally upregulated in all cases. We also detected repression of genes involved in DNA replication in autophagy mutants compared with control animals. The expression of Rack1 (receptor of activated protein kinase C 1) increased 4.1- to 5.5-fold during nutrient deprivation in all three genotypes. The scaffold protein Rack1 plays a role in a wide range of processes including translation, cell adhesion and migration, cell survival and cancer. Loss of Rack1 led to attenuated autophagic response to starvation, and glycogen stores were decreased 11.8-fold in Rack1 mutant cells. Endogenous Rack1 partially colocalized with GFP-Atg8a and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Endogenous Rack1 also showed a high degree of colocalization with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of glycogen synthase kinase 3B (GSK-3B). Our results, for the first time, demonstrated the fundamental role of Rack1 in autophagy and glycogen synthesis.

Keywords: Atg8; Drosophila; GSK-3B; Rack1; antimicrobial peptides; autophagy; fat body; glycogen; microarray; starvation.

Figure 1. Transcriptional analysis of starvation responses. The number of gene ontology (GO) terms enriched (p < 0.05) among the significantly upregulated (A) and downregulated (B) genes (minimum two-fold change in expression level during the 4-h complete starvation, p < 0.05) in control, Atg7 and Atg1 mutant larvae. See text and Tables S4 and S5 for details. (C) lists starvation-induced transcriptional changes (gene expression levels in starved animals relative to the expression levels detected in fed animals) for Drosophila homologs of yeast and human Atg genes, involved in the core mechanism of autophagy. Atg8a encoding a ubiquitin-like protein showed the highest induction during starvation. Interestingly, the highest average upregulation was observed for genes encoding members of the Atg9 cycling complex. (D) Quantitative real-time (QT) PCR analysis of fat bodies dissected from fed and starved control larvae. Numbers represent fold change values, and nonsignificant changes are crossed out in (C and D).

Figure 2. Rack1 is required for the full autophagic response to starvation, and for efficient glycogen synthesis. (A) Knockdown of Rack1 results in reduction of autophagy in response to starvation. Please compare the number of mCherry-positive dots in RNAi cells vs. control cells in each panel (red; the red channel is also shown in greyscale in insets). RNAi expressing cells are marked by coexpression of GFP (green). Cell nuclei are labeled with DAPI (blue). (B) Numerous small Lamp1-GFP dots are seen in fat body cells of well-fed control larvae, which are not co-labeled by Lysotracker Red. (C) In contrast, large Lamp1-GFP and Lysotracker-positive autolysosomes are formed during starvation. (D) Silencing of Rack1 strongly reduces the number of Lysotracker particles that are formed during starvation; note that the size of Lamp1-GFP dots is also more similar to fed control cells. (E and F) The number of starvation-induced mCherry-Atg8a dots is reduced in Rack1 null mutant fat bodies compared with controls. (G and H) Similarly, the number of Lysotracker-positive autolysosomes is reduced in Rack1 mutants compared with controls. (I) Starvation-induced autophagy is rescued by heat shock-mediated expression of a Rack1 transgene in Rack1 null mutant animals. (J) Transmission electron microscopy shows the presence of large glycogen stores (g), numerous double-membrane autophagosomes (arrowheads) and autolysosomes (arrows) in fat body cells of control animals starved for 4 h in 20% sucrose. (K) A portion of (J) is shown enlarged. (L) Fewer autophagic structures and glycogen particles are detected in Rack1 mutants. (M) Heat-shock mediated expression of a Rack1 transgene in Rack1 null mutant animals partially rescues the reduced autophagy and glycogen accumulation phenotypes of the mutants. (N–S) Statistical analyses of mutant and RNAi phenotypes on autophagy and glycogen stores. Graphs represent dots/cell (N–P), average dot size (Q) and normalized cytoplasmic area (R and S), **p < 0.01 (two-tailed, two-sample unequal variance Student’s t-test). (T) Knockdown of Rack1 expression cell-autonomously decreases glycogen stores. (U and V) No specific Rack1 labeling is observed in Rack1 mutants, and glycogen stores are strongly decreased compared with heterozygous controls. Scale bars: 30 μm for all fluorescent images (indicated in A, E and T–V), 10 μm for (J, L and M, indicated in J) and 2 μm for (K).

Figure 3. Localization of endogenous Rack1. (A and B) Knockdown of Rack1 reduces anti-Rack1 immunostaining in both midgut and fat body cells in a cell-autonomous manner. (C) Rack1 shows a partial colocalization with the (pre-) autophagosome reporter GFP-Atg8a. (D) In contrast, no overlap was found between Rack1 and the lysosome reporter Lamp1-GFP. Boxed areas in (C and D) are shown enlarged. (E) Immunogold labeling of Rack1 is associated with a pre-autophagosomal phagophore near the growing edges (asterisk) and an autophagosome (red arrowhead). Please note the characteristic cleft (empty-looking space) between the two membrane sheets of phagophores and autophagosomes, which facilitates their recognition in glutaraldehyde-fixed samples. In contrast, no labeling of a lysosome is observed (arrow in E). Some of the gold particles are highlighted by yellow arrowheads. (F) Gold particles representing endogenous Rack1 are frequently associated with the peripheral parts of glycogen stores (g) in fat body cells. (G) Rack1 is also present in cytoplasmic clusters (c); an autophagosome (red arrowhead) is also labeled (yellow arrowhead). (H and I) Rack1 is closely associated with glycogen granules in the fat body, and it colocalizes with Shaggy, the Drosophila homolog of GSK-3B. Boxed areas of (A and B) are shown enlarged. Scale bars: 10 μm for (A–D, H and I), 5 μm for (C’–D” and H’–I”) and 1 μm for (E–G). All colocalization experiments were performed using confocal laser scanning microscopy.