The appearance of cytoplasmic cytochrome C precedes apoptosis during Drosophila salivary gland degradation.

Apoptosis is an important process for organism development that functions to eliminate cell damage, maintain homeostasis, and remove obsolete tissues during morphogenesis. In mammals, apoptosis is accompanied by the release of cytochrome C (Cyt-c) from mitochondria to the cytoplasm. However, whether this process is conserved in the fruit fly, Drosophila melanogaster, remains controversial. In this study, we discovered that during the degradation of Drosophila salivary gland, the transcription of mitochondria apoptosis factors (MAPFs), Cyt-c, and death-associated APAF1-related killer (Dark) encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes. The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation. Meanwhile, the Cyt-c protein colocates with mito-GFP, the marker indicating cytoplasmic mitochondria, and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm. Moreover, impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland, and accordingly decreases or increases the mitochondrial membrane potential, respectively. Our research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation, shedding light on partial conserved mechanism in apoptosis between insects and mammals.

Atg1 phosphorylation is activated by AMPK and indispensable for autophagy induction in insects.

Phosphorylation is a key post-translational modification in regulating autophagy in yeast and mammalians, yet it is not fully illustrated in invertebrates such as insects. ULK1/Atg1 is a functionally conserved serine/threonine protein kinase involved in autophagosome initiation. As a result of alternative splicing, Atg1 in the silkworm, Bombyx mori, is present as three mRNA isoforms, with BmAtg1c showing the highest expression levels. Here, we found that BmAtg1c mRNA expression, BmAtg1c protein expression and phosphorylation, and autophagy simultaneously peaked in the fat body during larval-pupal metamorphosis. Importantly, two BmAtg1c phosphorylation sites were identified at Ser269 and Ser270, which were activated by BmAMPK, the major energy-sensing kinase, upon stimulation with 20-hydroxyecdysone and starvation; additionally, these Atg1 phosphorylation sites are evolutionarily conserved in insects. The two BmAMPK-activated phosphorylation sites in BmAtg1c were found to be required for BmAMPK-induced autophagy. Moreover, the two corresponding DmAtg1 phosphorylation sites in the fruit fly, Drosophila melanogaster, are functionally conserved for autophagy induction. In conclusion, AMPK-activated Atg1 phosphorylation is indispensable for autophagy induction and evolutionarily conserved in insects, shedding light on how various groups of organisms differentially regulate ULK1/Atg1 phosphorylation for autophagy induction.

Transcriptional and Post-Transcriptional Regulation of Autophagy.

Autophagy is a widely conserved process in eukaryotes that is involved in a series of physiological and pathological events, including development, immunity, neurodegenerative disease, and tumorigenesis. It is regulated by nutrient deprivation, energy stress, and other unfavorable conditions through multiple pathways. In general, autophagy is synergistically governed at the RNA and protein levels. The upstream transcription factors trigger or inhibit the expression of autophagy- or lysosome-related genes to facilitate or reduce autophagy. Moreover, a significant number of non-coding RNAs (microRNA, circRNA, and lncRNA) are reported to participate in autophagy regulation. Finally, post-transcriptional modifications, such as RNA methylation, play a key role in controlling autophagy occurrence. In this review, we summarize the progress on autophagy research regarding transcriptional regulation, which will provide the foundations and directions for future studies on this self-eating process.

The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate.

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. Here we found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, our study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding our understanding of the comprehensive regulatory mechanisms of final body size in animals.

Can arbuscular mycorrhizal fungi reduce Cd uptake and alleviate Cd toxicity of Lonicera japonica grown in Cd-added soils?

A greenhouse pot experiment was conducted to study the impact of arbuscular mycorrhizal fungi--Glomus versiforme (Gv) and Rhizophagus intraradices (Ri) on the growth, Cd uptake, antioxidant indices [glutathione reductase (GR), ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), ascorbate (ASA), glutathione (GSH) and malonaldehyde (MDA)] and phytochelatins (PCs) production of Lonicera japonica in Cd-amended soils. Gv and Ri significantly increased P acquisition, biomass of shoots and roots at all Cd treatments. Gv significantly decreased Cd concentrations in shoots and roots, and Ri also obviously reduced Cd concentrations in shoots but increased Cd concentrations in roots. Meanwhile, activities of CAT, APX and GR, and contents of ASA and PCs were remarkably higher in Gv/Ri-inoculated plants than those of uninoculated plants, but lower MDA and GSH contents in Gv/Ri-inoculated plants were found. In conclusion, Gv and Ri symbiosis alleviated Cd toxicity of L. japonica through the decline of shoot Cd concentrations and the improvement of P nutrition, PCs content and activities of GR, CAT, APX in inoculated plants, and then improved plant growth. The decrease of shoot Cd concentrations in L. japonica inoculated with Gv/Ri would provide a clue for safe production of this plant from Cd-contaminated soils.