Characterization of the Intracellular Distribution of Adenine Nucleotide Translocase (ANT) in Drosophila Indirect Flight Muscles

Background: The high power output necessary for insect flight has driven the evolution of muscles with large myofibrils (primary energy consumers) and abundant mitochondria (primary energy suppliers). The intricate functional interrelationship between these two organelles remains largely unknown despite its fundamental importance in understanding insect flight bioenergetics. Unlike vertebrate muscle that relies on a phosphagen (creatine phosphate/creatine kinase) system to regulate high energy phosphate flux, insect flight muscle has been reported to lack mitochondrial arginine kinase (analogous to creatine kinase), a key enzyme that enables intracellular energy transport. Creatine kinase is known to interact with mitochondrial adenine nucleotide translocase (ANT) in the transfer of ADP and ATP into and out of the mitochondria. Results: Here, we use quantitative immunogold transmission electron microscopy to show that in Drosophila melanogaster indirect flight muscles (IFM), ANT is present in the mitochondria as well as throughout the myofibril. To confirm this unexpected result, we created a transgenic line that expresses a chimeric GFP-ANT protein and used an anti-GFP antibody to determine the intracellular distribution of the fusion protein in the IFM. Similar to results obtained with anti-ANT, the fusion GFP-ANT protein is detected in myofibrils and mitochondria. We confirmed the absence of arginine kinase from IFM mitochondria and show that its sarcomeric (i.e., intramyofibrillar) distribution is similar to that of ANT. Conclusions: These results raise the possibility that direct channeling of nucleotides between mitochondria and myofibrils is assisted by an ANT protein thereby circumventing the need for a phosphagen shuttle in the IFM. The myofibrillar ANT may represent a unique adaptation in the muscles that require efficient exchange of nucleotides between mitochondria and myofibrils.

Adenine nucleotide translocase 2(Ant2)is required for individualization of spermatogenesis of Drosophila melanogaster

Successful completion of spermatogenesis is crucial for the perpetuation of the species.In Drosophila,spermatid individualization,a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm.Ant2,encoding a mitochondrial adenine nucleotide translocase,is highly expressed in male testes and plays a role in energy metabolism in the mitochondria.However,its molecular function remains unclear.Here,we identified an important role of Ant2 in spermatid individualization.In Ant2 knockdown testes,spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution,and mature sperms were absent in the seminal vesicle,thus leading to male sterility.The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function.Excessive apoptotic cells were also observed in Ant2-knockdown testes.To further investigate the phenotype of Ant2 knockdown in testes at the molecular level,complementary transcriptome and proteome analyses were performed.At the mRNA level,868 differentially expressed genes were identified,of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown.iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins,of which 117 proteins were upregulated and 233 were downregulated.The expression of glutathione transferase(GstD5,GstE5,GstE8,and GstD3),proteins involved in reproduction were significantly regulated at both the mRNA and protein levels.These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis.

Antioxidant defense mechanisms and fatty acid catabolism in Red-billed Leiothrix(Leiothrix lutea)exposed to high temperatures

Extreme hot weather is occurring more frequently due to global warming,posing a significant threat to species survival.Birds in particular are more likely to overheat in hot weather because they have a higher body temperature.This study used a heat stress model to investigate the antioxidant defense mechanisms and changes in fatty acid catabolism in Red-billed Leiothrix(Leiothrix lutea)to gain an understanding of how birds adapt to high temperatures.The birds were divided into five groups:a control group(30℃for 0 days),1 D group(40℃for 1 day),3 D group(40℃for 3 days),14 D group(40℃for 14 days)and recovery group(40℃for 14 days,then 30℃for 14 days).Our results indicated that when Red-billed Leiothrix are subjected to heat stress,malondialdehyde(MDA)content in the liver significantly increased,as did the enzyme activities of catalase(CAT),glutathione-SH-peroxidase(GSH-PX)and total antioxidant capacity(T-AOC)in the liver.Furthermore,there was a significant increase in heat shock protein 70(HSP70)expression in the liver,while avian uncoupling protein(avUCP)expression in muscle was significantly reduced.Additionally,there was a significant reduction in fatty acid catabolism enzyme activity such as 3-hydroxyacyl-CoAdehydrogenase(HOAD)activity in the heart,and carnitine palmitoyl transferase 1(CPT-1)and citrate synthase(CS)activity in the heart and liver.Furthermore,fatty acid translocase(FAT/CD36)in the heart,heart-type fatty acid binding protein(H-FABP)and fatty acid binding protein(FABP-pm)in the liver and heart were also significantly decreased.These changes reverted after treatment,but not to the same level as the control group.Our results indicated that when Red-billed Leiothrix are exposed to heat stress their internal antioxidant defense system is activated to counteract the damage caused by high temperatures.However,even with high antioxidant levels,prolonged high temperature exposure still caused some degree of oxidative damage possibly requiring a longer recovery time.Additionally,Red-billed Leiothrix may be able to resist heat stress by reducing fatty acid transport and catabolism.