Screening Novel Genes Involved In Mitochondrial Function In Drosophilia

Mitochondria are important organelles generally existing in eukaryotic cells.Mitochondria regulate critical physiological processes.For example,they provide most of the cellular ATP by oxidative phosphorylation and regulate cell apoptosis;In addition,mitochondria are involved in regulating cytosolic Ca2+ level and the aging process.Structures determine functions.Therefore,the integrity of the mitochondrial morphology is closely related to its function.Mitochondria are dynamic organelles,the balance of mitochondrial fusion and fission contributes to cleaning damaged mi-tochondria,avoiding exerting an influence cells.Mitochondrial dysfunction can also lead to many diseases,mitochondrial DNA mutations can cause mitochondrial respir-atory chain diseases;defects in mitochondrial protein transport mechanisms can also result in related diseases.Besides,there are dysfunctional mitochondria in patients of neurodegenerative diseases such as Parkinson's disease.The minute mitochondrial structure makes it difficult to study in vivo,but they generally accumulate in tissues such as muscles,which demand abundant energy to meet their needs.So we consider muscular tissues as carriers to study the mitochon-drial function.Drosophilia melanogaster has many advantages of being a model ani-mal.Its short life cycle,abundant phonotypes,amenability to genetic and molecular analysis offer an ideal tool for biological study.In addition,the muscular structure of adult flies is similar to vertebrata skeletal muscle in morphology and physiology.One example of these muscles is indirect flight muscles(IFMs).The IFMs have a similar striated structure to mammalian skeletal muscle and have large tubular mitochondria typically arranged between myofibrils,thus providing a valuable in vivo model to re-search mitochondrial dynamics and function.In this study,we regarded the fruit flies motor ability as the primary standard of screening mitochondrial dysfunction.To disrupt gene functions exclusively in the muscle,we crossed the muscle-specific Mef2-Gal4 to each of the UAS-IR transgenic RNAi lines in our library.We conducted 6972 RNAi fruit flies.The primary screening results offered 141 UAS-RNAi strains,corresponding to the 139 conserved genes.And then we examined the Flybase database to assess the representation of various gene classes in our phenotypic categories.We selected 62 UAS-RNAi strains for the second screening.Alternations in Mito-GFP between myofibrils were our major criterion.Besides,difference in the morphology of myofibrils in IFMs was secondary screening standard.Taken together,we ultimately chose CG2508 RNAi,CG3356 RNAi to analyze their effect on mitochondrial function.CG2508,CG3356 perform as E3 ligases,thus taking part in ubiquitination.Ubiq-uitination is an enzymatic process by which proteins are modified with ubiquitin chains.The final aim of ubiquitination is to target proteins for degradation by the proteasome or lysosome.We studied the two gene functions from various levels.Knocking down CG2508,CG3356 in muscle could lead to alternations in mitochondrial morphology.Histological analysis of IFMs of the Mef2-Gal4>CG2508 RNAi flies and Mef2-Gal4>CG3356 RNAi flies revealed a little disruption of muscle integrity.The defects in IFMs of Mef2-Gal4>CG2508 RNAi flies were age-dependent.We also measured the mitochondrial function of CG2508 RNAi,CG3356 RNAi by considering ATP levels as an index.These flies showed severe reduction of thoracic muscular ATP levels.Mef2-Gal4>CG2508 RNAi flies exhibit more drastic reduction in ATP levels,which was also age-dependent.In our study,via RNAi-mediated screening,we can find some novel genes in-fluencing mitochondrial function.These genes mostly are conserved from insects to mammalian.These genes can affect mitochondrial morphology,numbers and function.We need to gain further insights into their molecular mechanisms by studying their characterized motifs and crosstalk with other molecules.