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Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase-mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain-mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting., (© 2015 Dong et al.)

Numerous mitochondrial DNA mutations cause mitochondrial encephalomyopathy: a collection of related diseases for which there exists no effective treatment. Mitochondrial encephalomyopathies are complex multisystem diseases that exhibit a relentless progression of severity, making them both difficult to treat and study. The pathogenic and compensatory metabolic changes that are associated with chronic mitochondrial dysfunction are not well understood. The Drosophila ATP6(1) mutant models human mitochondrial encephalomyopathy and allows the study of metabolic changes and compensation that occur throughout the lifetime of an affected animal. ATP6(1)animals have a nearly complete loss of ATP synthase activity and an acute bioenergetic deficit when they are asymptomatic, but surprisingly we discovered no chronic bioenergetic deficit in these animals during their symptomatic period. Our data demonstrate dynamic metabolic compensatory mechanisms that sustain normal energy availability and activity despite chronic mitochondrial complex V dysfunction resulting from an endogenous mutation in the mitochondrial DNA. ATP6(1)animals compensate for their loss of oxidative phosphorylation through increases in glycolytic flux, ketogenesis and Kreb's cycle activity early during pathogenesis. However, succinate dehydrogenase activity is reduced and mitochondrial supercomplex formation is severely disrupted contributing to the pathogenesis seen in ATP6(1) animals. These studies demonstrate the dynamic nature of metabolic compensatory mechanisms and emphasize the need for time course studies in tractable animal systems to elucidate disease pathogenesis and novel therapeutic avenues.

Mitochondrial encephalomyopathies are common and devastating multisystem genetic disorders characterized by neuromuscular dysfunction and tissue degeneration. Point mutations in the human mitochondrial ATP6 gene are known to cause several related mitochondrial disorders: NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh's syndrome), and FBSN (familial bilateral striatal necrosis). We identified a pathogenic mutation in the Drosophila mitochondrial ATP6 gene that causes progressive, adult-onset neuromuscular dysfunction and myodegeneration. Our results demonstrate ultrastructural defects in the mitochondrial innermembrane, neural dysfunction, and a marked reduction in mitochondrial ATP synthase activity associated with this mutation. This Drosophila mutant recapitulates key features of the human neuromuscular disorders enabling detailed in vivo studies of these enigmatic diseases.

Single-strand conformational polymorphism analysis has been used successfully to identify single nucleotide changes within sequences based on the fact that multidetection enhancement gels will separate molecules based on their conformation rather than their size. We have expanded the utility of this technique to analyze easily the alternative splicing of pre-mRNAs containing multiple mutually exclusive exons of the same size. We have used this technique to study the Caenorhabditis elegans let-2 gene containing two alternative exons and the Drosophilia melanogaster Dscam gene, which contains 12 mutually exclusive exons. The ease and the quantitative nature of this technique should be very useful.

RNA interference (RNAi) is a useful tool for degrading targeted messenger RNAs (mRNAs) and thus "knocking down" the abundance of the encoded protein. We have been using RNAi in cultured Drosophila cells to evaluate the effect of "knocking down" numerous mRNA processing factors on the alternative splicing of specific pre-mRNAs. This relatively simple technique has allowed us to identify a number of splicing factors that impact the alternative splicing of particular alternatively spliced exons. This approach can be extended to examine the splicing of nearly any gene.

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.