Chaohong Wu, Joost Schulte, Katharine J Sepp, Troy J Littleton, and Pengyu Hong. 2010. “
Automatic robust neurite detection and morphological analysis of neuronal cell cultures in high-content screening.” Neuroinformatics, 8, 2, Pp. 83-100.
AbstractCell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutamine-mediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington's Disease (HD) model.
2010_Neuroinfor_Wu.pdf Amy M Wiles, Mark Doderer, Jianhua Ruan, Ting-Ting Gu, Dashnamoorthy Ravi, Barron Blackman, and Alexander JR Bishop. 2010. “
Building and analyzing protein interactome networks by cross-species comparisons.” BMC Syst Biol, 4, Pp. 36.
AbstractBACKGROUND: A genomic catalogue of protein-protein interactions is a rich source of information, particularly for exploring the relationships between proteins. Numerous systems-wide and small-scale experiments have been conducted to identify interactions; however, our knowledge of all interactions for any one species is incomplete, and alternative means to expand these network maps is needed. We therefore took a comparative biology approach to predict protein-protein interactions across five species (human, mouse, fly, worm, and yeast) and developed InterologFinder for research biologists to easily navigate this data. We also developed a confidence score for interactions based on available experimental evidence and conservation across species. RESULTS: The connectivity of the resultant networks was determined to have scale-free distribution, small-world properties, and increased local modularity, indicating that the added interactions do not disrupt our current understanding of protein network structures. We show examples of how these improved interactomes can be used to analyze a genome-scale dataset (RNAi screen) and to assign new function to proteins. Predicted interactions within this dataset were tested by co-immunoprecipitation, resulting in a high rate of validation, suggesting the high quality of networks produced. CONCLUSIONS: Protein-protein interactions were predicted in five species, based on orthology. An InteroScore, a score accounting for homology, number of orthologues with evidence of interactions, and number of unique observations of interactions, is given to each known and predicted interaction. Our website http://www.interologfinder.org provides research biologists intuitive access to this data.
2010_BMC Sys Bio_Wiles.pdf Supplemental Files.zip Andrés Dekanty, Nuria M Romero, Agustina P Bertolin, María G Thomas, Claudia C Leishman, Joel I Perez-Perri, Graciela L Boccaccio, and Pablo Wappner. 2010. “
Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.” PLoS Genet, 6, 6, Pp. e1000994.
AbstractHypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.
2010_PLOS Gen_Dekanty.pdf Supplement.pdf Lutz Kockel, Kimberly S Kerr, Michael Melnick, Katja Brückner, Matthias Hebrok, and Norbert Perrimon. 2010. “
Dynamic switch of negative feedback regulation in Drosophila Akt-TOR signaling.” PLoS Genet, 6, 6, Pp. e1000990.
AbstractAkt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)-dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K-independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt-TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo.
2010_PLOS Gen_Kockel.pdf Supplement.pdf Sheng Zhang, Richard Binari, Rui Zhou, and Norbert Perrimon. 2010. “
A genomewide RNA interference screen for modifiers of aggregates formation by mutant Huntingtin in Drosophila.” Genetics, 184, 4, Pp. 1165-79.
AbstractProtein aggregates are a common pathological feature of most neurodegenerative diseases (NDs). Understanding their formation and regulation will help clarify their controversial roles in disease pathogenesis. To date, there have been few systematic studies of aggregates formation in Drosophila, a model organism that has been applied extensively in modeling NDs and screening for toxicity modifiers. We generated transgenic fly lines that express enhanced-GFP-tagged mutant Huntingtin (Htt) fragments with different lengths of polyglutamine (polyQ) tract and showed that these Htt mutants develop protein aggregates in a polyQ-length- and age-dependent manner in Drosophila. To identify central regulators of protein aggregation, we further generated stable Drosophila cell lines expressing these Htt mutants and also established a cell-based quantitative assay that allows automated measurement of aggregates within cells. We then performed a genomewide RNA interference screen for regulators of mutant Htt aggregation and isolated 126 genes involved in diverse cellular processes. Interestingly, although our screen focused only on mutant Htt aggregation, several of the identified candidates were known previously as toxicity modifiers of NDs. Moreover, modulating the in vivo activity of hsp110 (CG6603) or tra1, two hits from the screen, affects neurodegeneration in a dose-dependent manner in a Drosophila model of Huntington's disease. Thus, other aggregates regulators isolated in our screen may identify additional genes involved in the protein-folding pathway and neurotoxicity.
2010_Genetics_Zhang.pdf Supplemental Files