Identification of in vivo Protein Targets of Nitric Oxide in Drosophila melanogaster

Nitric Oxide (NO) is known to alter cell proliferation and growth. This project investigated the molecular targets of NO, using the salivary glands of Drosophila melanogaster third instar larvae. A screen of 75 transgenic Drosophila lines expressing Yellow Fluorescent Protein tagged proteins, for those that showed a rapid alteration in sub-cellular localization after organ culture with an NO donor was undertaken. This screen revealed Mi-2 as a target of NO action. This was confirmed in vivo as localization of Mi-2-YFP was altered in whole salivary glands expressing Nitric Oxide Synthase (NOS2). The localization of the endogenous Mi-2 protein was similarly altered in NOS2 expressing single cells. A quantitative analysis using the image analysis software “Velocity” demonstrated an increase in the nuclear concentration of Mi-2 protein in these cells. Targeted expression of RNAi-Mi-2 as well as analysis of Mi-2 transheterozygous mutant larvae revealed that NO can impart its reduced growth phenotype independently of Mi-2. Dref can associate with Mi-2 and can control cell proliferation and growth. Localisation of Dref did not show any noticeable change in single cells expressing NOS. However when these cells were double labelled both with anti-Dref and anti-Mi-2 antibodies, the anti-Dref staining was altered. This indicates that NO can alter the availability of the Dref antigen by reorganising the Mi-2/Dref complex. Mi-2 is a component of the NuRD complex. The effect of NO in altering the Mi-2/Dref complex was exploited to investigate the effect of simj (a regulatory component of NuRD), on either of the proteins after potential disassociation of the complex. The data demonstrated an independent effect of simj on the localization of each protein. NO and FOXO regulate the expression of a common set of genes and FOXO is required for the anti growth properties of NO. Thus the effect of FOXO expression on Mi-2 and Dref protein distribution was determined. Although no consistent alteration of Mi-2 localization was observed, a quantitative analysis showed a large increase of Dref protein concentration in FOXO expressing cells. Double antibody staining of these cells with both anti-Dref and anti-Mi-2 antibodies showed a novel nuclear localization of the proteins imparted by FOXO. Thus this study has identified the regulation of the Mi-2/Dref protein complex as a possible growth control mechanism by NO and FOXO