The activation of the heat shock response by Cyclin G/ cdk5 complex after cold stress and the Wolbachia influences on cellular stress response in Drosophila melanogaster

Abstract

The environmental changes caused by the human activities continue to threaten the health of the biosphere with heavy impact on natural ecosystems affecting all trophic levels from plant to insect and destabilizing the population dynamics that can lead to the extinction of the species. The temperature has influenced the distribution and the abundance of all species. As regards ectotherms, the temperature affects their distribution, development and growth in addition to physiological and metabolic processes. Increasing evidences have shown that, in all organisms, environmental, physiological and pathological stresses evoke the cellular stress response (CSR) involving as example the autophagy, the unfolded protein response, the DNA damage response, the mitochondrial unfolded protein response and the heat shock response to maintain and restore or re-establish the cellular homeostasis. Evidences showed that these pathways are regulated independently as well as co-ordinately as network of interlinked pathways. The CSR is considered the guardian of cellular homeostasis, it is a complex mechanism that involves many cellular pathways and cross-talker proteins. Afterward, the identification of the linker proteins that coordinate and regulate these interactions is very important. Nowadays many genes implicated in the CSR have been identified, but a lot of gaps need to be filled clarifying the determined functions and the interaction between genes of CSR pathways. In this Era, the CSR captures big attention because, the organisms are continually threatened by different stresses conditions. Within this context, this research project was thought in order to find new candidates genes involved in these cross-talk pathways, with the aim to understand how the organisms react to environmental factors e.g. cold stress and to deepen the influence of infection on the cellular stress response. We choose D. melanogaster as experimental model organism to study some keys genes involved in cellular stress response e.g. the Atg1 gene for the autophagy, the Xbp1 for the endoplasmic reticulum unfolded protein response, and the Hsp70Aa for the heat shock response after the cold stress. All the genes studied, were found to be involved in the stress response after cold stress. Hence, it was decided to focus the attention on the heat shock response and in particular on the heat shock proteins of the Hsp70 family since have a key role in the coordination of the CSR. The research continued searching a link between the cell cycle checkpoints, where cyclin/cdk complexes are involved, and the cellular stress response. With this aim it was done a time-course analysis of the expression of Hsp70Aa, HSF (the transcriptional factor of the heat shock proteins), Cyclin G and cdk5 upon different protocols of stress induction and recovery. All the analysed genes changed in gene expression more during the recovery phases than the stressing period except for HSF. Next step was to verify the interaction between the cyclin G and the cdk5. For this purpose co-immunoprecipitation experiments were set up. The interaction between these two proteins was confirmed. Then it was studied the possible involvement of the cdk5 in the activation of heat shock response after cold stress. Through RNAi experiment, using the GAL4/UAS system, it was demonstrated that cdk5 regulates the Hsp70Aa transcriptional activation. Moreover it was investigated on the possible involvement of Wolbachia infection (the most widespread endosymbiotic microbe found in arthropods) on the cellular stress response. It is already known that the infection by this bacterium affects the metabolism, the immunity system and reproduction. The gene expression of Atg1, Xbp1 and Hsp70Aa was studied on D. melanogaster strains infected or not by Wolbachia upon cold stress. Results showed that Wolbachia infection affects the expression levels of these genes and the recovery phase after cold stress treatments. At last it was studied the effect of the cold stress on the Wolbachia infection by monitoring the expression of the gene encoding for a Wolbachia surface protein (wsp). It was seen that the cold stress affects negatively the wsp gene expression up to 8 hours of recovery after cold stress, indicating that Wolbachia is very sensitive to cold stress and that its activity is hampered even after recovery of the host.