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During the last decade, a novel concept of cellular architecture and organization has re-emerged with the recognition of high-order compartmentalization via non membrane-bound macromolecular structures. These membrane-less structures are in most of the cases the result of several stress conditions. The aim of this thesis is to depict the mechanisms behind the formation of Sec bodies and Stress granules in Drosophila S2 cells upon amino-acid starvation. These stress assemblies are formed as a result of the stalled of protein transport and protein translation respectively. First we investigate the behavior of ERES components upon amino-acid starvation. This nutrient stress leads to the stalling of protein secretion, which results in the formation of a novel membrane-less stress assembly that we have named the Sec body. This assembly is mainly composed of COPII proteins as well as the large scaffold protein Sec16. We demonstrate that Sec bodies are reversible, pro-survival structures with liquid droplets properties. Sec bodies act as reservoir for ERES components for the duration that this nutrient stress is taking place. Therefore, upon the relief of the stress the secretory pathway is rapidly reconstituted and functional. Post-translational modifications Mono and Poly-ADP-ribosylation have been proposed to be involved in the formation of aggregates and RNA granules. For this purpose, we designed and build specific fluorescent probes that allow us to follow these post-translational modifications in vivo. We discover that mono-ADP-ribosylation by the mono-ADP-ribose dPARP16 plays a crucial role in Sec body formation, as it modifies the Sec body component Sec16 on a very specific sequence in its C–terminus. In summary, we demonstrate that dPARP16 catalytic activity on Sec16 is a necessary and sufficient step to induce Sec body assembly and cell survival. Furthermore, we propose that Sec16 is a key factor for the stress response to amino acid starvation. Further we investigate the post-translation mechanism behind the formation of stress granules upon amino-acid starvation. We report that mono-ADP-ribosylation by dPARP16 is necessary but not sufficient for their formation. Our experimental data points also towards a role for Poly-ADP-ribosylation. In this regard, we show that the nuclear resident, poly-ADP-ribose dPARP1 plays a crucial role. Interestingly, in order to induce stress granule formation dPARP1 has to localize at the cytoplasm. Critically, dPARP1 localization out of the nucleus depends on dPARP16, possibly through the mono-ADP-ribosylation of the nuclear exporter Karyopherin beta 3. These findings provide a link between the stress responses exerted at the secretory pathway, nuclear export and the translation turnover upon nutrient stress. And as last we approach the role of Sec16 in the formation of the stress granules upon amino-acid starvation. We have identified that Sec16 plays a crucial role in stress granule formation upon this nutrient stress. Using mass spectrometry and a plasma membrane anchor-away technique, we show that Sec16 specifically interacts with the phosphorylated-Ser-142 of Rasputin, the form that is exclusively required for stress granule formation upon this nutrient stress. These results reinforce the notion of Sec16 as a stress response protein upon amino-acid starvation.

The kinase TOR is found in two complexes, TORC1, which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function. We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules., Highlighted Article: The Drosophila TOR complex 2 is necessary for the formation of heat-induced cytoprotective stress granules, which can explain the heat sensitivity of TORC2 mutant flies.