Stress assemblies: Defining pathways leading to their formation and identifying their components

Abstract

After the presentation of some stress signaling pathways and stress assemblies (Chapters 1 and 2), the main scope of this thesis is to unravel the signaling pathways leading to the formation of stress assemblies, Sec bodies and Stress granules. It is articulated around 2 chapters. Chapter 3, describes the signaling pathways leading to Sec body formation. Here, we have found that amino acid starvation in KRB comprises two branches. First, KRB induces a NaCl stress due to its 2.6-fold increased (moderate) NaCl concentration when compared to the growing Schneider’s medium. Interestingly, a larger (4-fold) increase in NaCl concentration in Schneider's (SCH150) is sufficient to lead to Sec body formation. NaCl stress activates the Salt inducible Kinases (SIK) and their pharmacological inhibition (using the pan SIK inhibitor HG-9-91-01) reduces Sec body formation. The second pathway activated in KRB is due to amino acid starvation that elicits oxidative stress and ER stress. However, either ROS or ER stress or combined is not enough to trigger Sec body formation. To form Sec bodies, ER stress (through the activation of IRE1 and PERK) needs to be combined to moderate NaCl stress. Conversely, Chapter 4 describes the Signaling pathways leading to stress granule formation and we have compared these to Sec bodies. Here, we show that both KRB and high NaCl stress (SCH150) also lead to the formation of stress granules. However, neither SIKs, nor IRE1 nor PERK are involved in their formation. Instead, we found osmotic stress through the addition of any salt or sucrose leads to the formation of stress granules that is also partly modulated by calmodulin activation, in stark contrast to Sec body formation. In Chapter 5, we report how I have addressed the protein content of Sec body using APEX2 followed by mass spectroscopy. 52 proteins have been identified specifically enriched in Sec body, including a large proportion of ER and Golgi protein. We summary and discuss our findings in Chapter 6.