Temporal control of the integrated stress response by a stochastic molecular switch

Abstract

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

Activation of the mitochondrial unfolded protein response regulates the formation of stress granules

“…This effect was rescued in GADD34 KO cells, indicating that GADD34 upregulation is responsible for the inhibition of SG assembly following a second stressor. This result is in line with recent work demonstrating that sustained GADD34 levels upon ISR activation reduces cell responsiveness to a second stress pulse, promoting cell adaptation (Klein et al, 2022). Our data suggest that UPRmt employs GADD34 as a negative regulator of the ISR to fine-tune cellular stress responses and maintain mitochondrial function under these conditions.…”

“…Synthesis and purification of 200-bp dsRNA was described previously (Klein et al, 2022). In brief, a 200-bp region from the ampicillin resistance gene was used as template for in vitro synthesis of plus and minus strand transcripts, hybridized and purified.…”

Multiplexed dynamic control of temperature to probe and observe mammalian cells

“…Notably, SGs showed adaptation to heat stress, such that G3BP1 puncta spontaneously dissolved despite persistent heating. Adaptation of SGs has been previously reported in the context of oxidative stress 23 and viral infection 24 . Such adaptation is consistent with negative feedback, including through transcription of heat shock proteins (HSPs) [25][26][27][28][29] and of suppressors of the integrated stress response including GADD34 24,30,31 .…”

“…Adaptation of SGs has been previously reported in the context of oxidative stress 23 and viral infection 24 . Such adaptation is consistent with negative feedback, including through transcription of heat shock proteins (HSPs) [25][26][27][28][29] and of suppressors of the integrated stress response including GADD34 24,30,31 . Nevertheless, to our knowledge the adaptation of SGs to chronic heat stress has not been previously reported, demonstrating the utility of simultaneous thermal control and live-cell imaging.…”

eIF2B localisation and its regulation during the integrated stress response is cell type specific

“…Therefore, chronically ER stressed neurons redirect towards an eIF2α-independent mechanism when exposed to oxidative stress. These results are quite unexpected given that GADD34 expression levels are still elevated in these cells (Figure 3B), as GADD34 mRNA levels are known to serve as a molecular memory damper to subsequent stresses (Batjargal et al, 2022;Klein et al, 2022;Shelkovnikova et al, 2017). This apparent ability of (at least) glial cells to reset the ISR in the presence of GADD34 while neuronal cells seem to "forget" how to respond brings an important question:…”