Dataset Information

Identification and characterization of the heat-induced plastidial stress granules-like foci in Arabidopsis thaliana.

ABSTRACT: Plant cells, similar to animal cells repress translation under stressful conditions which leads to formation of cytosolic stress granules (SGs). Here, we report that in addition to cytosolic SGs plants also have plastidial SGs (pSGs). In order isolate and determine protein and metabolite composition of the pSG a combination of differential centrifugation and affinity purification methods was used. Plastidial Snowy Cotyledon 1 (SCO1) fused with GFP was used as a marker protein and its subcellular localization was confirmed by confocal microscope in a control as well as stress conditions. Multiple criteria were used to define pSG‐associated proteins. As the pSGs were only present in the heat/dark treated seedlings, we first selected for proteins that were either (i) respectively, present and absent in the pSGs isolate from the stress‐treated versus control seedlings, or (ii) significantly enriched (fold change > 2; t-test<0.05) in the SGs isolate from the stress‐treated versus the control seedlings. We then excluded cytosolic, nuclear, mitochondrial and membrane proteins, as these are unlikely to localize into plastidial SGs and can therefore be viewed as false positives. As an additional negative control, we compered obtained list of pSGs proteins with results gotten from independently performed experiment where we used seedlings over-expressing plastidial glyceraldehyde-3-phosphate dehydrogenase GAPCP1-GFP. In the absence of stress, GAPCP1-GFP aggregates into a dot-like condensate, which could be enriched and subsequently isolated using our protocol, but is clearly distinguishable from the SCO1-GFP pSGs. Interestingly, identified components of pSG are very similar to the ones from cytosolic SGs suggesting that the mechanism of their formation and function might be very similar.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Ewelina Sokolowska

LAB HEAD: Aleksandra Skirycz

PROVIDER: PXD018348 | Pride | 2020-09-24

REPOSITORIES: Pride

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Identification and Characterization of the Heat-Induced Plastidial Stress Granules Reveal New Insight Into <i>Arabidopsis</i> Stress Response.

Chodasiewicz Monika M Sokolowska Ewelina Maria EM Nelson-Dittrich Anna C AC Masiuk Aleksandra A Beltran Juan Camilo Moreno JCM Nelson Andrew D L ADL Skirycz Aleksandra A

Frontiers in plant science 20201030

Plants exhibit different physiological and molecular responses to adverse changes in their environment. One such molecular response is the sequestration of proteins, RNAs, and metabolites into cytoplasmic bodies called stress granules (cSGs). Here we report that, in addition to cSGs, heat stress also induces the formation of SG-like foci (cGs) in the chloroplasts of the model plant <i>Arabidopsis thaliana</i>. Similarly to the cSGs, (i) cpSG assemble rapidly in response to stress and disappear w ...[more]

PMID: 33224174

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Project description:The cellular stress response plays a vital role in regulating homeostasis by modulating cell survival and death. Stress granules (referred to as SGs) are cytoplasmic compartments that allow cells to survive various stressors. Defects in SG assembly and disassembly have been found to play important roles in neurodegenerative diseases, antiviral responses and cancer1–5. Inflammasomes are multi-protein heteromeric complexes that sense intracellular pathogen- and damage-associated molecular patterns and assemble into cytosolic compartments called ASC specks to facilitate caspase-1 (CASP1) activation. This activation of inflammasomes induces secretion of the leaderless proinflammatory cytokines IL-1β and IL-18 and also drives cell fate towards pyroptosis, a form of programmed inflammatory cell death that plays major role in health and disease6–12. Cellular stress sensing can trigger both SGs and inflammasomes; however, they drive contrasting cell fate decisions during stress conditions. Crosstalk between SGs and inflammasomes to decide cell fate has not been well studied. Here we show that the induction of SGs specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The SG protein DDX3X interacts with NLRP3 to drive inflammasome activation in the absence of SGs. Assembly of SGs leads to the sequestration of DDX3X to inhibit NLRP3 inflammasome function. SGs and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell fate decisions under stress conditions. Induction of SGs or loss of DDX3X in the myeloid compartment leads to decreased production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages utilize DDX3X availability to interpret stress signals and choose between pro-survival SGs and pyroptotic ASC specks. Together, our data show the role of DDX3X in driving NLRP3 inflammasome and SG assembly, informing a new rheostat-like mechanistic paradigm for regulating live or die cell fate decisions under stress conditions.

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