Biology subjects

Rabouille, C.

Publications and source records attributed to Rabouille, C..

5 recordsLinked to original sources

bioRxiv2022

Osmotic stress via calmodulin lead to the formation of stress granule in Drosophila S2 cells.

Cellular stress of S2 cells leads to the formation of stress assemblies by phase separation of cytoplasmic components. We have shown that the cellular stresses of either high increase of the NaCl concentration in the extracellular medium, or a moderate one combined to amino acid starvation, leads to the formation of Sec bodies where components of the endoplasmic Reticulum exit sites (ERES) coalesce. These extracellular stresses lead to both the activation of salt inducible kinase (SIK), and to ER stress triggering the activation of the two downstream kinases IRE1 and PERK. Interestingly, the same stresses also result in the formation of a second stress assembly, the stress granules, which stores specific RNAs and RNA binding proteins. Here we asked whether stress granule formation is governed by the same pathways as Sec bodies. However, we found that the inhibition of SIK, IRE1 and PERK does not affect stress granule formation. Instead, we found that osmotic stress through the addition of either salts (including calcium chloride) or sucrose leads to the formation of stress granules. Interestingly, stress granule formation is partly modulated by calmodulin activation, suggesting the involvement of calcium signaling. Furthermore, as Sec body formation is driven by entirely different pathways, these results show that the same cells under the same stress, form two different stress assemblies by non-overlapping downstream pathway activation, perhaps explaining that they do not coalescence into a single structure.

cell biology↗

bioRxiv2022

Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.

Phase separation of ER-exit-sites (ERES) components into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon specific cellular stressors, i.e., salt stress and amino acid starvation, and their formation is linked to the inhibition of the early secretory pathway. Here, we show Sec bodies also form in secretory mammalian INS-1 cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both isoforms of Sec16 (A and B) and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation. We show that the coalescence of ERES components into Sec bodies occurs by fusion, in line with their liquid-droplet properties. Lastly, we demonstrate that stress-induced ER-exit inhibition is a consequence of the significant coalescence of Sec16A into Sec bodies, leading to its depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of protein exit from the ER. We propose a model in which dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

cell biology↗

bioRxiv2021

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

Stress granules are phase separated assemblies formed around mRNAs whose identities remain elusive. The techniques available to identify the RNA content of stress granules rely on their physical purification, and are therefore not suitable for single cells and tissues displaying cell heterogeneity. Here, we adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress granule RNAs by fusing a stress granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We first optimized the expression of this fusion protein so that RNA editing preferentially occurs in stress granules. We then show that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells, and in Drosophila tissues, such as 398 neuronal stress granule mRNAs encoding ATP binding, cell cycle and transcription factors. This new method opens the possibility to identify the RNA content of stress granules as well other RNA based assemblies in single cells derived from tissues.

cell biology↗

bioRxiv2021

Salt Inducible Kinase activation and IRE1-dependent intracellular ATP depletion to form Sec bodies in Drosophila cells.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the ER exit sites under the stress of amino-acid starvation. Here we address which signaling pathways cause Sec body formation. We find that two pathways are critical. The first is a SIK dependent pathway induced by salt (NaCl) stress in a necessary and sufficient manner. The second is the activation of IRE1 (one of the key kinases mediating the Unfolded Protein Response) by absence of amino- acids, which partly leads to the depletion of intracellular ATP. However, IRE1 activation is not sufficient to induce Sec body formation and needs to be combined to salt stress. This works pioneers the role of SIK in phase transition and re-enforces the role of IRE1 as a metabolic sensor for the level of circulating amino-acids.

cell biology↗

bioRxiv2020

GORASPs link Golgi cisternae laterally to stabilize the rims and prevent vesiculation

In vitro experiments have shown GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans have given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other and the cisternal cross-sectional diameters are significantly reduced compared to their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks and we suggest that unlinking of stacks affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.

cell biology↗