Abstract
Cells are under threat of osmotic perturbation; cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge. After osmotic stress-enforced cell swelling or shrinkage, the cells regulate intracellular osmolality to recover their volume. However, the mechanisms recognizing osmotic stress remain obscured. We previously clarified that apoptosis signal-regulating kinase 3 (ASK3) bidirectionally responds to osmotic stress and regulates cell volume recovery. Here, we show that macromolecular crowding induces liquid-demixing condensates of ASK3 under hyperosmotic stress, which transduce osmosensing signal into ASK3 inactivation. A genome-wide small interfering RNA (siRNA) screen identifies an ASK3 inactivation regulator, nicotinamide phosphoribosyltransferase (NAMPT), related to poly(ADP-ribose) signaling. Furthermore, we clarify that poly(ADP-ribose) keeps ASK3 condensates in the liquid phase and enables ASK3 to become inactivated under hyperosmotic stress. Our findings demonstrate that cells rationally incorporate physicochemical phase separation into their osmosensing systems.
Highlights
Cells are under threat of osmotic perturbation; cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge
Through analyses of apoptosis signal-regulating kinase 3 (ASK3), we found that the subcellular localization of ASK3 drastically changes under hyperosmotic stress: a part of ASK3 diffuses throughout the cytosol, while the other forms granule-like structures, ASK3 condensates (Fig. 1a)
The number of ASK3 condensates increased in a hyperosmolality strength-dependent manner (Fig. 1b) and gradually decreased several dozen minutes after hyperosmotic stress (Supplementary Fig. 1a, b), which corresponds to the time range of cell volume recovery[9,18]
Summary
Cells are under threat of osmotic perturbation; cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge. Similar to the osmosensors proposed in bacteria, yeasts and plants, mechanical changes in/on the cell membrane have recently drawn attention in mammalian cells; for example, membrane stretching under hypoosmotic stress activates mechanosensitive channels, such as the transient receptor potential channel V410,11 This mechanism can be illustrated by an easy-to-understand signaling schematic with arrows directed from the extracellular side to the intracellular side. Lots of questions remain to be elucidated[12,13,14,15]; for instance, physicochemical theories and in vitro experiments suggest that macromolecular crowding is a driving force for the phase separation of biomolecular condensates, but the cellular system is detached from the classic thermodynamic equilibrium and the cell-based investigations have begun just recently[16]. Our findings demonstrate that cells recognize osmotic stress through liquid–liquid phase separation (LLPS) of ASK3 with the support of PAR
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