WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Single molecule Lipid Biosensors Mitigate Inhibition of Endogenous Effector Proteins.

Genetically encoded lipid biosensors are essential cell biological tools. They are the only technique that provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools also carry significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with their effectors, causing inhibition. Here, we show that although PI 3-kinase (PI3K)-mediated activation of Akt is not significantly reduced in a cell population transfected with a PH-Akt1 PIP3/PI(3,4)P2 biosensor, single cells expressing the PH-Akt at visible levels (used for live-cell imaging) have no activated Akt at all. Tagging endogenous AKT1 with neonGreen at its genomic locus reveals its EGF-mediated translocation to the plasma membrane, accumulating at densities of ~0.3 molecules/µm2. Co-transfection with the PH-Akt biosensor or other PIP3 biosensors completely blocks this translocation, despite robust recruitment of the biosensors. A partial inhibition is even observed with PI(3,4)P2-selective biosensor. However, we found that expressing lipid biosensors at low levels, comparable with those of endogenous AKT, produced no such inhibition. Helpfully, these single-molecule biosensors revealed improved dynamic range and kinetic fidelity compared with over-expressed biosensor. This approach represents a less invasive way to probe spatiotemporal dynamics of the PI3K pathway in living cells.

Nir1-LNS2 is a novel phosphatidic acid biosensor that reveals mechanisms of lipid production.

Despite various roles of phosphatidic acid (PA) in cellular functions such as lipid homeostasis and vesicular trafficking, there is a lack of high-affinity tools to study PA in live cells. After analysis of the predicted structure of the LNS2 domain in the lipid transfer protein Nir1, we suspected that this domain could serve as a novel PA biosensor. We created a fluorescently tagged Nir1-LNS2 construct and then performed liposome binding assays as well as pharmacological and genetic manipulations of HEK293A cells to determine how specific lipids affect the interaction of Nir1-LNS2 with membranes. We found that Nir1-LNS2 bound to both PA and PIP2 in vitro. Interestingly, only PA was necessary and sufficient to localize Nir1-LNS2 to membranes in cells. Nir1-LNS2 also showed a heightened responsiveness to PA when compared to biosensors using the Spo20 PA binding domain (PABD). Nir1-LNS2's high sensitivity revealed a modest but discernible contribution of PLD to PA production downstream of muscarinic receptors, which has not been visualized with previous Spo20-based probes. In summary, Nir1-LNS2 emerges as a versatile and sensitive biosensor, offering researchers a new powerful tool for real-time investigation of PA dynamics in live cells.

Molding a PI(3,5)P2 biosensor.

The lipid phosphatidylinositol 3,5-bisphosphate-PI(3,5)P2-is known to be a key regulator of cellular traffic in health and disease, but its cellular localization was somewhat enigmatic until now, with the discovery of a new PI(3,5)P2 biosensor reported in this issue of JCB by Vines et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202209077).

PIP3 abundance overcomes PI3K signaling selectivity in invadopodia.

PI3Kß is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kß and its coupling to SHIP2 to produce PI(3,4)P2 . We now test whether selectivity for PI3Kß is preserved under conditions of mutational increases in PI3K activity. In breast cancer cells where PI3Kß is inhibited, short-chain diC8-PIP3 rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P2 is SHIP2-independent. Surprisingly, the expression of either activated PI3Kß or PI3Kα mutants rescued the effects of PI3Kß inhibition. In both cases, gelatin degradation was SHIP2-dependent. These data confirm the requirement for PIP3 conversion to PI(3,4)P2 for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.

An update on genetically encoded lipid biosensors.

Specific lipid species play central roles in cell biology. Their presence or enrichment in individual membranes can control properties or direct protein localization and/or activity. Therefore, probes to detect and observe these lipids in intact cells are essential tools in the cell biologist's freezer box. Herein, we discuss genetically encoded lipid biosensors, which can be expressed as fluorescent protein fusions to track lipids in living cells. We provide a state-of-the-art list of the most widely available and reliable biosensors and highlight new probes (circa 2018-2021). Notably, we focus on advances in biosensors for phosphatidylinositol, phosphatidic acid, and PI 3-kinase lipid products.