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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 PIP 3 /PI(3,4)P 2 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/ μ m 2 . Co-transfection with the PH-Akt biosensor or other PIP 3 biosensors completely blocks this translocation, despite robust recruitment of the biosensors. A partial inhibition is even observed with PI(3,4)P 2 -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.

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)., (© 2023 Weckerly and Hammond.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

PI3Kβ is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kβ and its coupling to SHIP2 to produce PI(3,4)P 2 . We now test whether selectivity for PI3Kβ is preserved under conditions of mutational increases in PI3K activity. In breast cancer cells where PI3Kβ is inhibited, short-chain diC8-PIP 3  rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P 2  is SHIP2-independent. Surprisingly, the expression of either activated PI3Kβ or PI3Kα mutants rescued the effects of PI3Kβ inhibition. In both cases, gelatin degradation was SHIP2-dependent. These data confirm the requirement for PIP 3 conversion to PI(3,4)P 2 for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway., (© 2022 Federation of European Biochemical Societies.)