Biology subjects

Weckerly, C. C.

Publications and source records attributed to Weckerly, C. C..

4 recordsLinked to original sources

bioRxiv2026

In Cellulo pharmacological profiling and genomic editing reveals paralog-specific targets for PA generation during PLC signaling

Phosphatidic acid (PA) is an essential intermediate generated during phospholipase C (PLC) signaling, but its regulation is complex. PA can be generated by ten different diacylglycerol kinase paralogs (DGKs) and two different phospholipase D paralogs (PLDs) in mammals. Because these enzymes are activated under diverse conditions and at various membranes, understanding paralog-specific contributions to PA production is critical for therapeutic development of drugs that modulate the PLC pathway. To address this, we aimed to characterize the paralog specificity of the DGK inhibitors R59022 and BMS-502 against individual endogenously-tagged DGK paralogs in live cells. We found that R59022 and BMS-502 both recruited endogenous DGKalpha to the plasma membrane, and inhibited the catalytic fragment of DGKalpha when ectopically localized to the mitochondrial outer membrane. However, at its effective dose, R59022 paradoxically increased PA levels, while BMS-502 functioned as a potent inhibitor. Live-cell imaging experiments using BMS-502 with carbachol stimulation of endogenous muscarinic receptors showed that inhibition of both DGKalpha and the PLDs is needed to substantially reduce PA levels during PLC activation. Our findings both identify paralog-specific druggable targets for modulating PLC signaling events, and establish a new platform for characterizing DGK and PLD activity in living cells.

biochemistry↗

bioRxiv2024

Single molecule Lipid Biosensors Mitigate Inhibition of Endogenous Effector Proteins

Genetically encoded lipid biosensors uniquely provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools have significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with effectors, potentially inhibiting signaling. 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 PH-Akt at visible levels have reduced activation. Tagging endogenous AKT1 with neonGreen reveals its EGF-mediated translocation to the plasma membrane. Co-transfection with the PH-Akt1 or other PIP3 biosensors eliminates this translocation, despite robust recruitment of the biosensors. Inhibition is even observed with PI(3,4)P2-selective biosensor. However, 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 non-invasive way to probe spatiotemporal dynamics of PI3K signaling in living cells. eTOC summaryHolmes and colleagues show that AKT activation is blocked by commonly used PIP3 biosensors, but that this can be overcome by expressing biosensors at single molecule levels - which also improves kinetic fidelity and sensitivity.

cell biology↗

bioRxiv2024

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 analyzing the predicted structure of the LNS2 domain from the lipid transfer protein Nir1, we suspected that this phosphatidic acid-interacting Lipin-like sequence of Nir1 (PILS-Nir1) could serve as a novel PA biosensor. We then performed liposome binding assays as well as pharmacological and genetic manipulations of HEK293A cells expressing a fluorescent PILS-Nir1 to determine how specific lipids affect the interaction of PILS-Nir1 with membranes. We found that PILS-Nir1 bound to both PA and PIP2 in vitro. However, only PA was necessary and sufficient to localize PILS-Nir1 to membranes in cells. PILS-Nir1 also showed a heightened responsiveness to PA produced in various organelles when compared to biosensors using the Spo20 PA binding domain. PILS-Nir1s 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, PILS-Nir1 emerges as a versatile and sensitive biosensor, offering a new powerful tool for real-time investigation of PA dynamics in live cells.

cell biology↗

bioRxiv2022

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

When challenged by hypertonicity, dehydrated cells must defend 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 droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates 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 WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

cell biology↗