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Summary Eicosanoid lipids play important roles in cellular signaling as second messengers in inflammation, immune response, vascular tone, and the CNS. Biosynthesis of eicosanoid lipids proceeds via hydrolysis of esterified arachidonic acid from phospholipids followed by oxidation of the released arachidonic acid by a variety of enzymes including cyclooxygenases (COX). Herein, we demonstrate the remarkable ability of COX-2, but not COX-1, to directly oxidize 2-arachidonoyl-lysolipids, resulting in the generation of previously unknown classes of eicosanoid-lysolipids, and provide evidence that intracellular lipases can release eicosanoids from their eicosanoid-lysolipid precursors. Importantly, genetic ablation of a phospholipase, iPLA 2 γ, significantly reduced the amounts of these eicosanoid-lysolipids in murine hepatic tissue and fibroblasts. Furthermore, calcium stimulation of wild-type murine lung fibroblasts produced robust increases in these eicosanoid-lysolipids, which were markedly attenuated in iPLA 2 γ −/− fibroblasts. Collectively, these results identify an iPLA 2 γ-initiated pathway generating new classes of lipid metabolites with potential signaling functions resulting from the direct COX-2 catalyzed oxidation of 2-arachidonoyl-lysolipids. [ABSTRACT FROM AUTHOR]

Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5–2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction. [Display omitted] • ForceChrono probe quantifies the "temporal codes" of mechanotransduction in cells • Reveals force duration from tens of seconds to 100 s in adhesion sites • Measures integrin force loading rates from 0.5 to 2 pN/s • ForceChrono probe reveals in-cell protein roles by measuring force dynamics Development of the ForceChrono probe allows for precise measurement of duration and loading rates of single-molecule forces in living cells, revealing protein roles in force dynamics. [ABSTRACT FROM AUTHOR]