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      Visualizing Cardiolipin In Situ with HKCL-1M, a Highly Selective and Sensitive Fluorescent Probe

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          Abstract

          Reliable probing of cardiolipin (CL) content in dynamic cellular milieux presents significant challenges and great opportunities for understanding mitochondria-related diseases, including cancer, neurodegeneration, and diabetes mellitus. In intact respiring cells, selectivity and sensitivity for CL detection are technically demanding due to structural similarities among phospholipids and compartmental secludedness of the inner mitochondrial membrane. Here, we report a novel “turn-on” fluorescent probe HKCL-1M for detecting CL in situ. HKCL-1M displays outstanding sensitivity and selectivity toward CL through specific noncovalent interactions. In live-cell imaging, its hydrolyzed product HKCL-1 efficiently retained itself in intact cells independent of mitochondrial membrane potential (Δψ m). The probe robustly co-localizes with mitochondria and outperforms 10- N-nonyl acridine orange (NAO) and Δψ m-dependent dyes with superior photostability and negligible phototoxicity. Our work thus opens up new opportunities for studying mitochondrial biology through efficient and reliable visualization of CL in situ.

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          Protein and polymer analyses up tom/z 100 000 by laser ionization time-of-flight mass spectrometry

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            Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells

            Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased mitochondrial delivery to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1-light chain-3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modeling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an “eat-me” signal for the elimination of damaged mitochondria from neuronal cells.
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              The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin.

              Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.
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                Author and article information

                Journal
                J Am Chem Soc
                J Am Chem Soc
                ja
                jacsat
                Journal of the American Chemical Society
                American Chemical Society
                0002-7863
                1520-5126
                27 April 2023
                24 May 2023
                : 145
                : 20
                : 11311-11322
                Affiliations
                []Guangdong Provincial Key Laboratory of Optical Fiber Sensing and Communication, Institute of Photonics Technology, Jinan University , Guangzhou 510632, China
                []Clinical Pharmacology Section, Department of Pharmacology, Shantou University Medical College , Shantou 515041, China
                [§ ]Morningside Laboratory for Chemical Biology, Department of Chemistry, The University of Hong Kong , Hong Kong 999077, China
                []Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University , Hangzhou 310024, China
                []Qingdao Institute for Theoretical and Computational Sciences, Institute of Frontier and Interdisciplinary Science, Shandong University , Qingdao 266237, China
                [# ]School of Life Sciences, Westlake University , Hangzhou 310024, China
                []Department of Cell Biology, University of Pittsburgh , Pittsburgh, Pennsylvania 15219, United States
                []Department of Environmental and Occupational Health, University of Pittsburgh , Pittsburgh, Pennsylvania 15219, United States
                []Center for Free Radical and Antioxidant Health, University of Pittsburgh , Pittsburgh, Pennsylvania 15261, United States
                []Department of Critical Care Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania 15213, United States
                [†† ]Westlake Laboratory of Life Sciences and Biomedicine , Hangzhou 310024, China
                Author notes
                Author information
                https://orcid.org/0000-0003-2950-9882
                https://orcid.org/0000-0003-2177-8463
                https://orcid.org/0000-0003-2361-4120
                https://orcid.org/0000-0002-7245-1885
                https://orcid.org/0000-0002-1726-9335
                Article
                10.1021/jacs.3c00243
                10214440
                37103240
                1694ac30-bfe5-4b56-8e16-5f84576d3c11
                © 2023 The Authors. Published by American Chemical Society

                Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works ( https://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 05 March 2023
                Funding
                Funded by: Morningside Foundation, doi 10.13039/100018362;
                Award ID: NA
                Funded by: Westlake Educational Foundation, doi NA;
                Award ID: NA
                Funded by: University of Hong Kong, doi 10.13039/501100003803;
                Award ID: NA
                Funded by: Research Grants Council, University Grants Committee, doi 10.13039/501100002920;
                Award ID: HKU17309018
                Funded by: Research Grants Council, University Grants Committee, doi 10.13039/501100002920;
                Award ID: AoE/P-705/16
                Categories
                Article
                Custom metadata
                ja3c00243
                ja3c00243

                Chemistry
                Chemistry

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