Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death

11 pags, 6 figsBackground: The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail. Methods: We use optical techniques, including fluorescence confocal microscopy and lifetime imaging microscopy (FLIM) both in model membranes built up as giant unilamellar vesicles (GUVs) and cultured cells. These experiments are complemented with computational studies to unravel the molecular mechanism that makes NAO cytotoxic. Results: We have obtained direct evidence that NAO promotes strong membrane adhesion of negatively charged vesicles. The attractive forces are derived from van der Waals interactions between anti-parallel H-dimers of NAO molecules from opposing bilayers. Semi-empirical calculations have confirmed the supramolecular scenario by which anti-parallel NAO molecules form a zipper of bonds at the contact region. The membrane remodeling effect of NAO, as well as the formation of H-dimers, was also confirmed in cultured fibroblasts, as shown by the ultrastructure alteration of the mitochondrial cristae. Conclusions: We conclude that membrane adhesion induced by NAO stacking accounts for the supramolecular basis of its cytotoxicity. General significance: Mitochondria are a potential target for cancer and gene therapies. The alteration of the mitochondrial structure by membrane remodeling agents able to form supramolecular assemblies via adhesion properties could be envisaged as a new therapeutic strategy.This work was supported by the ERC Starting Grant “mitochon” (ERC-StG-2013 338133) and the ERC Proof of Concept “mitozippers” (ERC-PoC-2017 780440), FIS2015-70339-C2-1-R from MINECO (I. L-M.and F.M.), FIS2015-70339-C2-2-R (M.P.L. and C.G.) and S2013/MIT-2807 from the Madrid Regional Government (F.M. and A. G-M.)

Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts

Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.Peer Reviewe

Enhanced Cytotoxic Activity of Mitochondrial Mechanical Effectors in Human Lung Carcinoma H520 Cells: Pharmaceutical Implications for Cancer Therapy

8 pags, 5 figsCancer cell mitochondria represent an attractive target for oncological treatment as they have unique hallmarks that differ from their healthy counterparts, as the presence of a stronger membrane potential that can be exploited to specifically accumulate cytotoxic cationic molecules. Here, we explore the selective cytotoxic effect of 10-N-nonyl acridine orange (NAO) on human lung carcinoma H520 cells and compare them with healthy human lung primary fibroblasts. NAO is a lipophilic and positively charged molecule that promotes mitochondrial membrane adhesion that eventually leads to apoptosis when incubated at high micromolar concentration. We found an enhanced cytotoxicity of NAO in H520 cancer cells. By means Fluorescence lifetime imaging microscopy (FLIM) we also confirmed the formation of H-dimeric aggregates originating fromopposing adjacent membranes that interfere with the mitochondrial membrane structure. Based on our results, we suggest the mitochondrial membrane as a potential target in cancer therapy to mechanically control the cell proliferation of cancer cells.This work was supported by the ERC Starting Grant MITOCHON (ERC-StG-2013-338133), ERC Proof of Concept mitozippers (ERC-PoC-2017-780440) and FIS2015-70339-C2-1-R from the Spanish Ministry of Economy MINECO (IL-M); and FIS2015-70339-C2-2-R (MPL and CG)

Self-assembly of 10-N-nonyl acridine orange (NAO) creates molecular zippers promoting cell death by adhesion of membranes

Joint 12th EBSA congress / 10th ICBP – IUPAP congress: Biophysics for life and technology, Madrid, Spain, July 20-24, 2019. -- http://www.ebsa2019.org/ . -- European Biophysics Journal (2019

Polar ammoniostyryls easily converting a clickable lipophilic BODIPY in an advanced plasma membrane probe

7 pags., 5 figs.A very simple, small and symmetric, but highly bright, photostable and functionalizable molecular probe for plasma membrane (PM) has been developed from an accessible, lipophilic and clickable organic dye based on BODIPY. To this aim, two lateral polar ammoniostyryl groups were easily linked to increase the amphiphilicity of the probe and thus its lipid membrane partitioning. Compared to the BODIPY precursor, the transversal diffusion across lipid bilayers of the ammoniostyryled BODIPY probe was highly reduced, as evidenced by fluorescence confocal microscopy on model membranes built up as giant unilamellar vesicles (GUVs). Moreover, the ammoniostyryl groups endow the new BODIPY probe with the ability to optically work (excitation and emission) in the bioimaging-useful red region, as shown by staining of the plasma membrane of living mouse embryonic fibroblasts (MEFs). Upon incubation, this fluorescent probe rapidly entered the cell through the endosomal pathway. By blocking the endocytic trafficking at 4 °C, the probe was confined within the PM of MEFs. Our experiments show the developed ammoniostyrylated BODIPY as a suitable PM fluorescent probe, and confirm the synthetic approach for advancing PM probes, imaging and science.Financial support from MICINN (PID2020-114755GB-C31, -C32 and C-33) and Basque Government (IT1639-22) is gratefully acknowledged by S. d. l. M., J. B. and I. G.-M. I. L.-M. acknowledge financial support from the Spanish Ministry of Science, Innovation and Universities through the grant PGC2018-097903-B-I00. This work was also supported by the TECNOLOGI´AS 2018 program, funded by the Regional Government of Madrid (Grant S2018/BAA-4403 SINOXPHOS-CM, to I. L-M.). S. S.-B. thanks the Regional Government of Madrid for an INVESTIGO contract. M. M.-U´. was recipient of a Sara Borrell fellowship (CD15/00190) financed by the Spanish Ministry of Health