Journal: Journal of Lipid Research

Article Title: Cadmium-cardiolipin disruption of respirasome assembly and redox balance through mitochondrial membrane rigidification

doi: 10.1016/j.jlr.2025.100750

Figure Lengend Snippet: Alterations in fluidity of isolated cellular membranes by cadmium. A: Depiction of membrane fluidity-reporting fluorophores (laurdan, diphenylhexatriene [DPH], di-4-ANEPPDHQ) interaction with a model membrane (not to scale). Human proximal tubule (HPCT) cells were exposed to 2.5 μM CdCl 2 (Cd) or 0.1 mM NiCl 2 (Ni) for 6 h in serum free medium (SFM). Plasma membranes (B), lysosomes (C), and mitochondria (D) were isolated as described in , loaded with 1:550 laurdan:lipid, incubated overnight at 37°C and measured at λ ex /λ em 340/440 nm and 340/490 nm. Laurdan generalized polarization (GP) was determined, where increased GP indicates liquid-ordered membranes and decreased GP indicates membrane fluidization. Data were measured in technical triplicate from at least two independent isolations. E: Quality control of mitoplast preparations from rat kidney cortex mitochondria (rKC mito ) as depicted by loss of outer mitochondrial membrane protein (OMM) VDAC and retention of inner mitochondrial membrane (IMM) cytochrome c (cyt. c ). Immunoblot is representative of 4 independent experiments. Mitoplasts were exposed to Cd for 1 h and loaded with laurdan (F) or DPH (G). Increased DPH anisotropy indicates membrane rigidification. Data were generated in technical triplicate from 6 to 8 independent mitoplast preparations.

Article Snippet: ANEPP-loaded HPCT cells in live-cell imaging chambers (ibidi GmbH) were spectral imaged (λ em 490-750 nm) with λ ex 488 nm using a Nikon A1R+ inverted confocal microscope equipped with epi-fluorescence Ti2 illuminator, high-resolution Galvano scanner, CFI Plan Apochromat Lambda 60x oil objective, and collected using high sensitivity photomultiplier tube detectors.

Techniques: Isolation, Membrane, Clinical Proteomics, Incubation, Control, Western Blot, Generated

Journal: Journal of Lipid Research

Article Title: Cadmium-cardiolipin disruption of respirasome assembly and redox balance through mitochondrial membrane rigidification

doi: 10.1016/j.jlr.2025.100750

Figure Lengend Snippet: Cadmium augments mitochondrial membrane rigidity in HPCT cells and CL nanoliposomes. After exposure to CdCl 2 for 1 h, HPCT cells were loaded for 15 min at 37°C with 1 μM laurdan (A) or 50 nM di-4-ANEPPDHQ (C) and imaged after washing. A: Laurdan-loaded cells were imaged at λ ex /λ em 340/440 nm and 340/490 nm using a multiphoton microscope. Elevated membrane rigidity is indicated by increased λ em 440 nm and decreased λ em 490 nm that was observed in intracellular vesicles (arrows) and in circular membrane structures (arrowheads). Scale bar = 20 μm. B: Quantification of single-cell laurdan GP from 90 (control), 68 (0.5 μM Cd), and 139 (1 μM Cd) cells from three independent experiments. C: Cells loaded with di-4-ANEPPDHQ were spectral imaged on a laser-scanning confocal microscope with a 488 nm excitation laser. A spectral shift occurs when di-4-ANEPPDHQ distributes to liquid-ordered (λ em 570 nm) or to liquid-disordered (λ em 620 nm) lipid domains. Plasma membrane (arrows) and mitochondria (arrowheads) are indicated in representative images. Scale bar = 20 μm. D and E: Fluorescent intensities from eight images per condition containing 339 (control) and 417 (Cd) cells were analyzed from two independent experiments and divided by cell number. F: Ratios of 570–/620 nm using the values obtained in (D and E) are plotted (n = 8 images). G: Fluorescence signals at λ em 620 nm were quantified in vesicular structures, plasma membrane (PM) and mitochondria of control and Cd-treated HPCT cells. At least 70 regions per condition in 40–60 cells from 4 independent images were used. H: Structures of tetraoleoyl-cardiolipin (TOCL) were drawn using ChemDraw. (left) Cadmium (Cd 2+ ) electrostatic interaction with each phosphate headgroup (monodentate binding) neutralizes negative charges dissipating charge repulsion. (right) Putative bidentate binding of Cd 2+ with both phosphate headgroups. Drawings are not to scale. I: Phase transition of pure TMCL nanoliposomes with laurdan. Nanoliposomes preloaded with laurdan were measured before and 5 min after Cd addition. J: Comparison of laurdan GP at CL phase transition temperature before and after Cd addition. Measurements were performed in triplicate from two independent nanoliposome preparations.

Article Snippet: ANEPP-loaded HPCT cells in live-cell imaging chambers (ibidi GmbH) were spectral imaged (λ em 490-750 nm) with λ ex 488 nm using a Nikon A1R+ inverted confocal microscope equipped with epi-fluorescence Ti2 illuminator, high-resolution Galvano scanner, CFI Plan Apochromat Lambda 60x oil objective, and collected using high sensitivity photomultiplier tube detectors.

Techniques: Membrane, Microscopy, Control, Clinical Proteomics, Fluorescence, Binding Assay, Sublimation, Comparison

Journal: Journal of Lipid Research

Article Title: Cadmium-cardiolipin disruption of respirasome assembly and redox balance through mitochondrial membrane rigidification

doi: 10.1016/j.jlr.2025.100750

Figure Lengend Snippet: Ablation of cadmium-perturbed mitochondrial respiratory supercomplexes assembly by MTP-131, Human proximal tubule (HPCT) cells (A, B) or isolated crude rat kidney mitochondria (rKC mito ) (C) were exposed to 1, 2.5 and 5 μM CdCl 2 (Cd) for 6 h in SFM or for 1 h in MSH buffer, respectively. Cell pellets and isolated organelles were processed as described in and separated by BN-PAGE using total monomeric complex IV to control for loading. Immunoblotting (IB) was used to visualize the mitochondrial supercomplexes (SCs). Representative immunoblots from n = 6 in (A) are shown. B: Densitometry analysis of complex I (CI), complex III (CIII) and complex IV (CIV) signals corrected for loading and normalized to control show a significant reduction of fully assembled SCs (n = 6). C: Representative BN-PAGE followed by IB from n = 3 of isolated rKC mito . Relative optical densities normalized to control show a reduction of fully assembled SCs following Cd exposure. D: The antioxidant α-tocopherol (α-toco; 50 μM) was preincubated for 1 h prior to 6 h Cd treatment. SC formation was assessed by BN-PAGE and IB. Representative immunoblots from n = 4 are shown. E: Densitometry analysis of IBs in (D) show no significant impact of α-tocopherol on SC loss by Cd. F: Laurdan GP in nanoliposomes (100 nm diameter) comprised of 40 mol % POPC, 35 mol % DOPE, 20 mol % TOCL and 5 mol % SAPI to mimic the IMM lipid composition. Laurdan was added after Cd addition. Means ± SD from replicate measurements from two independent nanoliposomes preparations are shown. G: At physiological 37°C, laurdan GP and membrane rigidification is increased by Cd (n = 6–9). H: HPCT cells were pretreated with 0.1 μM MTP-131 for 1 h and exposed to 5 μM Cd for 6 h. Cell pellets were subjected to BN-PAGE followed by IB to visualize the SCs. A representative experiment from n = 4–5 is shown. I: Quantitative analysis of band optical density normalized to control shows protection by MTP-131 pretreatment (n = 4–5). J: Cd exposure of HPCT cells significantly reduces the ATP concentration only at 5 μM after 24 h (n = 4). K: Pretreatment with 0.1 μM MTP-131 has no effect on ATP concentrations by 24 h Cd (n = 6).

Article Snippet: ANEPP-loaded HPCT cells in live-cell imaging chambers (ibidi GmbH) were spectral imaged (λ em 490-750 nm) with λ ex 488 nm using a Nikon A1R+ inverted confocal microscope equipped with epi-fluorescence Ti2 illuminator, high-resolution Galvano scanner, CFI Plan Apochromat Lambda 60x oil objective, and collected using high sensitivity photomultiplier tube detectors.

Techniques: Isolation, Control, Western Blot, Membrane, Concentration Assay

Journal: Journal of Lipid Research

Article Title: Cadmium-cardiolipin disruption of respirasome assembly and redox balance through mitochondrial membrane rigidification

doi: 10.1016/j.jlr.2025.100750

Figure Lengend Snippet: MTP-131 abolishes elevated mitochondrial H 2 O 2 by cadmium, Amplex UltraRed fluorescence of succinate-energized crude rKC mito suspended in iso-osmotic MSH buffer was monitored at λ ex /λ em 545/590 nm. A: Cd (1, 2.5, or 5 μM) was added 5 min after mitochondria were energized by 5 mM Na-succinate (succ). B: MTP-131 (0.1 μM) was preincubated for 15 min at RT. Representative experiments from n = 5 are shown in ( A ) and ( B ). C: Pretreatment with MTP-131 significantly reduced the rates of H 2 O 2 release, determined by the slope of normalized fluorescence intensities during the first 15 min after Cd addition (n = 5). D: HPCT cells exposed to 5 μM Cd for 3 h in SFM show a significant reduction of ROS formation measured by fluorescence intensities of oxidized rhodamine 123 + (Rh123 + ) at λ ex /λ em 485/535 nm (n = 4).

Article Snippet: ANEPP-loaded HPCT cells in live-cell imaging chambers (ibidi GmbH) were spectral imaged (λ em 490-750 nm) with λ ex 488 nm using a Nikon A1R+ inverted confocal microscope equipped with epi-fluorescence Ti2 illuminator, high-resolution Galvano scanner, CFI Plan Apochromat Lambda 60x oil objective, and collected using high sensitivity photomultiplier tube detectors.

Techniques: Fluorescence

Journal: Journal of Lipid Research

Article Title: Cadmium-cardiolipin disruption of respirasome assembly and redox balance through mitochondrial membrane rigidification

doi: 10.1016/j.jlr.2025.100750

Figure Lengend Snippet: Expression of cardiolipin synthase 1 (CRLS1) rescues cadmium cytotoxicity. A: CRLS1 mRNA abundancy after transient transfection of HPCT cells for 24 h with 0.5 μg Myc-DDK-hCRLS1 plasmid or pCMV6-entry empty vector (EV) was assessed by qPCR (n = 6). B: Overexpression of Myc-DDK-tagged CRLS1 protein was confirmed by immunoblotting of whole-cell lysates with anti-DDK antibody. Transcript variants 1 ( arrow ) and 2 ( arrowhead ) are seen. Anti-GAPDH antibody signal was used as a loading control. C: Analysis of CL extracted from whole cell HPCT pellets (50–100 μg protein) by LC-MS/MS confirmed functional CRLS1 in Myc-DDK-hCRLS1 transfected cells (n = 3). D: Viability of hCRLS1-HPCT cells exposed to 5 μM Cd for 24 h was determined by MTT assay relative to transfected untreated controls (n = 4). E: reversal and reduction of ROS formation by 5 μM Cd for 3 h in hCRLS1-transfected HPCT cells measured by oxidation of DHR123 to Rh123 + at λ ex /λ em 485/535 nm (n = 3) as compared to Cd-treated EV cells. F: HPCT cells were transiently transfected with 0.25 μg or 0.5 μg Myc-DDK-hCRLS1 plasmid for 24 h. After 1 μM Cd exposure for 1 h, cells were loaded with di-4-ANEPPDHQ and imaged at λ ex /λ em 485/550 nm, which detects liquid-ordered domains. Fluorescence intensity from >50 selected regions of mitochondria were analyzed from 50 cells per condition and two independent experiments.

Article Snippet: ANEPP-loaded HPCT cells in live-cell imaging chambers (ibidi GmbH) were spectral imaged (λ em 490-750 nm) with λ ex 488 nm using a Nikon A1R+ inverted confocal microscope equipped with epi-fluorescence Ti2 illuminator, high-resolution Galvano scanner, CFI Plan Apochromat Lambda 60x oil objective, and collected using high sensitivity photomultiplier tube detectors.

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Western Blot, Control, Liquid Chromatography with Mass Spectroscopy, Functional Assay, MTT Assay, Fluorescence

Journal: STAR Protocols

Article Title: In vitro cyst puncture and injury-induced tubule formation using renal epithelial cells

doi: 10.1016/j.xpro.2022.101874

Figure Lengend Snippet:

Article Snippet: Place the dish with the injected cysts into the live cell imaging chamber of the Keyence microscope.

Techniques: Recombinant, Modification, Software, Microscopy