Intracellular Heterogeneity of Mitochondrial Membrane Potentials: A Study Using JC-1 Dye, Study notes of Cell Biology

Download Intracellular Heterogeneity of Mitochondrial Membrane Potentials: A Study Using JC-1 Dye and more Study notes Cell Biology in PDF only on Docsity! Proc. Nail. Acad. Sci. USA Vol. 88, pp. 3671-3675, May 1991 Cell Biology Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1 (fluorescence microscopy/vital dye/respiration/5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) STEPHEN T. SMILEY*t, MARTIN REERStt, CRISTINA MOTTOLA-HARTSHORN*t, MEi LIN*t, ANN CHEN*t, THOMAS W. SMITHtt, GLENN D. STEELE, JR.t§, AND LAN Bo CHEN*t *Dana-Farber Cancer Institute, tBrigham and Women's Hospital, 1New England Deaconess Hospital, and tHarvard Medical School, 44 Binney Street, Boston, MA 02115 Communicated by Ruth Sager, December 28, 1990 (received for review September 7, 1990) ABSTRACT By using a potential-dependent J-aggregate- forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1), we rind that membrane potentials across mitochondria in a living cell can be heterogeneous. Remarkably, even within a long contiguous mitochondrion, regional heterogeneity in membrane potentials appears to be possible. cl Cl Cl < s,C2H5 C2H5CI \ / N NN \ / C2H5 C2H5 Delocalized lipophilic cations such as rhodamine-123 have been used to monitor mitochondrial membrane potential in a single living cell (1-4). However, two long-standing issues have not been resolved. Do all mitochondria within one cell necessarily adopt the same membrane potentials? Is mem- brane potential maintained uniformly throughout a long sin- gle mitochondrion? It has been known for more than a half-century that some dyes form J-aggregates in certain environments (5, 6). The formation of J-aggregates is accompanied by dramatic shifts in both the absorption and fluorescence maxima of the dye. A unique feature of these dyes potentially useful for cell biological studies is that J-aggregates may form rapidly at favorable sites and display "resonance fluorescence" (7). Their large size would cause them to diffuse slowly so they may be useful as reporter molecules for localized biochemical events. Surprisingly, this remarkable phenomenon has not been purposefully exploited by biologists. Here we report that J-aggregates may serve as reporter molecules for heter- ogeneity in mitochondrial membrane potentials in living cells. MATERIALS AND METHODS Cell Culture. 3T3, NRK, BHK, CCL22, CCL64, CCL146 (ATCC), FS-2 (R. Sager, Dana-Farber Cancer Institute), CX-1 (S. Bernal, Boston University), and MCF-7 (Michigan Cancer Foundation) were grown in Dulbecco's modified Eagle's medium supplemented with 10%o (vol/vol) calf serum (iron-supplemented, HyClone) at 37°C, 5% C02/95% air, and 100%6 humidity. Spectroscopic Analysis. Uptake of 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (Po- laroid) (JC-1) by CX-1 cells was examined by a Kontron SFM25 fluorescent spectrophotometer. Cells were washed with (5 ml) and incubated in (1 ml) low-K+ buffer containing 137 mM NaCI, 3.6 mM KCI, 0.5 mM MgCl2, 1.8 mM CaCl2, 4 mM Hepes, dextrose (1 mg/ml), and 1 x mixed amino acids of modified Eagle's medium (GIBCO) at pH 7.2 (8) and then incubated in 1 ml ofJC-1 in the same buffer at concentrations specified for 10 min. Cells were then washed with three 2-ml washes and left in 1 ml of trypsin (lx, Whittaker Bioprod- FIG. 1. Chemical structure of JC-1. ucts) in low-K+ buffer for 5 min. About 0.8 ml of cell suspension was mixed with 1.2 ml oflow-K+ buffer in a 1-cm quartz cuvette for 5 min. Fluorescence scans were made by synchronously varying both the emission and excitation wavelengths with a constant differential of 15 nm. Slit widths were 10 nm. Recordings of spectra from 550 to 620 nm were repeated at a higher detector sensitivity. Fluorescence Microscopy. All cells were grown on 12-mm square glass coverslips (Bradford Scientific, Epping, NH). A stock solution of JC-1 was made at 1 mg/ml in dimethyl sulfoxide. Fresh staining solution (10 ,ug/ml) was prepared by diluting the stock solution in warm (37°C) culture medium supplemented with 10% calf serum. Fifty microliters was immediately applied to a coverslip, which was then kept in a cell culture incubator for 10 min (or as specified). (Staining solution older than 3 min should be discarded.) Cells were rinsed in warm dye-free culture medium and mounted in a living-cell chamber made of 0.7-mm-thick silicon rubber (N.A. Reiss, Belle Mead, NJ) as described (9). To dissipate plasma membrane potentials, cells were stained and main- tained in high-K+ buffer (same as low K+ buffer except 137 mM KCI and 3.6 mM NaCl), instead of culture medium. A Zeiss Axiophot microscope or a Zeiss Photomicroscope III equipped with epifluorescence optics and a 100-W mercury lamp was used. Objective lenses used included Plan Apo 40x [numerical aperture (n.a.) 1.3], Plan Apo or Neofluor 100x (n.a. 1.2). For visualizing the green fluorescence of JC-1 monomer, any filter combinations (such as Zeiss' barrier 515-565 nm, dichroic mirror FT 510 nm, and exciter 450-490 nm) used for fluorescein dye are adequate. Likewise, any rhodamine filter sets (such as Zeiss' barrier LP 590 nm, dichroic mirror FT 580 nm, and exciter BP 546/12 nm) are suitable for detecting the red fluorescence of J-aggregates. However, to visualize green and red fluorescence simulta- neously, a long-pass filter system (such as Zeiss' barrier LP 520 nm, dichroic mirror FT 510 nm, and exciter 450-490 nm) is desirable. For Figs. 2, 4, 6, and 7A, fluorescent images were recorded on Kodak Professional Ektamatic P800/1600 positive films at exposure index (E.I.) 1600 for green and yellow fluorescence and at E.I. 6400 for red fluorescence (Kodak E-6 processing at Push 1) and printed on Ilford Abbreviation: FCCP, carbonylcyanidep-trifluoromethoxyphenylhy- drazone. 3671 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 88 (1991) Cibachrome A-II papers. Fig. 7B was recorded on Kodak Ektar 1000 negative films at E.I. 1600 (Kodak C-41 process- ing) and printed on Fuji color paper with a +4 neutral-density filter. Uptake of JC-1 by Mitochondria in Vitro. Rat-liver mito- chondria were isolated as described by Johnson and Lardy (10) and Reers et al. (11). Briefly, the initial homogenization was carried out at 40C in buffer I (210 mM sucrose/70 mM mannitol/3 mM Mops-KOH, pH 7.4) supplemented with 0.2 mM EGTA*KOH and 2% (wt/vol) bovine serum albumin. The final mitochondrial fraction was suspended at a concen- tration higher than 60 mg/ml in buffer I. The respiration control ratios with glutamate/malate as substrate were above 18. Mitochondria (0.25 mg/ml) were suspended in a cuvette a 3 ml of buffer II (200 mM sucrose/20 mM mannitol/1 mM EDTA.NaOH/20 mM Mops-NaOH, pH 7.5) at 37TC. After additions of rotenone (1 mM), oligomycin (2.5 mM), JC-1 (1.7 mM), and valinomycin (20 nM), subsequent additions of a 1 M KCI solution were applied to change the membrane potential across the inner mitochondrial membrane. The cuvette was placed in a sample compartment of a Spex Fluorolog (Edison, NJ) equipped with a magnetic stirrer and a temperature controlled cuvette holder. JC-1 was excited at 575 ± 1.8 nm. The J-aggregate fluorescence was monitored at C) C 0) C.)CO) 02 ML 100 FIG. 2. Localization of JC-1 in MCF-7 cells by epifluorescence microscopy. (A) Red fluorescence under green excitation correspond- ing to J-aggregates fluorescence. (B) Green fluorescence under blue excitation corresponding to monomer fluorescence. (C) Yellow fluo- rescence under blue excitation and a long-pass filter. (Bar = 10 Am.) 480 500 520 540 560 580 600 620 Wavelength, nm FIG. 3. Fluorescent spectra of JC-1 in CX-1 cells under various conditions. Recordings of spectra from 550 to 620 nm were also repeated at a higher detector sensitivity. (A) Uptake of JC-1 in low-K+ buffer by CX-1 cells in 5 ,ug/ml (solid line), 2.5 pg/ml (dashed line), or 1.25 ,ug/ml (dotted line). (B) CX-1 cells were incubated with JC-1 (10 ,g/ml) in the presence of 5 ,uM carbonyl- cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 0.5% eth- anol (dashed line) or 0.5% ethanol (solid line). (C) CX-1 cells were first incubated with JC-1 (10l g/ml) and then treated with FCCP (5 ,uM with 0.5% ethanol, dashed line) or 0.5% ethanol (solid line). 3672 Cell Biology: Smiley et al.