RAPID COMMUNICATION |
Correspondence to: David J. Vaux, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mitochondrial matrix contains endogenously biotinylated proteins. These proteins can cause unexpected background signal when biotin-avidin- or biotin-streptavidin-based detection systems are used in immunocytochemistry. Here we show that this reactivity can be deliberately exploited, using a simple anti-biotin reagent, to obtain strong and highly specific labeling of mitochondria by both light and electron microscopy. The signal is substantially stronger than when either avidin or streptavidin is used to detect the endogenous biotin. These results confirm the accessibility of protein-bound endogenous biotin to exogenous probes, and localize the biotinylated enzymes to the mitochondrial matrix. (J Histochem Cytochem 45:1053-1057, 1997)
Key Words: organelle-specific markers, rat kidney, confocal microscopy, electron microscopy, colloidal gold immunocytochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biotin-dependent carboxylases in mammalian cells are important regulators of lipogenesis and gluconeogenesis and are required for amino acid catabolism (
The presence of these endogenously biotinylated proteins in mammalian cells can lead to unexpected background problems when the biotin-avidin system is used in immunocytochemistry, e.g., for detection of in situ hybridization of biotinylated probes (
Here we describe the optimization of a system to exploit the presence of high concentrations of biotin-dependent carboxylases within mitochondria to develop a simple, strong, and highly organelle-specific labeling protocol for these organelles, which is equally effective at the light or EM level.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Normal rat kidney (NRK) cells were grown as an adherent cell line on glass coverslips in tissue culture dishes in DMEM with 10% (v/v) fetal calf serum at 37C in a 5% carbon dioxide atmosphere.
For vital labeling of the mitochondria, coverslips bearing subconfluent NRK monolayers were incubated for 60 min at 37C in normal growth medium containing 5 µg/ml of freshly prepared Mitotracker Green FM (M7514; Molecular Probes, Eugene, OR). The Mitotracker Green was prepared as a 1 mg/ml stock in dimethylsulfoxide and was diluted 1~200 in growth medium immediately before use. Labeled cells were rinsed twice in PBS and fixed in methanol at -20C for 5 min. The fixed coverslips were rinsed again in PBS and mounted in Moviol (
For detection of biotin, NRK cells were grown, fixed, and washed as above. For biotin detection with avidin, the coverslips were incubated in fluorescein-conjugated avidin (Vector Labs; Burlingame, CA) at a dilution of 1~100 of a 0.5 mg/ml stock in PBS at room temperature (RT) for 20 min. The coverslips were washed twice with PBS and mounted in Moviol. For streptavidin labeling the protocol was identical, but used 1~100 of a 0.5 mg/ml stock of Texas Red-conjugated streptavidin (Zymed Laboratories; San Francisco, CA).
For indirect immunodetection of biotin, coverslips were prepared as above and blocked in a solution of 0.2% (w/v) porcine gelatin in PBS for 5 min and then incubated with a monoclonal mouse anti-biotin antibody (200-002-096; Jackson ImmunoResearch Labs, West Grove, PA) diluted 1~100 in the blocking solution for 20 min at RT. After washing in PBS, the coverslips were incubated for 20 min in a 1~300 dilution of rhodamine-conjugated goat anti-mouse IgG (Cappell, distributed by Organon Teknika, Turnhout, Belgium) in blocking solution before washing in PBS and mounting in Moviol.
The tissue sections were obtained from normal rat kidney fixed in 4% (w/v) paraformaldehyde (PFA) in 250 mM HEPES buffer, pH 7.4, for 10 min on ice, followed by 50 min at RT in 8% PFA in the same buffer. After rinsing in PBS, the tissue blocks were cryoprotected in 2.1 M sucrose and frozen in liquid nitrogen. Sections were cut at approximately 100 nm thickness using a Reichert Ultracut S with the FCS attachment and collected onto gelatin-coated glass slides (for confocal microscopy) or Formvar-coated, glow-discharged EM grids (for electron microscopy). Tissue sections on glass slides were labeled using the monoclonal anti-biotin antibody exactly as described above.
All fluorescently labeled specimens were examined using a BioRad MRC 1024 confocal laser scanning microscope as described (
For electron microscopic immunocytochemistry, sections on grids were washed on droplets of PBS, blocked with 10% (v/v) fetal calf serum in PBS for 10 min, and incubated with the anti-biotin antibody at 1~10 dilution of the stock in 5% (v/v) fetal calf serum in PBS for 30 min. After washing the grids in PBS, they were floated for 20 min on drops of rabbit anti-mouse IgG (Cappell) diluted 1~50. The grids were then washed in PBS and incubated for 20 min with a 1~100 dilution of 9-nm protein A-gold stock (
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mitochondrial morphology in normal tissues and in tissue culture cell lines can be extremely complex. In many cell types the mitochondria form a dynamic branched network, constantly breaking and re-forming. The behavior of mitochondria in living cells can be demonstrated using vital dyes that are sequestered by active mitochondria in a membrane potential-dependent manner. These probes include rhodamine 123 (
Figure 1A shows that the Mitotracker probe Mitotracker Green FM decorates a complex, branched, reticular mitochondrial mass in methanol-fixed NRK cells. The dye gives a strong specific mitochondrial stain in living NRK cells (data not shown), and this staining pattern is not visibly altered when the cells are fixed in methanol at -20C.
|
Figure 1C illustrates the use of a fluorochrome-conjugated avidin to label NRK cells to which no bio-tinylated probe has been applied. Despite this lack of exogenous biotin, a strong labeling pattern is visible. The pattern is very reminiscent of the mitochondrial distribution in these cells (Figure 1A). Avidin is a 66-kD glycoprotein with a positive charge at physiological pH. Both its oligosaccharide moiety and its charge have been implicated in nonspecific background binding in indirect biotin-dependent detection systems (
To overcome the charge and oligosaccharide problems associated with avidin, it is possible to detect biotin with streptavidin, a nonglycosylated bacterial product of 60 kD with a near-neutral isolectric point and consequently very little net charge at physiological pH. Figure 1D illustrates the result when methanol-fixed NRK cells are incubated with a directly fluorochrome-conjugated streptavidin. Once again, no bio-tinylated probe has been used, and yet both the avidin and streptavidin probes are labeling similar widespread branched, tubular structures in the cytoplasm. In the case of the streptavidin there is an additional nuclear background.
The reason for this endogenous background reactivity with both avidin and streptavidin conjugates becomes apparent when an anti-biotin monoclonal antibody is used to display the endogenous biotin distribution in NRK cells (Figure 1B). A complex, branched tubular network of structures containing high levels of endogenous biotin is clearly labeled. Comparison of a specific mitochondrial probe (Figure 1A) with the anti-biotin labeling (Figure 1B) confirms that the high levels of biotinylated carboxylase enzymes within mitochondria make this organelle an excellent substrate for anti-biotin labeling. The anti-biotin labeling shown in Figure 1B is completely ablated if the monoclonal antibody is preincubated with excess free biotin, or if the specimen is preincubated with excess unconjugated streptavidin to block the endogenous biotin (data not shown), confirming that the mitochondrial staining by the monoclonal anti-biotin is indeed via endogenous biotin.
Figure 2A shows that the same anti-biotin labeling can be used to identify the mitochondria in sections of normal rat kidney that have been aldehyde-fixed and cryosectioned. Strong labeling of discrete cytoplasmic structures is seen in the cells lining the walls of the two tubules present in the section. The identification of these structures as mitochondria is confirmed by electron microscopic immunocytochemistry using immunogold labeling (Figure 2B). Prominent gold labeling is found within the mitochondria; the majority of the gold particles lie over the mitochondrial matrix. In some places an alignment of gold particles along the inner membrane and cristae is visible. These results are compatible with biochemical fractionation studies localizing these enzymes to the mitochondrial matrix compartment (
|
In this report we have demonstrated that avidin and streptavidin share the ability to recognize the endogenously biotinylated enzymes of the mitochondrion. This may give rise to a serious background problem and to false interpretation of the labeling produced by exogenous biotinylated probes if it is not taken into account. The problem has been recognized in nucleic acid hybridization experiments, and digoxigenin probes may be preferred over biotinylated probes for this reason (
Finally, we have shown that the background caused by endogenous biotin can easily be exploited to achieve highly specific mitochondrial labeling at both the light and the electron microscopic level. In this case, optimal labeling is achieved not with avidin or streptavidin but by using a monoclonal anti-biotin antibody to detect the biotinylated mitochondrial enzymes.
![]() |
Acknowledgments |
---|
Supported by a project grant from the Medical Research Council to DJV.
We are grateful to members of the laboratory for useful discussions and to Lance Tomlinson for excellent photographic assistance.
Received for publication March 31, 1997; accepted April 29, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fricker M, Hollinshead M, White NJ, Vaux DJ (1997) Interphase nuclei of many mammalian cell types contain deep, dynamic, tubular membrane-bound invaginations of the nuclear envelope. J Cell Biol 136:531-544
Grouselle M, Tueux O, Dabadie P, Georgescaud D, Mazat JP (1990) Effect of local anaesthetics on mitochondrial membrane potential in living cells. Biochem J 271:269-272 [Medline]
Hector ML, Cochran BC, Logue ER, Fall RR (1980) Subcellular localisation of 3-methyl-crotonyl coenzyme A carboxylase in bovine kidney. Arch Biochem Biophys 199:28-33 [Medline]
Kirkeby S, Moe D, BogHansen TC, Van Noorden CJF (1993) Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding. Histochemistry 100:415-421 [Medline]
LeonDelRio A, Leclerc D, Akerman B, Wakamatsu N, Gravel RA (1995) Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli. Proc Natl Acad Sci USA 92:4626-4630 [Abstract]
Longin A, Souchier C, Ffrench M, Bryon PA (1993) Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study. J Histochem Cytochem 41:1833-1840
Poot M, Zhang YZ, Kramer JA, Wells KS, Jones LJ, Hanzel DK, Lugade AG, Singer VL, Haugland RP (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem 44:1363-1372 [Abstract]
Reers M, Smith TW, Chen LB (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30:4480-4486 [Medline]
Satoh S, Tatsumi H, Suzuki K, Taniguchi N (1992) Distribution of manganese superoxide dismutase in rat stomach: application of Triton X-100 and suppression of endogenous streptavidin binding activity. J Histochem Cytochem 40:1157-1163
Slot JW, Geuze HJ (1985) A new method of preparing gold probes for multiple labelling cytochemistry. Eur J Cell Biol 38:87-93 [Medline]
Tokuyasu KT (1980) Immunocytochemistry in ultra-thin frozen sections. Histochem J 12:381-403 [Medline]
Varma VA, Cerjan CM, Abbott KL, Hunter SB (1994) Non-isotopic in situ hybridization method for mitochondria in oncocytes. J Histochem Cytochem 42:273-276
Wolf B (1995) Disorders of biotin metabolism. In Scriver CR, Beaudet AL, Sly WS, Valle D, eds. Metabolic and Molecular Basis of Inherited Disease. 7th Ed. New York, McGraw-Hill, 3151-3177
Wood GS, Warnke R (1981) Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems. J Histochem Cytochem 29:1196-1204 [Abstract]
Yi J, Michel O, SassyPrigent C, Chevalier J (1995) Electron microscopic location of mRNA in the rat kidney: improved post-embedding in situ hybridization. J Histochem Cytochem 43:801-809