ARTICLE |
Correspondence to: Hans-Peter Elsässer, Robert-Koch-Str. 5, Marburg, Germany. E-mail: elsaesse@mailer.uni-marburg.de
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Summary |
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The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids. (J Histochem Cytochem 48:251258, 2000)
Key Words: monodansylcadaverine, lysosomotropic agent, autophagic vacuole, solvent polarity probe, Stokes shift
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Introduction |
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A VARIETY OF REAGENTS have been described for in vivo labeling of acidic cellular compartments (
Like other lysosomal compartments, AVs possess an acidic pH which is generated by a V-type H+-ATPase (
The autofluorescent substance MDC is also a weak base. In addition to its ability to label AVs in vivo (
The fluorescence properties of MDC in solution are dependent on the polarity of the solvent (Narayanan and Balaram 1976). The effect of solvent polarity on fluorescence properties has been intensively investigated for 6-propionyl-2-(dimethyl-amino)naphthalene (PRODAN), which is structurally similar to MDC, but not for MDC itself (
It was assumed that the capability of MDC to label AVs in vivo depends also on an ion trap mechanism (
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Materials and Methods |
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Materials
Phosopholipon 90 was obtained from NattermannPhospholipid (Cologne, Germany). Phospholipon 90 is lecithin purified from soybean (USP XXIII) and contains 93 ± 3% phosphatidylcholine, 3 ± 3% lysophosphatidylcholine, and a minimum of 0.1%. d,I--tocopherol. The fatty acid composition is 12 ± 2% palmitic acid (16:0), 3 ± 1% stearic acid (18:0), 10 ± 3% oleic acid (18:1), 66 ± 5% linoleic acid (18:2), and 5 ± 2% linolenic acid (18:3). Acridine orange was purchased from Serva (Heidelberg, Germany). Destruxin B was prepared as described elsewhere (
Fluorescence Measurement and Scanning
All experiments were performed using the Fluorescence Photometer SFM 25 (Kontron Instruments; Zurich, Switzerland). For fluorescence measurement of MDC excitation wavelength was set to 335 nm and emission wavelength to 525 nm, unless otherwise indicated. Emission scanning was performed from 600 nm to 400 nm with an excitation wavelength of 335 nm and a scan speed of 100 nm/min. Excitation scans were performed in the same way, with emission wavelength set to 525 nm and scanning range from 450 nm to 200 nm. Amplification by high voltage setting was chosen individually in each experiment to record data within the linear scale of the photometer between relative fluorescence values (RF) of 10 and 160. Therefore, RF can differ in similar experiments.
Cell Culture Experiments
All experiments were performed using the cell line PaTu 8902, which was established from a human primary pancreatic adenocarcinoma (
Fluorescence Microscopy
Staining of PaTu 8902 cells with MDC and subsequent fixation of the cells were described elsewhere (
Preparation of Liposomes
For preparation of multilamellar vesicles, 30 mg of Phospholipon 90 was dissolved in 2 ml dichlormethane. The solution was dried under vacuum. The dried lipids were kept under vacuum for at least 45 min and then suspended in 2 ml 10 mM Tris-HCl, pH 8.0. These preliposomes were sonicated with a Labsonic U (B. Braun; Melsungen, Germany). After sonification, vesicles were centrifuged at 10,000 x g for 10 min at 4C and the supernatant was used for further investigations. The remaining supernatant was used only when a small pellet was visible and no changes in the phospholipid concentration could be observed according to
Purification and Analysis of Autophagic Vacuoles
PaTu 8902 cells were grown until confluence. Cells from eight 55-cm2 Falcon plastic dishes (BectonDickinson) were homogenized as described before (
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Results |
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In a previous study we showed by cell fractionation and ultrastructural studies that the autofluorescent substance MDC is capable of staining autophagic vacuoles in vivo (
One common and widespread hypothesis for the mechanism underlying this specific staining behavior is that an ion trapping mechanism is responsible for the accumulation of various substances in the acidic environment of lysosomal compartments (
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As mentioned above, isolated MDC-positive organelles are autophagic vacuoles (
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Similar fluorescence behavior had been shown for the structurally related solvent polarity probe 6-propionyl-2-(dimethyl-amino)naphthalene (PRODAN) in response to bovine serum albumin (BSA) (f of the solvents according to Lippert (
f values and thus polarity of a certain environment can be obtained by measuring the Stokes shift of MDC fluorescence (see below). MDC exhibited an increased relative fluorescence intensity under the influence of hydrophobic molecules as well as a Stokes shift dependent on the polarity of the solvent, characterizing the MDC molecule as a solvent polarity probe.
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These results suggest that, within the cell, a specific interaction of MDC with membrane lipids might be responsible for AV staining. To test this, we purified AVs on a sucrose step gradient. Organelles floating on 20% sucrose, containing the lysosomal marker acid phosphatase (data not shown; = 9280 cm-1;
f = 0.310), in isolated AVs (
= 8960 cm-1;
f = 0.303), and liposomes (
= 9070 cm-1;
f = 0.307). The obtained
f values are almost identical and are similar to those obtained for methanol and ethanol (Figure 3).
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Discussion |
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The term "lysosomotropic agent" was introduced by DeDuve and co-workers (1974) to designate substances that are taken up selectively into lysosomes. This definition leaves open the chemical nature of a lysosomotropic substance and the mechanism of its uptake. However, almost all agents with lysosomotropic properties based on cell permeation rather than endocytotic uptake belong to the class of weak bases. This has led to the assumption that the specific uptake of lysosomotropic substances into lysosomes depends on the acidic pH of these compartments, ion-trapping weak bases (
The autofluorescent substance MDC has been used as a lysosomotropic agent to study the function of lysosomes in different cell types (
AVs are characterized by a high content of lipids originating from membrane material regularly filling the internal space of these vacuoles as myelin-like whirls (f correlated with the Stokes shift according to the dipole interaction theory of
= 9070 cm-1) correlating with a
f of 0.307, which corresponds to
f values of ethanol. Finally, as mentioned in Results, a similar Stokes shift was also seen with isolated autophagic vacuoles and with a cell suspension stained with MDC in vivo. However, this effect could also have been induced by AV proteins. It has been shown that BSA acts similarly on PRODAN as lipids do, enhancing relative fluoresence intensity and blue-shifting the emission maximal wavelength (
= 8827 cm-1. Increase in relative fluorescence intensity depended on the amount of BSA added to a constant amount of MDC, and this was saturable with an 60-fold molar excess of BSA (Figure 2c). This finding appears to be in contradiction to data published earlier for MDC (Narayanan and Balaram 1976), in which an interaction of MDC with BSA could not be observed. However, in this report 60 µM MDC was incubated with only 3 µM BSA, a concentration that also gave no detectable increase in relative fluorescence intensity in our experiments (Figure 2c). The BSA effect occurred only at a 50-fold higher molar concentration than the effect lipids have on MDC fluorescence properties. Furthermore, isolated AV lipids had the same effect on Stokes shift and increased relative fluorescence intensity as entire isolated AVs (Figure 4a), indicating that AV proteins are not responsible for altering the fluorescent properties of MDC. Whether the observed effect is due exclusively to the interaction of lipid molecules with MDC or whether there is also a concentration effect of MDC in the lipid phase cannot be answered by the data presented. The partition coefficient for MDC between an aqueous phase and octanol was found to be 1:1 (not shown), which does not imply a higher affinity of MDC for a hydrophobic environment. However, because the lipids of the AVs are organized in regular bilayers with defined hydrophobic and hydrophilic domains, it is possible that the partition of a substance between two phases of opposite hydrophobicity does not properly reflect the interaction of a molecule with a lipid bilayer.
The question remains of why MDC stains only AVs and not the plasma membrane or other endomembranes. One reason could be the high concentration of membrane material in these organelles in which MDC molecules might be concentrated and its relative fluorescence intensity is increased in a small volume, so that the sensitivity of a fluorescent microscope is sufficient for MDC detection, whereas in other membranes the amount of emitted fluorescent light is below the threshold of fluorescent microscopic sensitivity. When cells were fractionated after MDC incubation, a weak fluorescent label could also be detected by the more sensitive fluorescent spectrophotometry in membranes other than in AVs (not shown).
Alternatively, the lipid composition of AV membranes or the presence of one AV-specific lipid with a high affinity for MDC binding could also account for the specific in vivo MDC staining of AVs. It has recently been shown that lysobisphosphatidic acid (LBPA), the antigen of the autoimmune disease antiphospholipid syndrome (
In conclusion, we provide evidence that the lysosomotropic agent monodansylcadaverine (MDC), which preferentially labels autophagic vacuoles, is not exclusively effective as a lysosomotropic agent according to an ion trapping mechanism that depends on the proton gradient between the cytosol and the lysosomal compartment. Instead, MDC, which might be effectively incorporated into at least some cell membranes because of its amphipathic structure, also functions as a solvent polarity probe, exhibiting a Stokes shift and an increased relative fluorescence intensity in a hydrophobic environment. This property depends on the interaction of MDC with lipid molecules. Whether the specificity of MDC staining of AVs depends only on the high content of lipids in these organelles, or whether MDC can interact specifically with lipid molecules unique for AVs, remains to be elucidated.
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Acknowledgments |
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Supported by Studienstiftung des Deutschen Volkes and by Deutsche Forschungsgemeinschaft grant EL 125/2-1.2.
We thank Dagmar Fischer for the introduction to liposome preparation and for providing Phospholipon 90. We thank Ursula Lehr for expert technical assistance, Volkwin Kramer for preparation of the microscopic reprints, and Annette Biederbick, Horst Franz Kern, and Roland Lill for critical comments and helpful discussions.
Received for publication April 9, 1999; accepted September 8, 1999.
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