ARTICLE |
Correspondence to: Hans-Peter Elsässer, Dept. of Cell Biology, Robert-Koch-Str. 5, University of Marburg, 35033 Marburg, Germany. E-mail: elsaesse@mailer.uni-marburg.de
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Summary |
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We have recently shown that monodansylcadaverine labels autophagic vacuoles. Analysis of the mechanism underlying the labeling revealed that monodansylcadaverine acts as a lysosomotropic agent, being concentrated into acidic compartments by an ion-trapping mechanism, and as a solvent polarity probe, increasing its relative fluorescence intensity by interacting with membrane lipids that are highly concentrated in the autophagic vacuoles. In this study, we synthesized three structurally related derivatives of monodansylcadaverine, replacing the primary amino group of monodansylcadaverine with a neutral (dansylamylamine; MDH), a polar (dansylaminopentanol; MDOH), or an acidic group (dansylaminovaleric acid; MDA), to replace the lysosomotropic character of the marker. Whereas MDH showed a specific staining of autophagic vacuoles, the polar and acidic derivatives did not show any staining. We further demonstrate that the MDH staining of autophagic vacuoles is independent on the acidic pH and thus on an ion-trapping mechanism, but it still shows the same preferences for autophagic membrane lipids as monodansylcadaverine. We propose that MDH can specifically interact with lamellar bodies of the autophagic type as a solvent polarity probe. Therefore, dansylated aminopentane can be used as a specific marker for autophagic vacuoles in vivo and in fixed cells.
(J Histochem Cytochem 49:177185, 2001)
Key Words: monodansylpentane, monodansylcadaverine, lysosomal marker, autophagic vacuole, multilamellar body, solvent polarity probe
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Introduction |
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MONODANSYLCADAVERINE (MDC) has been described as an in vivo marker of autophagic/lysosomal vacuoles (
In addition to autophagic vacuoles, several other intracellular vacuoles containing high amounts of lipids occur in different cell types. These organelles are called lamellar bodies because the lipids are mostly organized in lamellar whirl-like sheets, as are observed in autophagic vacuoles (
Because MDC associates with acidic and lipid-rich compartments, it is unlikely that this substance can clearly discriminate among lysosomes, autophagic vacuoles, and lamellar bodies. The aim of this study was to modify the MDC molecule so as to skip the lysosomotropic property but to preserve the function of a solvent polarity probe. To achieve this, we replaced the primary amino group of monodansylcadaverine with a neutral (dansylamylamine; MDH), a polar (dansylaminopentanol; MDOH), or an acidic group (dansylaminovaleric acid; MDA). We show that only MDH stains vacuolar structures that are autophagic vacuoles, that this staining occurs in vivo and in vitro, and that it is independent of pH gradients. Therefore, we propose that MDH is a specific marker for autophagic vacuoles and probably for lamellar bodies in general.
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Materials and Methods |
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Materials
Pentamines were purchased from Aldrich (Steinheim, Germany). All other chemicals used were obtained from Sigma (Diesenhofen, Germany).
Staining, Synthesis, and Characterization of Dansylated Pentamines
Dansylation of different pentamines was performed according to
All experiments were performed using the cell line PaTu 8902, which was established from a human primary pancreatic adenocarcinoma (
Fluorescence Microscopy
Staining and fixation of PaTu 8902 cells using MDC in a final concentration of 0.1 mM were described elsewhere (
Subcellular Fractionation of MDC/MDH-labeled Compartments
Fractionation was performed as described elsewhere (
Purification of Autophagic Vacuoles
Purification of autophagic vacuoles was performed as described previously (
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Results |
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The autofluorescent substance MDC specifically labels autophagic vacuoles (AVs) (
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To analyze the contribution of the primary amino group to these staining properties of MDC, we substituted this amino group with a hydrogen atom (dansylamylamine; MDH), a hydroxyl group (dansylaminopentanol; MDOH), or a carboxylic acid group (dansylaminovaleric acid; MDA). Synthesis was performed as described in Materials and Methods. Purity was determined by TLC. Plates were analyzed using a LCD video camera under UV light (Fig 2). The purity of the dansylated pentamines was determined by comparing the RF on the plate of dansyl hydroxide (unreacted dansyl chloride) with the RF of the dansylated derivative. Whereas the MDH and MDOH reaction led to a 98% pure product, MDA showed 86% purity (see Fig 2).
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Morphologically, MDH revealed in vivo fluorescence staining of AVs similar to that with MDC (Fig 3a and Fig 3b). In contrast, MDOH exhibited only weak and blurred vacuolar staining, which disappeared quickly when cells were washed (Fig 3c). MDA did not show any staining of vacuolar structures (Fig 3d). Finally, as a control, we stained the cells for 1 hr with 0.1 mM dansyl hydroxide. Even at this high concentration, dansyl chloride showed only weak, blurred staining of the cytoplasm (data not shown). Therefore, an effect of the dansyl hydroxide contamination on the synthesized dansyl derivatives can be excluded. Because only MDH revealed a staining pattern comparable to MDC, we further concentrated on the comparison of MDC and MDH.
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To further confirm the interaction of MDH with AVs, we used isolated AVs from cells that had not been incubated with either MDC or MDH. As previously shown for MDC (
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In the next experiment, we examined the influence of the length of the MDH acyl side chain. We synthesized derivatives of dansylpentamine, changing the length of the polycarbon chain from C2 to C8. Although morphologically all derivatives showed a staining pattern of the same quality as MDC or MDH (equal to the C5 derivative), quantitation of the fluorochrome uptake revealed that the acyl chain length is crucial for uptake of the dansyl derivatives, with an acyl chain of n = 5 (MDH) being superior to all other derivatives (Fig 5).
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To test the pH dependence of the accumulation of MDH within the cells, we measured the uptake of MDH in cells treated or untreated with ammonium chloride. As shown in Fig 6a, the disturbance of the lysosomal acidic pH had no effect on MDH uptake. This is in contrast to MDC uptake, which is inhibited by at least 30% with ammonium chloride. As a lysosomotropic agent, MDC itself is believed to affect the lysosomal pH in vivo in a dose-dependent manner. We used Acridine Orange as an alternative lysosomotropic agent and tested the effect of MDC and MDH on the Acridine Orange staining pattern. Cells incubated with 200 µM MDH for 1 hr showed the same Acridine Orange staining pattern as untreated cells (Fig 6b and Fig 6d), whereas cells treated with MDC did not show any vacuolar Acridine Orange staining (Fig 6c). This indicates that the uptake of MDH into subcellular compartments does not affect the pH of these compartments and that it is independent of the lysosomal pH. Therefore, MDH did not exhibit the properties of a classical lysosomotropic agent.
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Although MDH showed a comparable in vivo staining pattern compared with MDC (Fig 2a and Fig 2b), the question remained whether MDH really stains the same compartments. To test this, cell fractionation was applied to PaTu 8902 cells that had been incubated for 1 hr with either 0.1 mM MDC or MDH. The pellets of the postnuclear supernatants were separated by centrifugation on continuous sucrose gradients (1540% sucrose) as described elsewhere (
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Discussion |
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Monodansylcadaverine (MDC) has been shown to be a useful autofluorescent marker for lysosomal/autophagic vacuoles in in vivo and in vitro applications (
As a classical lysosomotropic agent, MDC is a weak base. Like other weak bases, such as ammonium chloride or chloroquine, MDC is believed to influence the lysosomal pH when it enters this compartment. This is shown in the experiment of Fig 6, in which cells first stained with MDC were subsequently treated with the lysosomotropic substance Acridine Orange, which did not result in an accumulation of Acridine Orange in lysosomes in MDC-pretreated cells. The same effect is observed when cells are treated with inhibitors of the +H-V-ATPase (
Although MDC and MDH stain the same subcellular structures when applied to fixed cells, there are some technical differences in their properties. When cells are again fixed with paraformaldehyde after MDC application, MDC is retained in the vacuolar structures even when longer staining protocols for immunohistochemistry follow. Under the same conditions, MDH fades, so that the background increases over time and the vacuolar structures become hazy. However, in freshly stained fixed cells MDH staining is more brilliant because MDC cause background by faint cytoplasmic staining, which does not occur with MDH. Nevertheless, MDH must be used with PBS as mounting medium, because with other mounting media, such as Mowiol, MDH is partly extracted from the stained structures, which also leads to increased background and decreased contrast. This has not been observed with MDC, most likely because MDC is immobilized by aldehyde fixation via the amino group. Under in vivo conditions, the staining with MDH is much more rapid than that observed with MDC. Whereas after a 10-min exposure to MDH cells are saturated with the fluorochrome (Fig 6a), MDC takes more than 45 min to reach the same level of saturation (
Our extended experiments with a variety of MDC derivatives revealed that both the charged end group and the alkyl side chain length are important for the interaction of these molecules with biological membranes (Fig 3 Fig 4 Fig 5). Interestingly, none of the compounds used led to fluorescent staining of single lipid bilayers, such as the plasma membrane or other endomembranes. In multilamellelar bodies such as AVs, many membrane bilayers are stacked with only a small, if any, amount of aqueous phase between them. Therefore, many fluorochrome molecules are tightly packed, emitting light that combines to yield a strong signal. The amount of fluorochrome molecules in a single membrane bilayer might be in a range at which they are not detectable by the fluorescence microscopy methods applied in this study.
Fluorescent probes that are structurally similar to MDH have been described previously. Two of them, Prodan and Laurdan (Fig 10), have been extensively used to study biophysical aspects of biological membranes. Whereas the alkyl side chain of MDH is linked to the dansyl moiety via a sulfonamide group, Prodan and Laurdan possess a carbonyl group to bridge the gap between the alkyl side chain and the fluorescent group. Furthermore, Prodan has a C2 side chain length, while Laurdan has a C11 side chain length (Fig 10). On the basis of the experiment shown in Fig 5, we assume that the association of Prodan and Laurdan with intracellular membranes of living cells is not very efficient. However, both probes have been used in vitro, either as single compounds or in combination with each other, predominantly to investigate different lipid phases in biological membranes, applying the method of generalized polarization (
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Acknowledgments |
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Supported by Studienstiftung des Deutschen Volkes and by P. E. Kempkes Stiftung Marburg.
We thank Ursula Lehr for expert technical assistance, Volkwin Kramer for preparation of the microscopic reprints, and Annette Biederbick for critical comments and helpful discussions.
Received for publication May 9, 2000; accepted August 31, 2000.
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