Fluorescence Properties and Staining Behavior of Monodansylpentane, a Structural Homologue of the Lysosomotropic Agent Monodansylcadaverine

Abstract

S U M M A R YWe 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 iontrapping 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.

The Dynamics of Autophagy Visualised in Live Cells: from Autophagosome Formation to Fusion with Endo/lysosomes

“…The pattern of MDH puncta suggested that it is loading into (or forming) very lipid rich environments. 21 Indeed, when we examined MDH staining in superior cervical ganglion neurons induced to accumulate lipid droplets, MDH staining corresponded to the staining seen with the lipophilic dye Oil-Red O (data not shown). Thus, MDH too is unlikely to be useful as a marker of nascent autophagosome formation.…”

“…Monodansylpentane (MDH) was synthesized as described previously. 21 pEGFP-LC3 was a kind gift of Tamotsu Yoshimori (National Institute for Basic Biology, Okazaki, Japan), pEGFP-CD63 was a kind gift of Paul Luzio (CIMR, University of Cambridge, UK), mRFP, kindly supplied by Roger Tsien (UCSD, Ca, USA) 37 was inserted in place of EGFP in the pEGFP-LC3 vector to create mRFP-LC3. The anti-LC3 antibody was a kind gift of Yasuo Uchiyama (Osaka University, Osaka, Japan).…”

“…21 However, although MDH did form puncta, these failed to localise with either EGFP-LC3 or EGFP-CD63 positive compartments, or to change their pattern of staining upon stimulation of autophagy (see supplemental Fig. S2).…”

“…20 Based on the propensity of MDC to stack into adjacent membranes, a neutral derivative, monodansylpentane, was synthesized and this has been suggested to locate more exclusively into double membraned structures. 21 No quantitative studies have been conducted alongside cell markers of other organelles to determine which fraction of the vesicles stained by MDC and MDH are true nascent autophagosomes and not autophagosomes that have fused with endosomes, multivesicular bodies and lysosomes. Munafo and Colombo 18 have performed the most critical assessment of MDC stained vacuoles, and have shown that a small proportion of MDC-stained vacuoles are also stained with the autophagic marker MAP-LC3, but the possibility that these are autolysosomes and not autophagosomes was not excluded.…”

7‐Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7‐ketocholesterol–induced apoptosis: Analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation

“…Therefore, during 7KC-induced apoptosis, these cytoplasmic structures, which were previously described to produce positive staining with monodansylcadaverine (a solvent polarity probe and a lysosomotropic agent that stains acidic compartments) (13,23,33,60,61), were characterized by flow cytometric and microscopic methods including FRET biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry after staining with NR.…”