Detergent-Triggered Membrane Remodelling Monitored via Intramembrane Fluorescence De-Quenching

Summary/Background:

The composition and functionality of biological membranes are of great importance for cell maintenance. Additionally, mimetic membranes are widely used for production of drugs and vaccines, and in in vitro studies with different purposes^1–3.

Integral membrane proteins (IMPs) are crucial for life, as they perform vital functions like transporting molecules, transmitting signals, and facilitating communication across cell membranes⁴. To study these IMPs, it is crucial to purify them from cellular membranes without compromising their functions. Moreover, membrane solubilization is important for biotechnological applications such as virus inactivation, cellular drug delivery and formation of niosomes in vaccine formulations^5–7.

Triton X-100 (TX-100) is a non-ionic detergent used for IMP isolation, while maintaining their structure and function⁸. However, the mechanisms of detergent-membrane solubilization are still poorly understood. Several aspects of their interaction, which depends on membrane fusion⁹, are still unclear — including how exactly the detergent acts at the molecular level, how it affects lipid–lipid interactions, and what the structure and morphology of the fused products are.

In recent years, a three-step model has been proposed: (1) detergent monomers, at concentrations below the critical micellar concentration (CMC), progressively saturate the membrane; (2) the formation of mixed detergent–lipid micelles lead to membrane disruption and fragmentation; and (3) these mixed micelles are subsequently released into the solution⁹. This preprint builds on the author’s previous study using Förster resonance energy transfer (FRET) and single-vesicle analysis to investigate how Triton X-100 (TX-100) interacts with synthetic large unilamellar vesicles (LUVs)¹⁰. In this preprint, the authors present a fluorescence de-quenching assay to confirm the presence of a vesicle swelling step that occurs prior to complete solubilization or micellization.

Significance/Why I think this preprint is important:

This study is highly significant, not only for elucidating how detergents interact with and solubilize membranes, but also for providing the scientific community with a robust technique to assess and characterize lipid–protein interactions. In the context of detergent–membrane solubilization, the approach presented in this preprint is both sensitive and relatively straightforward to apply and interpret, particularly when compared to methods such as FRET analysis. Collectively, these aspects underscore the study’s relevance for advancing our understanding of biological membrane behavior.

Key findings:

Fluorescence de-quenching tracks TX-100–induced membrane perturbations

In preprint Figure 1, the authors show that increasing concentrations of TX-100 led to an increase in DiI fluorescence emission, along with a rise in the average lifetime amplitude (τₐᵥ), suggesting that DiI fluorescence de-quenching occurs proportionally to the amount of TX-100 added to POPC membranes. The authors further compared the observed de-quenching effects with FRET data from their previous study, in which the FRET signal was shown to decrease following detergent–membrane interaction. Collectively, these results support the reliability of the de-quenching approach as a quantitative method for detecting nanoscale perturbations in vesicles.

Extending fluorescence dequenching to other detergent-induced membrane disturbances

With the goal of extending the applicability of their technique to experiments using other fluorescent probes, the authors used DiO and DiD dyes (shown in preprint Figure 2), which also had their emission intensities and ͳ_av increased. Variations in the magnitude of the lifetime changes across the dyes were correlated by the authors with potential differences in dye–lipid binding strength, intermolecular dye interactions within the membrane, and the extent of dye incorporation into the bilayer. Furthermore, the authors used other surfactants, such as sodium dodecyl sulfate (SDS) and Tween 20 to assess the utility of de-quenching measurements across a range of disruptors. With this data, the authors show that fluorescence de-quenching may be a generalizable assay for assessing detergent-membrane solubilization.

Vesicle imaging corroborates dequenching findings

The authors used fluorescence lifetime imaging (FLIM) to visualize vesicles de-quenching and assess lifetime distributions in Dil-labelled LUVs (preprint Figure 3). The authors observed a clear difference in the fluorophore decay time in the presence of the detergent, corroborating the DiI emission de-quenching and alterations in the lipid microenvironment, as previously shown in preprint Figure 1. As illustrated in preprint Figure S3, the total vesicle count remained stable, suggesting that the fluorescence variations result from morphological rearrangements rather than vesicle loss. To test this hypothesis, the authors employed scanning electron microscopy to visualize LUVs in the absence and presence of TX-100 (preprint Figure 4). The images revealed that exposure to TX-100 increased vesicle diameter and induced a morphological transition from spherical to toroidal-like structures. Taken together, the findings support the previously proposed solubilization model, in which TX-100 promotes vesicle swelling and morphological rearrangements preceding the full micellization and solubilization of LUV membranes.

Comments/Questions:

Major comments

1. The manuscript would benefit from the inclusion of a statistical analysis section within the Methods, specifying the tests used and the criteria for data significance. This addition would enhance the transparency and reproducibility of the results.
2. In the first paragraph of the results the authors mentioned that the distance between the probes inserted in POPC vesicles’ bilayers (DiI–DiI) was <2 nm. However, it is not clear how this distance was determined. Is there an optimal or required distance between the probes to assess its quenching mode? And it is not clear whether the distances were also determined for the other probes used (DiD and DiO), and whether these values were comparable to those observed for DiI–DiI. Including this information would enhance the reproducibility and demonstrate the broader applicability of the assay.
3. In preprint Figures 1 and 2, increasing concentrations of TX-100 were used. How do the authors ensure that concentrations below the CMC do not disrupt the membranes prior to micellization? It might be helpful to include vesicle morphology data at detergent concentrations below the CMC, obtained by scanning electron microscopy, to clarify this point. Moreover, it is not clear how the detergent concentrations used in preprint Figures 3 and 4 were selected, and how these differences could influence the interpretation of the results. Providing data or representative images that demonstrate the micellization process under the experimental conditions used in this study would further strengthen the work.
4. The authors could consider replacing the representative microscopy images in preprint Figure 3 with higher-resolution versions, particularly to improve the visibility of the scale bar.
5. Greater emphasis on the statistical analysis of preprint Figures 3 and 4 is recommended. While the authors report the number of vesicles analyzed in the graphs, it remains unclear whether the results were reproducible across independent vesicle preparations. Including such information would enhance the credibility and interpretability of the data.
6. The manuscript would benefit from a discussion on how its results align or contrast with other studies utilizing quenching techniques to assess LUV structure and surfactant interactions (g.: PMIDs: 36901959; 39132807; 8580339; 34150932). Such comparisons would provide important context for interpreting the findings.

Minor comments

1. Replace “remodelling” with “remodeling.”
2. Including page numbers throughout the document is recommended.
3. The experiments and figures in the preprint align well with the authors’ aims. However, improving the resolution of the figures would enhance the overall impact of the data presentation.
4. In preprint Figure 1e, the authors present the optimization of the probe concentration used in the study. The figure shows three concentrations (0.01%, 0.1%, and 1.5%), none of which correspond to the concentration used in the experiments, which may be confusing for readers. It would be helpful to include the curve corresponding to the concentration employed in most of the experiments (1%).
5. The meaning of the red and black lines in preprint Figure 1e is not clearly explained. Clarifying what these lines represent would help readers better interpret the data presented.
6. The use of different fluorescent probes supports the notion that the de-quenching assay can be generalized beyond DiI/TX-100, as shown in preprint Figure 2. However, the manuscript does not specify the sources of these probes or describe how they differ structurally or in their interactions with membranes. Including this information would provide stronger support for the discussion, as such differences could explain the variations in fluorescence emissions and quantum yield observed in Figure 2e.
7. Although the overall results support the idea that vesicle swelling occurs as an intermediate step before micellization, the use of terms such as “indicating” (page 7, 2^nd paragraph) and “assigned” (page 9) may give the impression that this phenomenon was directly observed in all figures, whereas it becomes visually evident only toward the end of the manuscript. Revising the text or adjusting the order of the figures could help highlight this key finding more clearly for the reader.
8. It appears that some references are missing in sections where the authors cite previous findings, particularly in the Discussion (page 11, 3^rd paragraph). Including these and other citations would provide proper context and strengthen the manuscript.
9. Including a graphical abstract illustrating the proposed vesicle swelling step could greatly aid readers in visualizing and understanding the central mechanistic concept of the study.

References:

1. Chugh, V., Vijaya Krishna, K. & Pandit, A. Cell Membrane-Coated Mimics: A Methodological Approach for Fabrication, Characterization for Therapeutic Applications, and Challenges for Clinical Translation. ACS Nano 15, 17080–17123 (2021).
2. Majeed, S., Ahmad, A. B., Sehar, U. & Georgieva, E. R. Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins. Membranes (Basel). 11, (2021).
3. Tan, S., Wu, T., Zhang, D. & Zhang, Z. Cell or cell membrane-based drug delivery systems. Theranostics 5, 863–81 (2015).
4. Whitelegge, J. P. Integral membrane proteins and bilayer proteomics. Anal. Chem. 85, 2558–68 (2013).
5. Lin, Q. et al. Sanitizing agents for virus inactivation and disinfection. View (Beijing, China) 1, e16 (2020).
6. Hammond, J. et al. Membrane Fusion-Based Drug Delivery Liposomes Transiently Modify the Material Properties of Synthetic and Biological Membranes. Small 21, e2408039 (2025).
7. Liga, S., Paul, C., Moacă, E.-A. & Péter, F. Niosomes: Composition, Formulation Techniques, and Recent Progress as Delivery Systems in Cancer Therapy. Pharmaceutics 16, (2024).
8. Koley, D. & Bard, A. J. Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM). Proc. Natl. Acad. Sci. U. S. A. 107, 16783–7 (2010).
9. Lichtenberg, D., Ahyayauch, H. & Goñi, F. M. The mechanism of detergent solubilization of lipid bilayers. Biophys. J. 105, 289–99 (2013).
10. Dresser, L. G. et al. Multiple intermediates in the detergent-induced fusion of lipid vesicles. Commun. Mater. 5, 195 (2024).

 

Language refinement was supported using ChatGPT (OpenAI), which assisted in improving clarity and readability.

Tags: detergent solubilization, fluorescence, lipid membrane

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