Skip to main content

Advertisement

SpringerLink
Log in

Spectral Phasor Analysis of Nile Red Identifies Membrane Microenvironment Changes in the Presence of Amyloid Peptides

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

The interaction of protein and peptide amyloid oligomers with membranes is thought to be one of the mechanisms contributing to cellular toxicity. However, techniques to study these interactions in the complex membrane environment of live cells are lacking. Spectral phasor analysis is a recently developed biophysical technique that can enable visualisation and analysis of membrane-associated fluorescent dyes. When the spectral profile of these dyes changes as a result of changes to the membrane microenvironment, spectral phasor analysis can localise those changes to discrete membrane regions. In this study, we investigated whether spectral phasor analysis could detect changes in the membrane microenvironment of live cells in the presence of fibrillar aggregates of the disease-related Aβ42 peptide or the functional amyloid neurokinin B. Our results show that the fibrils cause distinct changes to the microenvironment of nile red associated with both the plasma and the nuclear membrane. We attribute these shifts in nile red spectral properties to changes in membrane fluidity. Results from this work suggest that cells have mechanisms to avoid or control membrane interactions arising from functional amyloids which have implications for how these peptides are stored in dense core vesicles. Furthermore, the work highlights the utility of spectral phasor analysis to monitor microenvironment changes to fluorescent probes in live cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.

References

  1. Riek, R., & Eisenberg, D. S. (2016). The activities of amyloids from a structural perspective. Nature, 539, 227–235. https://doi.org/10.1038/nature20416

    Article  PubMed  Google Scholar 

  2. Knowles, T. P., Vendruscolo, M., & Dobson, C. M. (2014). The amyloid state and its association with protein misfolding diseases. Nature Reviews Molecular Cell Biology, 15, 384–396. https://doi.org/10.1038/nrm3810

    Article  CAS  PubMed  Google Scholar 

  3. Itoh, N., Takada, E., Okubo, K., Yano, Y., Hoshino, M., Sasaki, A., Kinjo, M., & Matsuzaki, K. (2018). Not oligomers but amyloids are cytotoxic in the membrane-mediated amyloidogenesis of amyloid-beta peptides. ChemBioChem, 19, 430–433. https://doi.org/10.1002/cbic.201700576

    Article  CAS  PubMed  Google Scholar 

  4. Sciacca, M. F. M., Tempra, C., Scollo, F., Milardi, D., & La Rosa, C. (2018). Amyloid growth and membrane damage: Current themes and emerging perspectives from theory and experiments on Abeta and hIAPP. Biochimica et Biophysica Acta - Biomembranes, 1860, 1625–1638. https://doi.org/10.1016/j.bbamem.2018.02.022

    Article  CAS  PubMed  Google Scholar 

  5. Walsh, P., Vanderlee, G., Yau, J., Campeau, J., Sim, V. L., Yip, C. M., & Sharpe, S. (2014). The mechanism of membrane disruption by cytotoxic amyloid oligomers formed by prion protein(106-126) is dependent on bilayer composition. Journal of Biological Chemistry, 289, 10419–10430. https://doi.org/10.1074/jbc.M113.515866

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chuang, E., Hori, A. M., Hesketh, C. D., & Shorter, J. (2018) Amyloid assembly and disassembly. Journal of Cell Science, 131, jcs189928. https://doi.org/10.1242/jcs.189928

  7. Maji, S. K., Perrin, M. H., Sawaya, M. R., Jessberger, S., Vadodaria, K., Rissman, R. A., Singru, P. S., Nilsson, K. P., Simon, R., Schubert, D., Eisenberg, D., Rivier, J., Sawchenko, P., Vale, W., & Riek, R. (2009). Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science, 325, 328–332. https://doi.org/10.1126/science.1173155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nespovitaya, N., Gath, J., Barylyuk, K., Seuring, C., Meier, B. H., & Riek, R. (2016). Dynamic assembly and disassembly of functional beta-endorphin amyloid fibrils. Journal of the American Chemical Society, 138, 846–856. https://doi.org/10.1021/jacs.5b08694

    Article  CAS  PubMed  Google Scholar 

  9. Jayawardena, B. M., Jones, M. R., Hong, Y., & Jones, C. E. (2019). Copper ions trigger disassembly of neurokinin B functional amyloid and inhibit de novo assembly. Journal of Structural Biology, 208, 107394 https://doi.org/10.1016/j.jsb.2019.09.011

    Article  CAS  PubMed  Google Scholar 

  10. Golfetto, O., Hinde, E., & Gratton, E. (2015). The Laurdan spectral phasor method to explore membrane micro-heterogeneity and lipid domains in live cells. Methods in Molecular Biology, 1232, 273–290. https://doi.org/10.1007/978-1-4939-1752-5_19

    Article  CAS  PubMed  Google Scholar 

  11. Fereidouni, F., Bader, A. N., & Gerritsen, H. C. (2012). Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images. Optics Express, 20, 12729–12741. https://doi.org/10.1364/OE.20.012729

    Article  CAS  PubMed  Google Scholar 

  12. Andrews, L. M., Jones, M. R., Digman, M. A., & Gratton, E. (2013). Spectral phasor analysis of Pyronin Y labeled RNA microenvironments in living cells. Biomedical Optics Express, 4, 171–177. https://doi.org/10.1364/BOE.4.000171

    Article  CAS  PubMed  Google Scholar 

  13. Sediqi, H., Wray, A., Jones, C. E., & Jones, M. R. (2018). Application of spectral phasor analysis to sodium microenvironments in myoblast progenitor cells. PLoS One, 13, e0204611 https://doi.org/10.1371/journal.pone.0204611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fereidouni, F., Bader, A. N., Colonna, A., & Gerritsen, H. C. (2014). Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin. Journal of Biophotonics, 7, 589–596. https://doi.org/10.1002/jbio.201200244

    Article  CAS  PubMed  Google Scholar 

  15. Malacrida, L., Astrada, S., Briva, A., Bollati-Fogolin, M., Gratton, E., & Bagatolli, L. A. (2016). Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures. Biochimica et Biophysica Acta, 1858, 2625–2635. https://doi.org/10.1016/j.bbamem.2016.07.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sameni, S., Malacrida, L., Tan, Z., & Digman, M. A. (2018). Alteration in fluidity of cell plasma membrane in huntington disease revealed by spectral phasor analysis. Scientific Reports, 8, 734 https://doi.org/10.1038/s41598-018-19160-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sena, F., Sotelo-Silveira, M., Astrada, S., Botella, M. A., Malacrida, L., & Borsani, O. (2017). Spectral phasor analysis reveals altered membrane order and function of root hair cells in Arabidopsis dry2/sqe1-5 drought hypersensitive mutant. Plant Physiology and Biochemistry, 119, 224–231. https://doi.org/10.1016/j.plaphy.2017.08.017

    Article  CAS  PubMed  Google Scholar 

  18. Shahzad, R., Jones, M. R., Viles, J. H., & Jones, C. E. (2016). Endocytosis of the tachykinin neuropeptide, neurokinin B, in astrocytes and its role in cellular copper uptake. Journal of Inorganic Biochemistry, 162, 319–325. https://doi.org/10.1016/j.jinorgbio.2016.02.027

    Article  CAS  PubMed  Google Scholar 

  19. Menon, R., Christofides, K., & Jones, C. E. (2020). Endocytic recycling prevents copper accumulation in astrocytoma cells stimulated with copper-bound neurokinin B. Biochemical and Biophysical Research Communications, 523, 739–744. https://doi.org/10.1016/j.bbrc.2019.12.087

    Article  CAS  PubMed  Google Scholar 

  20. Greenspan, P., & Fowler, S. D. (1985). Spectrofluorometric studies of the lipid probe, nile red. Journal of Lipid Research, 26, 781–789

    Article  CAS  PubMed  Google Scholar 

  21. Stringari, C., Cinquin, A., Cinquin, O., Digman, M. A., Donovan, P. J., & Gratton, E. (2011). Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue. Proceedings of the National Academy of Sciences of the United States of America, 108, 13582–13587. https://doi.org/10.1073/pnas.1108161108

    Article  PubMed  PubMed Central  Google Scholar 

  22. Mukherjee, S., Raghuraman, H., & Chattopadhyay, A. (2007). Membrane localization and dynamics of Nile Red: Effect of cholesterol. Biochimica et Biophysica Acta, 1768, 59–66. https://doi.org/10.1016/j.bbamem.2006.07.010

    Article  CAS  PubMed  Google Scholar 

  23. Zentgraf, H., Deumling, B., Jarasch, E. D., & Franke, W. W. (1971). Nuclear membranes and plasma membranes from hen erythrocytes. I. Isolation, characterization, and comparison. Journal of Biological Chemistry, 246, 2986–2995

    Article  CAS  PubMed  Google Scholar 

  24. Almeida, P. F., Pokorny, A., & Hinderliter, A. (2005). Thermodynamics of membrane domains. Biochimica et Biophysica Acta, 1720, 1–13. https://doi.org/10.1016/j.bbamem.2005.12.004

    Article  CAS  PubMed  Google Scholar 

  25. Wardhan, R., & Mudgal, P. (2017). Textbook of membrane biology. 2017. Singapore: Springer

    Book  Google Scholar 

  26. Raghuraman, H., & Chattopadhyay, A. (2007). Melittin: A membrane-active peptide with diverse functions. Bioscience Reports, 27, 189–223. https://doi.org/10.1007/s10540-006-9030-z

    Article  CAS  PubMed  Google Scholar 

  27. Sabapathy, T., Deplazes, E., & Mancera, R. L. (2020) Revisiting the interaction of melittin with phospholipid bilayers: The effects of concentration and ionic strength. International Journal of Molecular Sciences, 21, 746. https://doi.org/10.3390/ijms21030746

  28. van den Bogaart, G., Guzman, J. V., Mika, J. T., & Poolman, B. (2008). On the mechanism of pore formation by melittin. Journal of Biological Chemistry, 283, 33854–33857. https://doi.org/10.1074/jbc.M805171200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yang, Z., Choi, H., & Weisshaar, J. C. (2018). Melittin-induced permeabilization, re-sealing, and re-permeabilization of E. coli membranes. Biophysical Journal, 114, 368–379. https://doi.org/10.1016/j.bpj.2017.10.046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Xue, W. F., Hellewell, A. L., Gosal, W. S., Homans, S. W., Hewitt, E. W., & Radford, S. E. (2009). Fibril fragmentation enhances amyloid cytotoxicity. Journal of Biological Chemistry, 284, 34272–34282. https://doi.org/10.1074/jbc.M109.049809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jones, C. E., Underwood, C. K., Coulson, E. J., & Taylor, P. J. (2007). Copper induced oxidation of serotonin: analysis of products and toxicity. Journal of Neurochemistry, 102, 1035–1043. https://doi.org/10.1111/j.1471-4159.2007.04602.x

    Article  CAS  PubMed  Google Scholar 

  32. Mishra, R., Sjolander, D., & Hammarstrom, P. (2011). Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red. Molecular BioSystems, 7, 1232–1240. https://doi.org/10.1039/c0mb00236d

    Article  CAS  PubMed  Google Scholar 

  33. Segota, S., Vojta, D., Pletikapic, G., & Baranovic, G. (2015). Ionic strength and composition govern the elasticity of biological membranes. A study of model DMPC bilayers by force- and transmission IR spectroscopy. Chemistry and Physics of Lipids, 186, 17–29. https://doi.org/10.1016/j.chemphyslip.2014.11.001

    Article  CAS  PubMed  Google Scholar 

  34. Pinho, C. M., Teixeira, P. F., & Glaser, E. (2014). Mitochondrial import and degradation of amyloid-beta peptide. Biochimica et Biophysica Acta, 1837, 1069–1074. https://doi.org/10.1016/j.bbabio.2014.02.007

    Article  CAS  PubMed  Google Scholar 

  35. Mastroeni, D., Chouliaras, L., Grover, A., Liang, W. S., Hauns, K., Rogers, J., & Coleman, P. D. (2013). Reduced RAN expression and disrupted transport between cytoplasm and nucleus; a key event in Alzheimer’s disease pathophysiology. PLoS One, 8, e53349 https://doi.org/10.1371/journal.pone.0053349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors would like to acknowledge the Advanced Materials Characterisation Facility (AMCF) of Western Sydney University for access to its instrumentation and staff. R.M. acknowledges support from Australian Rotary Health. B.M.J. acknowledges the support of a Western to the World Postgraduate Scholarship. In memory of Fredrick Jones (9 March 1936 to 2 May 2022).

Author information

Author notes

  1. Resmi Menon

    Present address: PYC Therapeutics, QEII Medical Centre, 6 Verdun St, Nedlands, 6009, WA, Australia

Authors and Affiliations

  1. School of Science, Western Sydney University, Locked Bag 1797, Penrith, 2751, NSW, Australia

    Bhawantha M. Jayawardena, Resmi Menon, Mark R. Jones & Christopher E. Jones

Authors
  1. Bhawantha M. Jayawardena
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Resmi Menon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark R. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher E. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christopher E. Jones.

Ethics declarations

Conflict of Interest

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jayawardena, B.M., Menon, R., Jones, M.R. et al. Spectral Phasor Analysis of Nile Red Identifies Membrane Microenvironment Changes in the Presence of Amyloid Peptides. Cell Biochem Biophys 81, 19–27 (2023). https://doi.org/10.1007/s12013-022-01105-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-022-01105-0

Keywords

Search

Navigation