The innards of the cell: studies of water dipolar relaxation using the ACDAN fluorescent probe

Otaiza-González, Santiago N.; Cabadas, Manuel; Robert, Germán; Stock, Roberto P.; Malacrida, Leonel; Lascano, Ramiro; Bagatolli, Luís A.

doi:10.1088/2050-6120/ac8d4c

Cited by 4 publications

(2 citation statements)

References 42 publications

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“…Our results show that the nanoenvironment in the protein condensates is altered with salt concetration in the medium, leading to different confined water properties. These changes affect water dynamics which is sensed by ACDAN 28, 29, 30 , providing a means for analyzing physico-chemical changes in the condensates at nanometer scales.…”

Section: Introductionmentioning

confidence: 99%

Biomolecular condensates modulate membrane lipid packing and hydration

Mangiarotti

Siri

Zhao

et al. 2023

Preprint

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Membrane wetting by biomolecular condensates recently emerged as a critical phenomenon in cell biology, involved in a great diversity of processes across different organisms. However, understanding the molecular mechanism behind this process is still missing. Exploiting ACDAN and LAURDAN properties as nano-environmental sensors in combination with phasor analysis of hyperspectral and lifetime imaging microscopy, we obtained vital information on the process of condensate formation and membrane wetting. The results reveal that glycinin condensates display differences in water dynamics when changing the salinity of the medium as a consequence of rearrangements in the secondary structure of the protein. The analysis of membrane-condensates interaction indicated a correlation between increased wetting affinity and enhanced lipid packing, demonstrated by a decrease in water dipolar relaxation at both protein and polymer systems. These results suggest a general mechanism to tune membrane order by condensate wetting.

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Section: Introductionmentioning

confidence: 99%

Biomolecular condensates modulate membrane lipid packing and hydration

Mangiarotti

Siri

Zhao

et al. 2023

Preprint

Self Cite

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“…Based in its simple rules, such as the linear combination and reciprocity principle, the SP enables to identify the occurrence of different molecular environments for a dye of interest with no previous knowledge [14]. In the past, this approach has been used to understand the photophysics of LAURDAN or ACDAN in various systems such as cell, plants or animals [15][12] [16] . Using this approach, one can take full advantage of the hyperspectral imaging for live cell experiments [17][18][19] [20][21].…”

Section: Introductionmentioning

confidence: 99%

Spectral phasor plot for hyperspectral imaging of acridine orange photophysics within the cell

Díaz

Zolessi

Malacrida

2023

Preprint

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Acridine Orange (AO) has rich photophysics that has been exploited to study its interaction with RNA and DNA. Traditionally AO was used to stain cells differentiating nucleus and cytoplasm due to the differences in DNA and RNA differential composition. AO cell labeling shows complex behavior depending on several parameters, such as concentration, pH, etc. Such complex photophysics will benefit from a model-free analysis such as the spectral phasor (SP) for hyperspectral imaging. The SP linear combination and reciprocity principles allow us to understand better the AO spectral shift associated with its exploration of the different molecular environments in cells. Using this approach, we identified the SP fingerprints for RNA and DNA for in vitro experiments, which remain invariant for fixed and live cells. However, the SP analysis of the AO fluorescence from live cells shows the occurrence of a third spectral component at 640 nm. The linear properties of the SP analysis enable quantification of the AO bound to RNA, DNA, and a third component. By co-labeling with AO and LysoTracker, we associated part of these subcellular structures with acidic compartments. Nonetheless, we could differentiate AO self-interactions from its binding to RNA within cytoplasmic compartments by use of the SP analysis. This work establishes the use of SP for hyperspectral imaging of AO for the first time, expanding our knowledge of its complex photophysics and promoting further application for in vivo cell imaging.

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Biomolecular condensates modulate membrane lipid packing and hydration

Mangiarotti,

Siri,

Tam

et al. 2023

Nat Commun

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Membrane wetting by biomolecular condensates recently emerged as a key phenomenon in cell biology, playing an important role in a diverse range of processes across different organisms. However, an understanding of the molecular mechanisms behind condensate formation and interaction with lipid membranes is still missing. To study this, we exploited the properties of the dyes ACDAN and LAURDAN as nano-environmental sensors in combination with phasor analysis of hyperspectral and lifetime imaging microscopy. Using glycinin as a model condensate-forming protein and giant vesicles as model membranes, we obtained vital information on the process of condensate formation and membrane wetting. Our results reveal that glycinin condensates display differences in water dynamics when changing the salinity of the medium as a consequence of rearrangements in the secondary structure of the protein. Remarkably, analysis of membrane-condensates interaction with protein as well as polymer condensates indicated a correlation between increased wetting affinity and enhanced lipid packing. This is demonstrated by a decrease in the dipolar relaxation of water across all membrane-condensate systems, suggesting a general mechanism to tune membrane packing by condensate wetting.

show abstract

The innards of the cell: studies of water dipolar relaxation using the ACDAN fluorescent probe

Cited by 4 publications

References 42 publications

Biomolecular condensates modulate membrane lipid packing and hydration

Biomolecular condensates modulate membrane lipid packing and hydration

Spectral phasor plot for hyperspectral imaging of acridine orange photophysics within the cell

Biomolecular condensates modulate membrane lipid packing and hydration

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