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Transient state imaging of intermittent interactions between lipids and receptor proteins in artificial and live cell membranes
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.ORCID iD: 0000-0002-6191-9921
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.ORCID iD: 0000-0002-4762-4887
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.ORCID iD: 0000-0003-3200-0374
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Transient collisional interactions between lipids and membrane proteins play an important role in modulating cellular functions but occur at frequencies too low to be readily observable via fluorescence imaging or quenching studies. We used transient state imaging (TRAST) to quantify those interaction in living cells. This method combines sensitive detection of fluorescence from fluorophore marker molecules with the ability to monitor their long-lived dark triplet states, highly sensitive to molecular interactions in artificial and live cell membranes.

By TRAST we first determined the dark transient state kinetics of 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), an extensively used biomembrane fluorophore, available as a label on a wide range of lipids and sterols. We then measured quenching of NBD triplet states by spin-labels, in the membranes of small unilamellar vesicles (SUVs), and studied how it depends on the fluorescent lipid-derivative type and on the position of the spin label in the membranes. By the same strategy, we then quantified the collisional quenching of NBD-lipid derivatives and spin-labelled stearic acids in live cell plasma membranes.

Finally, we extended the method to study the collisional interactions between G-Protein Coupled Receptors (GPCRs), covalently labelled with the spin label TEMPO, and NBD-lipid derivatives, in the plasma membranes of living cells. Thereby, we could resolve transient interactions between the GPCRs and lipids with different hydrophilic heads or sterols, and how these interactions were changed upon activation of the GPCR by an agonist. The presented approach offers a straightforward and widely applicable means to characterize and image transient interactions in live cell membranes, of large biomedical relevance.

National Category
Other Physics Topics
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-246017OAI: oai:DiVA.org:kth-246017DiVA, id: diva2:1295288
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20190312

Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-03-12Bibliographically approved
In thesis
1. Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
Open this publication in new window or tab >>Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorophore blinking dynamics are highly sensitive to the local environment and can be used as an additional readout parameter to increase the information gained from existing fluorescence techniques.The origin of these blinking patterns are photophysical transitions to and from a manifold of non-luminescent states. The long lifetime of these dark transient states, typically 103 to 106 times longer than the fluorescent state, gives them correspondingly more time to sense their environment. For this reason, fluorophore blinking dynamics are particularly sensitive to low frequency events, such as diffusion-mediated interactions between the fluorophore and dilute species.

Transient State (TRAST) monitoring has been developed to quantify fluorophore blinking dynamics in a simple and widely applicable manner. TRAST does not need to resolve individual blinking events, but instead monitors the average fluorescence intensity in response to a modulated excitation. By systematically varying the modulation parameters, the transient state kinetics of the sample are mapped out. Without the need for time-resolved detection, a regular camera can be used to image blinking dynamics with high spatial resolution.

This thesis presents TRAST characterizations of common autofluorescent compounds and demonstrates their ability to sense relevant biological parameters such as oxygen concentration and redox potential. In Papers I and II, the autofluorescent co-enzymes flavin and NAD(P)H were studied, and label-free imaging of local redox variations within cells was demonstrated. Perturbing the cells, through dilute additions of mitochondrial uncouplers, revealed a strong andlocalized response in the TRAST images. In Paper III we studied tryptophan autofluorescence and used it to detect conformational changes in an unlabeled spider silk protein.

Labeling with external fluorophores can add further specificity to the TRAST measurements. In Paper IV, TRAST was used to monitor diffusion-mediated interactions between lipids and receptors in a cell membrane, including the influence of receptor activation. In Paper V we tracked folding of RNA into G-quadruplexes in live cells, monitored via the isomerization properties of an attached cyanine dye.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. i-vi; 116
Series
TRITA-SCI-FOU ; 2019:13
National Category
Other Physics Topics
Research subject
Biological Physics; Physics
Identifiers
urn:nbn:se:kth:diva-246020 (URN)978-91-7873-142-8 (ISBN)
Public defence
2019-04-05, FB53, KTH, Roslagstullsbacken 21, Stockholm, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Note

QC 20190312

Available from: 2019-03-12 Created: 2019-03-11 Last updated: 2019-03-13Bibliographically approved

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Tornmalm, JohanPiguet, JoachimChmyrov, VolodymyrWidengren, Jerker

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