Trans–Cis Isomerization of Lipophilic Dyes Probing Membrane Microviscosity in Biological Membranes and in Live Cells

Abstract

PostprintThis is the accepted version of a paper published in Analytical Chemistry. This paper has been peerreviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Chmyrov, V., Spielmann, T., Hevekerl, H., Widengren, J. (2015) Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells. Corresponding AuthorJerker Widengren: jerker@biomolphysics.kth.se.ABSTRACT Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins, and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540) was first analyzed by Fluorescence Correlation Spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics could then also be monitored of MC540 in lipid vesicles, and the influence of lipid polarity, membrane curvature and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photo-induced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. Based on these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics, and that the TRAST imaging technique can provide spatial maps of photo-induced isomerization as well as both photo-induced and thermal back-isomerization, resolving differences in local membrane micro-viscosity in live cells.

Molecular rotors report on changes in live cell plasma membrane microviscosity upon interaction with beta-amyloid aggregates

“…37 The effects of temperature on membranes seen in our experiments are in accordance with previous fluorescence spectroscopy studies, which demonstrated that membrane fluidity is reduced with decreasing temperature. [46][47][48][49][50] Our results confirm the expectation that plasma membrane viscosity of live SH-SY5Y cells responds to temperature and that BODIPY 1 is capable of measuring these changes.…”

Characterization of the Role of the Malate Dehydrogenases to Lung Tumor Cell Survival

“…TRAST measurements were performed on a home-built, epi-illuminated, microscope, the basic configuration and operation principles of which has been described elsewhere 20 . Cytosolic fractions were isolated from cells are described previously 21 , in brief cells were permeabilized using 0.005% digitonin in PBS, incubated on ice for 5min and centrifuged.…”

“…Data analysis: As previously described 20 , the redox kinetics of NADH were determined from so-called TRAST curves, showing how the detected time-averaged NADH fluorescence varied with the excitation pulse-train characteristics (pulse duration)]. In the cellular lysates, 355 nm irradiation resulted in fluorescence from both NADH and flavin compounds in the samples.…”

Isomerization and Properties of Isomers of Carbocyanine Dyes

“…While trans-cis (photo)isomerization of various compounds is widely used in molecular photoswitches, over the past decades, most works have been focused mainly on azobenzene derivatives [63], spiropyrans [64], diarylethenes [65], and stilbenes [66], and, until recently, this property of cyanine dyes has not been applied in practice. The use of cyanine photoisomerization in research is mainly for probing microviscosity of microheterogeneous media [67,68]. On the contrary, rigidization of the polymethine chain of dyes is often carried out by introducing "bridges" that suppress internal conversion and photoisomerization processes to increase the stability of the dyes and the fluorescence quantum yield [17,18,69,70].…”