Ordered Lipid Domains Assemble via Concerted Recruitment of Constituents from Both Membrane Leaflets

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Ordered Lipid Domains Assemble via Concerted Recruitment of Constituents from Both Membrane Leaflets

Ali Saitov, Sergey A. Akimov, Timur R. Galimzyanov, Toma Glasnov, and Peter Pohl

Phys. Rev. Lett. 124, 108102 – Published 12 March 2020

Abstract

Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment.

Received 5 June 2019
Accepted 26 February 2020

DOI:https://doi.org/10.1103/PhysRevLett.124.108102

Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.

Published by the American Physical Society

Physics Subject Headings (PhySH)

Mechanical properties of membranes Membrane structure

Physics of Living Systems

Authors & Affiliations

Ali Saitov¹, Sergey A. Akimov², Timur R. Galimzyanov², Toma Glasnov³, and Peter Pohl ¹

¹Institute of Biophysics, Johannes Kepler University Linz, Gruberstraße 40, Linz 4020, Austria
²A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, 31/5 Leninskiy prospekt, Moscow 119071, Russia
³Institute of Chemistry, University of Graz, Heinrichstr. 28, 8010 Graz, Austria

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Issue

Vol. 124, Iss. 10 — 13 March 2020

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Images

Figure 1
Domains of all sizes from the two membrane leafs are in register. The bright spots from the two monolayers always coincide: the left column displays micrographs that were obtained by exciting Atto565–DPPE in the cis monolayer; the middle column shows the fluorescence of Atto633–PPE in the trans monolayer of the same membrane at the same time; the right column shows perfect overlap of both channels. The upper and lower rows were obtained in two subsequent experiments at room temperature ( $T = 295 K$ ). The lipid composition was diphytanoyl phosphatidylcholine (DPhPC): dipalmitoyl phosphatidyl choline (DPPC): photoswitchable diacylglycerol (PhoDAG–1): cholesterol $2 ∶ 1 ∶ 1 ∶ 2$ plus 0.004 mol% Atto565–DPPE in the cis monolayer and 0.004 mol% of Atto633-PPE in the trans monolayer. The buffer contained 20 mM HEPES and 20 mM KCl ( $pH = 7.0$ ). The scale bar has a length of $20 μ m$ .
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Figure 2
Photoinduced appearance of LODs and LDDs. Illuminating PhoDAG–1 at 460 nm via a xenon lamp coupled to a monochromator (Polychrome V, TILL Photonics GmbH, Germany) switched the lipid into its trans configuration. Dimmer LODs appeared that were surrounded by bright LDDs (upper row). Back switching of PhoDAG–1 into its cis configuration by exposure to light at a wavelength of 365 nm resulted in the appearance of bright LDDs within dimmer LODs. The number in the upper right corner of each frame indicates the time (in seconds) that has elapsed from the moment of photoswitching. The panels represent a superposition (merger) of the Atto565–DPPE and Atto633–PPE channels. For other conditions see Fig. 1. The scale bar has a length of $5 μ m$ .
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Figure 3
Domain size is dynamic. The growth of a LOD from $d_{a} \approx 1.5$ to $d_{a} \approx 4.5 μ m$ occurred without merger with other domains (upper row, white arrows). Shrinkage of an LDD from an initial $d_{a} \approx 14$ to $d_{a} \approx 11 μ m$ (white arrows, lower row) took place without visible domain patches pinching-off. The numbers in the upper left corner of each frame indicate the time (in seconds) that has elapsed after the photoswitch has been initiated. The panels represent a superposition (merger) of the Atto565–DPPE and Atto633–PPE channels. Experimental conditions were as in Fig. 1. The scale bars have a length of $5 μ m$ .
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Figure 4
Domain tracing by confocal laser scanning microscopy. A representative trajectory (domain, see arrow) is placed between the first and the last images. The time (in s) is indicated in the upper right corner. The scale bars of the images and the trajectory are 20 and $5 μ m$ in length, respectively.
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Figure 5
Size dependence of (i) LOD mobility in LDDs and (ii) LDD mobility in LODs. The symbols indicate experimental data, the lines represent the fits of Eq. (2) to the data. The lipid composition was (i) DPhPC:DPPC: PhoDAG–1:cholesterol $2 ∶ 1 ∶ 1 ∶ 2$ plus 0.004 mol% Atto565–DPPE in the cis monolayer and DPhPC:DPPC: cholesterol $2 ∶ 2 ∶ 2$ plus 0.004 mol% of Atto633-PPE in the trans monolayer (filled circle), and (ii) DPhPC:DPPC:PhoDAG–1: cholesterol $2 ∶ 1 ∶ 1 ∶ 2$ plus 0.004 mol% Atto565–DPPE in the cis monolayer and DPhPC:DPPC:PhoDAG–1:cholesterol $2 ∶ 1 ∶ 1 ∶ 2$ plus 0.004 mol% of Atto633-PPE in the trans monolayer (circle).
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Figure 6
The mobility of lipid molecules in LDDs and LODs measured by fluorescence correlation spectroscopy. Dipalmitoyl-phosphatidyl-ethanolamine anchored ATTO–488 served as the probe. Representative autocorrelation functions of measured fluorescence intensities are shown as a function of time $τ$ in logarithmic scale. $D$ derived from fitting a two-dimensional diffusion model to the autocorrelation function for the lipid probe in LDDs and LODs agreed reasonably well with values that were predicted based on the data in Fig. 5. The inset shows an image of a planar bilayer that has been subjected to fluorescence correlation spectroscopy. The LDD is bright, while the LOD exhibits a dimmer fluorescence. The membrane consisted of one-third of DPhPC, one-third of cholesterol, and one-third of DPPC.
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