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. 2018 Nov;10(11):1118-1125.
doi: 10.1038/s41557-018-0127-3. Epub 2018 Aug 27.

A Fluorescent Membrane Tension Probe

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Free PMC article

A Fluorescent Membrane Tension Probe

Adai Colom et al. Nat Chem. .
Free PMC article

Abstract

Cells and organelles are delimited by lipid bilayers in which high deformability is essential to many cell processes, including motility, endocytosis and cell division. Membrane tension is therefore a major regulator of the cell processes that remodel membranes, albeit one that is very hard to measure in vivo. Here we show that a planarizable push-pull fluorescent probe called FliptR (fluorescent lipid tension reporter) can monitor changes in membrane tension by changing its fluorescence lifetime as a function of the twist between its fluorescent groups. The fluorescence lifetime depends linearly on membrane tension within cells, enabling an easy quantification of membrane tension by fluorescence lifetime imaging microscopy. We further show, using model membranes, that this linear dependency between lifetime of the probe and membrane tension relies on a membrane-tension-dependent lipid phase separation. We also provide calibration curves that enable accurate measurement of membrane tension using fluorescence lifetime imaging microscopy.

Conflict of interest statement

Competing Interests.

The authors declare no competing financial and non-financial interest.

Figures

Figure 1
Figure 1. The FliptR probe.
(a) Chemical structure. The carbon bond around which the fluorescent groups (green) can twist is shown in red. (b) Pressure along the axis of the FliptR probe can planarize the two fluorescent groups, leading to changes in excitation maxima and fluorescence lifetime (see text). (c) Fluorescence lifetime τ1 of FliptR as a function of lipid composition in GUVs, from liquid-disordered membrane (Ld) to increasingly liquid-ordered membranes (Lo). Compositions are: DOPC (N=5, R=15), DOPC:CL 60:40 (N=5, R=25), phase-separated DOPC:SM:CL 25:58:17 (N=4, R=5) and SM:CL 70:30 (N=5, R=25). Mean ± SD.
Figure 2
Figure 2. Different FliptR lifetimes correspond to different lipid compositions in cells.
(a) Labelling of MDCK and HeLa cells with time. FliptR mostly stays in the plasma membrane. However, after 2h of incubation, bright spots with low lifetime values (i.e. more blue colors) are seen, probably corresponding to endosomes (N=4). (b) MDCK epithelial cells show longer lifetimes on the apical side (red on the side view) than on the basolateral part (green). The top view corresponds to a single confocal plane (between dashed lines in the left side view schematics), and depending on the height of the cells, either the apical side (red) or the basolateral part (green) are visible (N=4).
Figure 3
Figure 3. Response of FliptR fluorescence lifetime to osmotic shocks on cells and GUVs.
(a) FLIM images of MDCK and GUVs (POPC:SM:CL 57:14:29) in isoosmotic buffer, and after hyper- or hypoosmotic shocks. (b) FliptR fluorescence lifetime τ1 as a function of the osmotic pressure Π applied to Hela cells (formula image, slope hypo= -0.27 ± 0.13 ns·Osm–1, slope hyper: -0.59 ± 0.04 ns·Osm–1, Mean ± SD as in the rest of the measurements, N=20), MDCK cells (formula image, slope hypo= -0.50 ± 0.33 ns·Osm–1, slope hyper: -0.88 ± 0.07 ns·Osm–1, N=6) and GUVs (formula image, slope hypo= -0.19 ± 0.18 ns·Osm–1, slope hyper: -0.22 ± 0.03 ns·Osm–1, N=4), with linear curve fit, black line represents the initial state. (c) To correlate osmotic pressure Π with membrane tension σ, cells were connected to optical tweezers through pulled-out lipid nanotubes, and σ was calculated from the tube force measured in response to osmotic shocks applied. (d) FliptR fluorescence lifetime τ1 as a function of the membrane tension σ applied by osmotic shocks to Hela (formula image, slope hypo= 0.26 ± 0.06 ns·m·mN–1, slope hyper: 0.78 ± 0.14 ns·m·mN–1, N=7) and MDCK cells (formula image, slope hypo= 0.16 ± 0.07 ns·m·mN–1, slope hyper: 2.38 ± 0.18 ns·m·mN–1, N=11) with pulled-out tubes connected to optical tweezers (c), with fit to the linear range before the onset of saturation above τ1 = 5.5 ns).
Figure 4
Figure 4. Possible structural changes of lipid bilayer membranes in response to tension reported by FliptR.
(a) Initial state where FliptR molecules are partially planarized in the membrane. Upon increasing tension, three structural changes could conceivably occur, namely (b) a loosening of the packing with increasing distance between less ordered lipid acyl chains, leading to FliptR deplanarization, (c) a tilting and stretching of more ordered acyl chains, which should lead to more compression of the FliptR probe and thus planarization, and (d) phase separation, in which case, FliptR molecules are more planarized in the Lo phase and less planarized in the Ld phase.
Figure 5
Figure 5. Response of FliptR lifetime to membrane tension in GUVs with and without phase separation induced by micropipette aspiration.
(a) Schematics of the micropipette tube-pulling assay. (b) FLIM images and dependence of lifetimes τ1 on membrane tension σ for SM/CL 70:30 (▲, slope = –1.32 ± 0.19 ns·m·mN–1, N=6 GUVs) and POPC:SM:CL 57:14:29 (■, slope = –0.27 ± 0.05 ns·m·mN–1, N=7 GUVs). (c) Left Panels: FLIM images of DOPC:SM:CL 30:30:40 GUVs aspirated in micropipettes and undergoing phase separation at high tension. Middle Panel: Distribution of the fluorescence lifetimes with Gaussian fits at low tension (Black) and high tension (red). Right panel: average lifetimes (N=8 GUVs) for GUVs at low tension (Black) and at high tension (Red) Error bars and the measure of center is the mean ± SD. (d) Lifetime images of the membrane (confocal image of the lamellipodium) of a Hela cell in isoomosmotic condition (Iso) and after hyperosmotic shock (Hyper), and the corresponding lifetime histograms (right panel) (N=3).
Figure 6
Figure 6

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