Photoinduced Flip-Flop of Amphiphilic Molecules in Lipid Bilayer ...

VOLUME 89, N UMBER 28

PHYSICA L R EVIEW LET T ERS

31 DECEMBER 2002

Photoinduced Flip-Flop of Amphiphilic Molecules in Lipid Bilayer Membranes Thomas Pons, Laurent Moreaux, and Jerome Mertz Laboratoire de Neurophysiologie et Nouvelles Microscopies, INSERM EPI00-02,CNRS FRE 2500, ESPCI, 10 rue Vauquelin, 75005 Paris, France (Received 24 July 2002; published 31 December 2002) We demonstrate localized photoinduced flip-flop of stilbazolium markers in model lipid bilayer membranes. The flip-flop mechanism and dynamics are determined by combined two-photon excited fluorescence and second-harmonic generation microscopy. Upon illumination of labeled membranes with a femtosecond laser beam, two-photon absorption induced photoisomerization provokes a significant increase in the cis- marker population whose flip-flop rate was determined to be at least a thousand times faster than that for transmarkers, allowing the possibility of fast targeted control of the local interleaflet distribution of markers in biological membranes. DOI: 10.1103/PhysRevLett.89.288104

PACS numbers: 87.16.Dg, 42.65.Ky, 82.30.Qt, 87.64.Vv

Azobenzene and stilbene derivatives have generated considerable interest because of their photoisomerization properties and their potential use in the design of optically oriented materials. The photoassisted cycling between trans and cis isomers provokes angular redistributions that allow these molecules to be oriented in polymer matrices. This has led to techniques such as angular hole burning, photoassisted electrical poling, and alloptical poling [1–3], which have been applied to inorganic materials specifically aimed at the development of electro-optical devices. We demonstrate for the first time the application of photoinduced molecular reorientation to biological membranes. Our technique is based on the excitation of membrane markers by two-photon absorption (TPA). It is well known that nonlinear absorption is confined to the focal center of an excitation beam, leading to the spatial localization of fluorescence or photoinduced polymerization. We exploit the inherent confinement of TPA to spatially localize the photoisomerization of amphiphilic stilbazolium markers, leading to their targeted flip-flop in membranes. The flip-flop mechanism is determined from an analysis of the two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) signals produced by the markers in a laser scanning microscope configuration [4]. These signals, acquired simultaneously, provide a direct measurement of the marker distributions in the inner and outer membrane leaflets, with high spatial and temporal resolution. The observed possibility of fast, localized, optically controlled flip-flop of amphiphilic molecules in lipid bilayers leads to a wide range of potential applications including the study of membrane asymmetry dynamics, photoassisted molecular trafficking in membranes, and localized membrane permeabilization (nonlocalized permeabilization, for example, has been reported in [5]). The amphiphilic stilbazolium markers we use here are (E)-4-[2-[4- (dihexylamino)phenyl]ethenyl]-1(4-sulfobutyl)-pyridinium (Di-6-ASPBS for short — see Fig. 1), whose nonlinear properties are well characterized

[4]. The membrane hosts are giant unilamellar vesicles (GUV’s) in water, typically 50–100 m in diam, consisting of dioleoylphosphatidylcholine (DOPC) bilayers that are electroformed and labeled according to a previously described protocol [6]. As opposed to bulk polymer hosts that are isotropic and impart no inherent ordering to guest chromophores, lipid-bilayers impose well-defined alignment directions to amphiphilic markers. The most energetically favorable marker configuration is the trans conformation with molecular axis aligned roughly perpendicular to the membrane plane. Passive (thermally induced) flipping of markers from one leaflet to another is infrequent since it requires the passage of a polar headgroup through the hydrophobic membrane core, which is energetically costly. The time constant for such flipping is given by m eE=kT , where E is the associated energy cost and m is a characteristic time governing molecular orientation fluctuations in a membrane, typically a nanosecond [7]. For Di-6-ASPBS in a DOPC membrane, this time constant was determined to be 2 h at room temperature [4], allowing one to infer E 30kT. Such a barrier is large compared to thermal energies, however it is somewhat smaller than the electronic transition energy of Di-6-ASPBS, which corresponds to optical frequencies. We can therefore expect the possibility of significantly increasing the flip-flop rate of Di-6ASPBS by optical excitation. This possibility is, in fact, borne out. We observed that when we increased illumination intensity the flip-flop rate of Di-6-ASPBS increased more than a thousandfold. Several preliminary observations strongly suggested that photoisomerization was the likely cause of this increase. First, no photoinduced flip-flop was observed when the excitation laser was continuous (de-mode-locked) rather than pulsed, indicating that the flip-flop mechanism depends nonlinearly on intensity and cannot be driven by linear effects such as sample heating caused by linear absorption. Second, we found that the TPEF emission spectrum of Di-6-ASPBS was altered at high excitation

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 2002 The American Physical Society

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FIG. 1. Top: typical SHG image of a GUV segment, and transcis conformations of Di-6-ASPBS. Bottom: TPEF and SHG signals following the application of a flip-flop pulse (solid bar). The recovery of the asymmetry ratio R (gray trace) is due to diffusional exchange of the markers with the un-flip-flopped membrane reservoir.

intensities (data not shown), suggesting that molecular conformations were modified. Third, stilbazolium is already well known to photoisomerize in solution [8]. Fourth, we observed no photoinduced flip-flop when we replaced Di-6-ASPBS by the corresponding ethynyl compound with a triple-bonded linker arm, which cannot be isomerized. We emphasize that a flip-flop mechanism driven by photoisomerization has the peculiarity that it does not involve an applied orientational torque. In this regard it is similar to angular hole burning, however in our case the resulting molecular dynamics differ fundamentally owing to the orientational constraints imposed by the membrane. To fully characterize our flip-flop dynamics, we define an asymmetry parameter Rt Next Nint =Next Nint , where Next and Nint are the number of marker molecules in the external and internal membrane leaflets, respectively. We demonstrate that the relative partitioning of markers in either leaflet can be both controlled and monitored with high spatial resolution using a mode-locked Ti:sapphire laser beam ( 100 fs pulses at 80 MHz; wavelength 830 nm; focus waist w 0:5m). In particular, we studied the dynamics of photoinduced flip-flop using a series of illumination protocols applied to freshly labeled GUV’s. Since our GUV’s were labeled by external perfusion, the markers initially populated only the external leaflet of the membrane bilayer [i.e., R0 1]. Our laser power at the sample was con288104-2

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trolled by an electro-optic modulator, and could be rapidly toggled between low ( < 2 mW) and high (typically 10–20 mW), corresponding, respectively, to ‘‘observation’’ and ‘‘flip-flop’’ powers. The laser power was continuously monitored by a photodiode to correct for ringing or thermal drift in the modulator (i.e., since the TPEF and SHG signals both scaled with the laser power squared, we normalized these to the square of the photodiode signal). A first illumination protocol consisted in rapidly and repetitively scanning over a small segment of GUV membrane, typically 20 m in arc length, using low excitation power with the laser polarization directed perpendicular to the patch. A specific point in the sample was then illuminated for a duration Tw2 =2S for every scan frame, where T was the frame duration ( 20 ms) and S the frame area ( 200 m2 ). After an initial observation period, the laser power was switched to high for approximately 1 sec, and then returned to low (Fig. 1). Immediately following the application of the high power flip-flop pulse, both the TPEF and SHG signals were observed to have been significantly reduced. The TPEF signal rapidly recovered to its initial prepulse level with a time constant 0 200–300 ms, whereas the SHG signal required a much longer time to recover, typically several seconds. The interpretation of these results is as follows: before the application of the flip-flop pulse, the Di-6ASPBS markers essentially all resided in their trans conformation (a 99% trans fraction has been measured in water [8], and can be expected to be even higher in membrane). During high power illumination the marker equilibrium was presumably displaced toward the cis conformation because of photoisomerization, leading to a reduction in the TPEF signal since the cis isomer was both less fluorescent than the trans isomer and less likely to be parallel to the excitation polarization. Upon completion of the flip-flop pulse, the TPEF rapidly recovered because of fast cis ! trans marker relaxation, as favored by the membrane environment, with rate constant ct 1 0 . In the specific case when all markers are definitely trans, then the TPEF signal scales as the total number of markers N Next Nint , and the SHG signal scales as Next Nint 2 (we will consider the more general case below). We emphasize that the rapid and full recovery of TPEF shown in Fig. 1 indicates that the application of the flip-flop pulse provoked no observable photodegradation of the markers, meaning that Nt remained effectively unchanged during the illumination protocol. The asymmetry parameter is then simply Rt p SHGt=SHG0, and is plotted in Fig. 1. The observed drop in Rt is evidence that the application of the flip-flop pulse provoked a substantial equilibration of the external and internal leaflet marker populations. The slow recovery of Rt resulted from the fact that the photoinduced marker redistribution was confined to only the small portion of the membrane that was illuminated, whose 288104-2



surface area was typically 20–30 m2 (TPA was axially confined here to about 2–3 m). When the flip-flop pulse was turned off, Rt gradually returned to its original level because of diffusional exchange between this small surface area and the remainder of the GUV membrane that was not illuminated (i.e., whose markers had not undergone flip-flop), which effectively played the role of an asymmetry reservoir. Numerical solutions to the diffusion equation in two dimensions indicated that the observed recovery of Rt corresponded to a marker diffusion constant of about 109 cm2 =s, in accord with previous measurements [9]. We note that while the entire GUV surface was large compared to the illuminated patch, it was not infinite. In particular, the repeated application of flip-flop pulses over the entire equator of small GUV’s was observed to eventually deplete the reservoir of un-flip-flopped markers, leading to an extinction of diffusional recovery. The above illumination protocol allowed us to study the marker recovery dynamics immediately following the application of a flip-flop pulse. The study of marker dynamics during a flip-flop pulse was more difficult because high illumination intensities possibly led to saturation of the marker excitation or signal detection, leading to artificially increased excitation volumes [10]. To ensure that the marker observation was always conducted under identical low-intensity illumination conditions, we introduced observation ‘‘windows’’ within our flip-flop pulse by alternating our laser power between high and low every image frame, allowing us to effectively probe the trans-cis transition dynamics. An example of the TPEF-SHG signals acquired during these observation windows is shown in Fig. 2. We distinguish two time scales: a rapid decrease in both TPEF and SHG with a time constant typically less than 200 ms at the onset of the flip-flop pulse, followed by a slower continued decrease in the SHG signal. To interpret these dynamics we must take into account the contribution of a cis population to both TPEF and SHG during the application of the flip-flop pulse. Cis isomers are known to fluoresce [11], as was verified by an observed change in the TPEF spectrum upon high intensity illumination. We did not determine whether cis isomers also produced SHG, however this possibility could not be neglected. Referring to Fig. 2, we define the TPA rate constants for the trans and cis isomers as t I 2 and c I 2 , where t;c are the respective TPA cross sections, and I2 is the temporal average of the excitation intensity squared (I 2 gI 2 , where g 4 104 for our configuration, taking into account laser pulse broadening in our optics). We further denote t;c as the fluorescence quantum efficiencies for the corresponding isomers. Finally, we define the effective SHG hyperpolarizabilities of the isomers to be t;c (see [12]). Over time scales longer than 0 , the markers in both membrane leaflets are assumed to have attained the same trans-cis equilibrium. 288104-3

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FIG. 2. Top: TPEF and SHG signals, and inferred asymmetry ratio R (gray trace) before, during, and after the application of a flip-flop pulse (protocol with alternating high and low illumination powers). Bottom: Energy level diagram depicting flipflop mechanism (variables defined in text).

That is, if we denote the net fraction of markers in the cis conformation as xc , the numbers of cis isomers in the external and internal leaflets becomes xc Next and xc Nint respectively, and the TPEF and SHG signals are TPEFt Nt 1 xc t 1 c c ; (1) TPEF0 N0 t t s SHGt RtNt c 1 xc t 1 : SHG0 N0 t

(2)

In the case of no photodegradation, Nt remains fixed and a general expression for the asymmetry parameter is p SHGt=SHG0 Rt ; (3) 1 1 TPEFt=TPEF0

where the constant 1 c =t = 1 c c =t t was readily determined from Eq. (3) by examining the TPEF and SHG signals shortly after the onset of the flip-flop pulse, that is, after the trans-cis equilibrium had been attained but before significant flip-flop had occurred, in which case Rt 1. We found 1:7. The dynamics of Rt for longer times are plotted in Fig. 2, and illustrate the progressive equilibration of the external and internal populations during the course of the flip-flop pulse. After completion of the flip-flop pulse, Rt underwent a slow diffusional recovery to its prepulse value of 1, as before. 288104-3



The flip-flop pulse protocol described above allowed us to quantify the experimentally relevant parameters governing photoinduced flip-flop. The coarse-grained dynamics (i.e., over time scales longer than 0 ) of Rt upon laser illumination is approximated by the simple decay law dR=dt kf R, where kf kc xc is the net rate constant for flip-flop from one leaflet to another, mediated in turn by kc , the cis isomer flip-flop rate constant. We note that for all flip-flop pulse intensities used in our experiments, at least a few scan frames were required for the TPEF signal to reach equilibrium, indicating that the isomerization dynamics were slow enough to allow the approximation: xc

t I 2 ; I 2 Is2

(4)

where t t tc =t tc c ct , tc;ct are the trans ! cis and cis ! trans photoisomerization quantum efficiencies, and Is2 T=g ct =t tc c ct . The experimental values of kf , obtained by measuring the decay of Rt provoked by the application of flip-flop pulses of different intensities, are shown in Fig. 3. We observe that kf initially increases quadratically with intensity, as expected since it is based on TPA, and then plateaus, in accord with Eq. (4). A numerical fit indicates that t kc 0:5 s1 and Is 13 mW=m2 . We point out that Is corresponds to the frame-averaged illumination intensity beyond which the flip rate is at its maximum (if the excitation beam were not scanning and parked on the membrane then Is2 would be about 400 times smaller). Finally, t and kc can be estimated separately by noting that kf is proportional to 1-TPEFt=TPEF0, from Eq. (1). We deduce from this relation that kc = 1 c c =t t 1:3 s1 . Since the TPEF signal was observed to equilibrate to as low as 60% of its initial value, we infer that 0 < c c =t t < 0:4, and hence 1:3 < kc < 0:8 s1 . We then conclude that 0:4 < t < 0:6. The parameter t represents the maximum equilibrium fraction of cis markers that can be produced by photoisomerization, and corresponds here to a roughly balanced distribution between cis and trans populations. We note that our recovery of photoinduced flip-flop is in many ways reminiscent of the well-known technique of fluorescence recovery after photobleaching (FRAP) [13]. In both cases, a localized perturbation is provoked in the sample, and the dynamics of the recovery can provide information on the local sample environment. In the case of photoinduced flip-flop, the perturbation is in the local molecular asymmetry in a membrane. Other techniques exist that can modify molecular asymmetry, however these involve chemical agents [14]. As shown in this report, a unique advantage of photoinduced flip-flop is that it can be localized and targeted, in principle to micron resolution. A further advantage specific to non-

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FIG. 3. Measured flip-flop rate constant as a function of flipflop pulse illumination intensity at sample, and fit from model.

linear excitation, aside from its being intrinsically localized, is that the concomitant TPEF and SHG signals provide a direct measure of the molecular asymmetry dynamics, which neither signal can provide alone. Our use of these signals has allowed us to validate for the first time, to our knowledge, the occurrence of photoinduced flip-flop of amphiphilic molecules in biological membranes. The observed dynamics of this flip-flop entirely agree with a model based on a mechanism of photoisomerization. We are very grateful to M. Blanchard-Desce (l’Universite´ de Rennes 1) for having provided us with the marker molecules used in these experiments.

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