Evaluation of lateral diffusion of lipids in continuous membrane

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Evaluation of lateral diffusion of lipids in continuous membrane between freestanding and supported areas by fluorescence recovery after photobleaching Azusa Oshima, Hiroshi Nakashima, and Koji Sumitomo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01595 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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Title page

Evaluation of lateral diffusion of lipids in continuous membrane between freestanding and supported areas by fluorescence recovery after photobleaching

Azusa Oshima,*,‡ Hiroshi Nakashima,* and Koji Sumitomo† *NTT Basic Research Laboratories, NTT Corporation, 3-1 Morinosato Wakamiya, Atsugi, Kanagawa 243-0198, Japan †University

of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan

‡Corresponding

author.

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Abstract The lateral diffusion of lipids is a key factor when functionalizing artificial planar bilayer lipid membranes (BLMs). Fluorescence recovery after photobleaching (FRAP) is an established method for evaluating the fluidity of BLMs by the quantitative determination of the diffusion coefficient. However, a BLM with a uniform diffusion coefficient is usually assumed for analysis. In addition, when the BLM to be evaluated is small, the spread of a bleached circle caused by lateral diffusion during the bleaching process and the divergence of the laser used for bleaching interfere with the quantitative analysis. In this study, a numerical calculation was adopted to make it possible to analyze the continuous BLM between freestanding and supported areas, where the diffusion coefficients change depending on the presence or absence of an interaction with the substrate. A quantitative evaluation independent of such bleaching conditions as the bleaching diameter was also ensured by incorporating the spreading effect of the bleached circle in the calculation, which was employed to analyze a freestanding BLM with a diameter of only a few micrometers. By comparing calculations and experiments on FRAP recovery curves, we found that there are multiple diffusion elements and high diffusion barriers at the boundary between the freestanding and supported areas in a BLM over a SiO2/Si microwell substrate.

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I. INTRODUCTION Artificial bilayer lipid membranes (BLMs) have been studied as a simple cell model, since they can be observed under chemical or physical control [Ref.1-3]. BLMs have been combined with a substrate in two different forms. One is where the BLM is supported on a solid substrate, and the other is a freestanding BLM, which covers a hole in the substrate. Because the supported BLMs remain very stable for a long time, their dynamic behavior has

been

investigated

with

various

analytical

methods,

including

fluorescence microscopy and atomic force microscopy (AFM) [Ref.4-6]. The lateral diffusion of lipids in BLMs is an important factor as regards the quantitative evaluation of membrane fluidity [Ref.7-10]. The interaction between BLMs and solid substrates can also be evaluated by observing the lateral diffusion of lipids. On the other hand, a freestanding BLM has been developed for the functional analysis of membrane proteins inserted in it because it separates the surrounding fluid environment into two compartments [Ref.11]. The problem of the low stability of freestanding BLMs was solved by modifying the shape and surface state of the substrate. In recent years, freestanding BLMs on patterned substrates have been extensively studied by combining


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them with semiconductor nanofabrication technology [Ref.12-15]. Ion channel proteins reconstituted in freestanding BLMs on a micropatterned substrate have sufficient space beneath the proteins to allow ions to pass through them, and so their function can be analyzed. We have reported that a freestanding BLM over Si microwells is stable and useful for examining the activity of ion channel proteins such as -hemolysin by using a fluorescent indicator [Ref.15]. Dynamic behavior, such as the phase separation of a freestanding BLM, has also been investigated [Ref.16]. The properties of freestanding BLMs on a patterned substrate, such as fluidity and stability, are dependent on the properties of the surrounding supported BLMs [Ref.17]. This is because it is a continuous BLM consisting of a part supported by the substrate and a freestanding part. The lateral diffusion through the boundary between a freestanding BLM and a supported BLM plays a very important role as regards dynamic behavior. However, thus far artificial BLMs have been characterized separately for the supported type and the freestanding type [Ref.17-19,21]. The evaluation of a continuous BLM consisting of freestanding and supported BLMs is important if we are to develop a platform for analyzing membrane proteins. Fluorescence recovery after photobleaching (FRAP) has been commonly used to quantitatively evaluate the diffusion coefficient of lipids in a supported BLM


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[Ref.19,20,22,]. The analytical method for FRAP is established, and the recovery curves can be explained with a simple equation, if certain experimental conditions are satisfied. On the assumption that the lateral diffusion of lipids in a BLM is isotropic, it can be described by the following diffusion equation[Ref.23].

{

}

∂𝐶(𝑟,𝑡) 1∂𝐶(𝑟,𝑡) ∂2𝐶(𝑟,𝑡) =𝐷 + ∂𝑡 𝑟 ∂𝑟 ∂𝑟2

(1) C(r,t) is the concentration of unbleached fluorophores at position r and time t, and D is the diffusion coefficient. Here, the center of the bleached circle is the origin of the coordinate r. Furthermore, assuming that the bleaching is performed with a uniform circular disk profile beam, the recovery curve can be described by the following equation [Ref.24]. 𝐹N = A ∙ exp (

―2𝜏D

{ (

)

(

)}

𝑡) ∙ 𝐼0 2𝜏D 𝑡 + 𝐼1 2𝜏D 𝑡

(2) FN is the normalized fluorescence intensity 𝐹N(𝑡) = {𝐹(𝑡) ― 𝐹0}/{𝐹 ― ― 𝐹0}. Here, F(t), F0, and F- are the total fluorescence intensities in the bleached area at time t, immediately after bleaching, and before bleaching, respectively. A is a fitting parameter defined as a mobile fraction. I0 and I1 are modified Bessel functions. D is the characteristic diffusion time, which has the following relationship with the diffusion 5 ACS Paragon Plus Environment

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coefficient D. 𝐷 = 𝑤2/𝜏D (3) where w is the radius of the bleached circle. The diffusion coefficient D can be quantitatively determined from the best fit of Eq. (2) with the experimental recovery curves. However, Eq. (2) assumes that the diffusion coefficient of the BLM is uniform over the entire area. With a continuous BLM, it is expected that a freestanding BLM and a supported BLM will have different diffusion coefficients [Ref.19]. Previously, Kocun et al. employed FRAP analysis to evaluate the fluidity of a continuous BLM over a porous substrate. However, they bleached many pores simultaneously when they analyzed the average fluidity of a freestanding BLM and a supported BLM [Ref.25]. In this study, we adopted a numerical calculation to solve the diffusion equation Eq. (1) to take account of the fact that the diffusion coefficient changes from D1 to D2 at the boundary between a freestanding BLM and a supported BLM. The numerical calculation enables us to analyze the fluorescent recovery of a single freestanding BLM. We focused on the lateral diffusion of lipids passing through the boundary between a freestanding BLM and a supported BLM. Based on this analysis, we determined the presence of multiple diffusion elements and high diffusion


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barriers at the boundary between a freestanding BLM and a supported BLM on a SiO2/Si substrate.

II. EXPERIMENT A. Supported BLMs on Si substrate 　 All the lipid molecules were purchased from Avanti Polar Lipids (Alabaster AL). Supported type planar BLMs were prepared by the vesicle fusion method [Ref.26] with large

unilamellar

vesicles

(LUVs)

1,2-dioleoyl-sn-glycero-3-phosphocholine

consisting

of (DOPC).

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rhod-DOPE) at 0.5 mol% was used for labeling the lipid membrane. A chloroform solution of lipids was dried in a glass vial and suspended in PBS buffer solution (pH7.4) at a lipid concentration of 5 mM. The lipid suspension, which consisted of multi-lamellar vesicles, was frozen and thawed five times to reduce the vesicle diameter and to form unilamellar vesicles [Ref.27]. A Si(001) wafer covered with a thermal silicon dioxide layer (120 nm) was used as a substrate. The substrate was cleaned by chemically treating it with piranha solution (H2SO4: H2O2 = 2:1), followed by NH4F for 3 min, and then piranha solution again. Several microliters of the LUVs dispersed in PBS solution was placed on the substrate, which was then incubated for 7 ACS Paragon Plus Environment

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more than 2 h at room temperature to obtain uniformly supported BLMs [Ref.6].

B. Freestanding BLMs over Si microwell Freestanding type planar bilayer lipid membranes were prepared by rupturing giant unilamellar vesicles (GUVs) formed by the electro-swelling method [Ref.28]. The GUVs consisted of DOPC with 0.5-mol% Rhod-DOPE for labeling. A chloroform solution of a lipid mixture with a final concentration of 2.5 mM was spread evenly on an indium tin oxide (ITO)-coated glass slide. The slide was dried in a vacuum for 2 h, and then a chamber was constructed from a lipid-coated slide and an uncoated slide that were coupled with 1-mm-thick silicone rubber. The chamber was filled with sucrose solution (200 mM). The GUVs were grown by supplying an AC voltage of 1 V at 10 Hz for 2 h at room temperature. Microwell patterns were fabricated on a silicon substrate, which was covered with a 120-nm-thick thermal oxide layer, using a conventional photolithographic and dry etching technique. The microwells were circular, 4 or 8 m in diameter, and 1 m deep. Several microliters of the GUV dispersion were added to the PBS solution placed on a substrate. The GUVs sank to the microwell surface and then ruptured to form freestanding BLMs [Ref.29].


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C. Fluorescence imaging and FRAP measurement The sample was observed from the top by using a confocal laser scanning microscope FV1200-BX61 (Olympus) with a 40× objective lens. To acquire fluorescence images, we used a laser light source emitting at 559 nm for excitation, and 575-675 nm band-pass filters to detect the fluorescence from rhodamine. For photobleaching, we used laser light sources emitting at 471 nm and 559 nm, which we scanned in a circle. The typical parameters for the FRAP measurements were as follows; a sampling speed of 0.429 s/frame (2.0 s/pixel; image size, 256×256 pixels), a bleaching time of 0.263 ~ 0.858 s, and bleaching radiuses of 2.48, 4.96, 9.92 and 19.98 m. (These sizes were obtained from pixel units.) Each radius was 80 pixels with a different digital zoom level. In our system, the control software was FV10-ASW, and we used the same laser for bleaching and imaging. Therefore, it was impossible to obtain an image while bleaching. The SiO2 substrate was placed on a microscope slide, and covered with a chamber consisting of silicone rubber and a cover slip. The chamber contained about 300 L of PBS solution. In all the FRAP experiments, it is confirmed that the recovery curves obtained by the three or more independent experiments for each condition were reproduced well.


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III. RESULTS AND DISCUSSION A. Numerical calculation parameters for FRAP analysis First, by analyzing a supported BLM, which was uniform and much larger than the bleaching area, we confirmed the effectiveness of our numerical calculation in this work. We also determined the parameters needed to calculate FRAP recovery curves for comparison with recovery curves obtained experimentally. The analysis of FRAP recovery curves is established, and in many cases the diffusion coefficient can be estimated using Eqs. (2) and (3). Uniform circular bleaching is assumed in this analysis. To minimize the effect of lateral diffusion during bleaching, it is recommended that the bleaching time be sufficiently small relative to the recovery time [Ref.24]. In the experiments, we used the minimum time for bleaching with our system except in special cases. The laser scanned the bleaching area just once and as fast as possible. Figure 1 shows the FRAP analysis using Eqs. (2) and (3) for various bleaching radiuses in the supported BLM on SiO2. Figure 1(a) shows typical fluorescence images obtained during the FRAP measurements: before bleaching, immediately after bleaching, and after almost complete recovery. The experimental fluorescence recovery curve (circles) and the best-fit curve (solid line) of Eq. (2) at that time are shown in Fig. 1(b). The experimental and calculated curves were in good


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agreement. In addition, the mobile fraction, which is defined as the ratio of the fluorescence intensity after a sufficient elapsed time to the intensity before bleaching, was almost 100%. This good fitting and almost complete recovery indicate that the supported BLM was continuous and composed of uniformly diffusing lipids without any obstructions. This good agreement between experiment and fitting was obtained for any bleaching radius. However, the diffusion coefficients estimated with Eq. (3) using D obtained by the best-fit curve of Eq. (2) were strongly dependent on the bleaching radius, as shown in Fig. 1(c). The diffusion coefficients should be the same regardless of the measurement conditions, if they are measured on supported BLMs with the same properties. However, as the bleaching radius decreases, the estimated diffusion coefficients become smaller. This is due to the ambiguity of the bleached radius. The lateral diffusion during bleaching and the divergence of the laser used for bleaching cannot be ignored, especially when the bleaching radius is small. When a small bleached area is required for a size limited BLM such as a living cell, powerful and short laser pulses generated with a special machine setting are often used instead of laser scanning [Ref.30]. In such cases, w, which is treated as the effective “spot size” of the bleached area for a Gaussian beam, causes frequent ambiguities in the analysis. In addition, in some cases the parameter  is used to correct the ambiguity of


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the bleaching profile in the relationship between the diffusion coefficient and the radius of the effective bleached circle as 𝐷 = 𝛽𝑤2/𝜏D, [Ref.19,20].  depends on the bleaching depth and is defined based on a numerical calculation. In this study, instead of analysis with Eq. (2), we adopted a numerical calculation to deal with the different diffusion coefficients for the supported BLM and the freestanding BLM in the continuous membrane. Therefore, we also calculated numerically the ambiguity of the bleaching radius, which is caused by lateral diffusion during bleaching and the divergence of the laser used for bleaching. Based on Eq. (1), we used the following equation to calculate the changes in the concentration of unbleached fluorophores caused by lateral diffusion with respect to both the distance (i) from the center of the bleaching circle and elapsed time (j). 𝐶𝑖,𝑗 = 𝐶𝑖,𝑗 ― 1 + 𝐷

{

1 (𝐶𝑖 + 1,𝑗 ― 1 ― 𝐶𝑖 ― 1,𝑗 ― 1) (𝐶𝑖 + 1,𝑗 ― 1 ― 2𝐶𝑖,𝑗 ― 1 + 𝐶𝑖 ― 1,𝑗 ― 1) + ∆𝑡 ∆𝑟 ∙ 𝑖 2∆𝑟 ∆𝑟2

}

(4) Here, ∆r and ∆t are increments of the distance and the elapsed time in the numerical calculation, and they are set at sufficiently small values to allow us to assume that the fluorophore concentrations are uniform in time and space. In this study, except in special cases, ∆r=0.01 nm and ∆t=1×10-5 s were used. The changes in the unbleached fluorophore concentration were calculated not only in the recovery process but also in


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the bleaching process by adding the bleaching term to Eq. (4) as follows. 𝐶𝑖,𝑗 = 𝐶𝑖,𝑗 ― 1 + 𝐷

{

1 (𝐶𝑖 + 1,𝑗 ― 1 ― 𝐶𝑖 ― 1,𝑗 ― 1) (𝐶𝑖 + 1,𝑗 ― 1 ― 2𝐶𝑖,𝑗 ― 1 + 𝐶𝑖 ― 1,𝑗 ― 1) + ∆𝑡 ∆𝑟 ∙ 𝑖 2∆𝑟 ∆𝑟2

}

―K0 ∙ 𝐼𝑖𝐶𝑖,𝑗 ― 1∆𝑡 (5) Here K0 is a constant indicating bleaching efficiency and Ii is the laser irradiation intensity. As a result, the lateral diffusion during bleaching is incorporated into the calculation. The effect of the divergence of the laser used for bleaching was included in the laser irradiation intensity Ii. The laser intensity ID (r) at a distance r from the center of the bleaching circle is given as follows when we assume that the laser has an ideal intensity distribution. 𝐼D(𝑟) =

𝑃0 𝜋𝑤2

(𝑟 ≤ 𝑤)

𝐼D(𝑟) = 0 (𝑟 > 𝑤) (6) Here, P0 is a constant and it indicates the laser power irradiating in the area w2. However, since the actually used laser has beam divergence, the laser irradiation intensity I(r) diverges from the ideal profile ID(r). Here, we approximate the profile of the laser with a Gaussian distribution as in Eq. (6).


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𝑓(𝑥) =

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( ) 𝑥2

1

𝑒𝑥𝑝 ― 2 2𝜎 2𝜋𝜎2

(7) Here, σ is the standard deviation. Since bleaching is performed by scanning a laser, the laser irradiation intensity I(r) is described by the following equation. 𝐼(𝑟) =

∫

3𝜎

𝐼D(|𝑟 + 𝑥|)𝑓(𝑥)𝑑𝑥 ―3𝜎

(8) The fluorophores are bleached with a certain probability by the irradiation of a laser whose intensity profile is I(r). ∂𝐶(𝑟,𝑡) = ― 𝐾0 ∙ 𝐼(𝑟)𝐶(𝑟,𝑡) ∂𝑡 (9) The laser irradiation intensity 𝐼𝑖 = 𝐼(𝑖∆𝑟) was used in the numerical calculation of bleaching performed with Eq. (5). Since the measured fluorescence intensity is proportional to the unbleached fluorophore concentration C(r,t), the calculated intensity F(t) is obtained by integrating C(r,t) in a bleaching circle with diameter w. F(𝑡) =

1

𝑤

∫ 2𝜋𝑟C(𝑟,𝑡)𝑑𝑟

𝜋𝑤2

0

(10) The fluorophore concentration before bleaching was defined as C(r,t)=1,t

Evaluation of lateral diffusion of lipids in continuous membrane

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