Systematic Variation of Gel-Phase Texture in Phospholipid

Aug 14, 2014 - One obvious question to ask is whether or not such a relationship between texture and domain shape also persists for domains grown to m...
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Systematic Variation of Gel-Phase Texture in Phospholipid Membranes Jes Dreier,† Jonathan Brewer,‡ and Adam Cohen Simonsen*,† †

MEMPHYS - Center for Biomembrane Physics, †Department of Physics Chemistry and Pharmacy and ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark ABSTRACT: The tilted gel phase of lipid bilayers can display in-plane orientational texture due to long-range alignment of the molecular director. We explore systematic variations of texture defects in a series of binary phospholipid membranes. Using polarized two-photon fluorescence microscopy, the texture pattern of single domains is revealed. The appearance of a central vortex-type defect in each domain correlates with a particular range of hydrophobic mismatch values h > 1 nm at the domain border while domains with h < 1 nm correlate with uniformly aligned texture. The central vortex defect is characterized by a defect angle, indicating its bend or splay nature. Using image analysis, we measure the defect angle and find that it has primarily bend character for small mismatch values (h ≈ 1 nm) and primarily splay nature for larger values of h. For domains containing a vortex, the domain shape is decoupled from the texture while for uniformly textured domains there is a preferred texture orientation of ≃45° along the domain border. The results establish a foundation for understanding texture phenomena in compositionally complex membranes.

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INTRODUCTION The lateral organization of biomembranes is closely linked to their biological function and emerges as an interplay between the membrane composition,1 the coupling to external scaffolds,2 and the system dynamics.3 The influential raf thypothesis states that domains play a functional role as platforms for components such as GPI-anchored proteins in biomembranes.4,5 However, a comprehensive understanding of the mechanisms that give lateral structure to biomembranes has not yet been established.6,7 A powerful reductionistic approach is the construction of simplified membrane models to reveal the underlying physical principles governing biomembrane structure.8 The phases of lipid bilayers are characterized by different degrees of order in the acyl chain conformation as well as the in-plane lipid mobility. The liquid disordered (ld) phase has a large acyl chain conformational freedom and high lateral diffusion coefficient (D ≃ (1−2) × 10−11 m2 s−1).9 Conversely, the gel phase is characterized by a slower lateral diffusion (D ≃ (0.01−1) × 10−13 m2 s−1)10,11 and high acyl chain order with a conserved polar tilt angle normal to the bilayer. Specifying the equilibrium phase state of a membrane does not fully account for its lateral structure. Although the area fraction of phases is typically governed by the lever rule,12,13 the domain sizes, shapes, and textures are much more slowly equilibrated. The gel phase, in particular, can display a rich pattern of domain shapes and sizes regulated by the growth kinetics and the spatial arrangement of nucleation points.14 With respect to biological significance, the gel phase has received less attention than the fluid membrane phases, but © 2014 American Chemical Society

studies have indicated that gel domains may have importance for the structure of the skin15 and act as signaling platforms in programmed cell death (apopotosis).16 We have previously established that the tilted gel phase may contain long-range orientational texture patterns in the projection (director) of the acyl chains on the bilayer plane.17−19 The texture of gel domains can have topological defects including integer and half-integer point disclinations as well as line defects. For comparison, textures in domains of Langmuir monolayers are well characterized20−22 and have been modeled by Landau-type expressions.23 Bilayer and monolayer textures share certain similarities with texture defects in liquid crystals in general24 and defects in smectic-C liquid crystals in particular.25,26 Free-standing thin films of smectics can be stabilized over millimeter-sized openings in a solid substrate and may display orientational textures when observed using depolarized reflected light microscopy (DRLM).27−29 The topological defects observed in such Sm-C systems include integer and fractional vortices as well as string defects and combinations of these. An important difference between texture in liquid crystals and lipid domains is that membrane domains are formed through nucleation and growth and have a finite size and a boundary to a surrounding isotropic membrane phase. Membrane textures can be caught in deep metastable states and the observed defect patterns are often linked to details of the nucleation and growth processes. Received: June 12, 2014 Revised: August 11, 2014 Published: August 14, 2014 10678

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glycero-3-phosphocoline (DMPC, C14:0/C14:0, Tm = 23 °C), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, C16:0/C16:0, Tm = 41 °C), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, C18:0/18:0, Tm = 55 °C). The low-Tm lipids were 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC, C18:1/C18:1, Tm = −20 °C), 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, C16:0/ C18:1, Tm = −2 °C), and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC, C12:0/C12:0, Tm = −2 °C). All lipids were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). For texture visualization we use the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan, Invitrogen) at 0.5 mol %. Spin-Coated Supported Bilayers. Binary supported membranes on mica were fabricated by spin-coating, using a previously established procedure.37 Briefly, a dry spin-coated lipid film was prepared using a stock solution of 10 mM total lipid in isopropanol, hexane, and water (60%:20%:20% volume ratio). A droplet (30 μL) of the lipid stock was applied to freshly cleaved mica (Plano GmbH) and spun on a Chemat Technology, KW-4A spin-coater at 3000 rpm for 40 s. The sample was placed under vacuum for minimum 1 h to maximize evaporation of solvents. The freshly coated dry lipid film contains multiple bilayers and exhibits a characteristic dewetting pattern.37,38 The dry sample was hydrated in a HEPES buffer at pH = 7 and heated to above the phase transition temperature. This is followed by a washing procedure to remove all but the proximal bilayer.37,39 Above the phase transition temperature the supported bilayer is uniform and featureless as observed in fluorescence microscopy.14 Subsequently, the sample was cooled at a controlled rate into the two-phase coexisting region. Gel domains nucleate when the temperature crosses the phase boundary to the gel−fluid coexistence region. The number of nucleation sites per unit area increases with an increasing cooling rate.14 As the sample is further cooled, the gel domains grow in size according to the lever rule, while the number of domains remains constant.14,40 The temperature ramp determines the density of nucleation sites and was 0.8 °C/min for all membrane compositions expect those containing DMPC where the ramp rate was 0.1 °C/min. The lower temperature ramp employed for the systems containing DMPC was used to ensure that sufficiently few and large domains would form in these systems. Microscopy. Atomic force microscopy (AFM) was performed in soft contact mode using a JPK, Nanowizard (JPK Instruments AG, Berlin, Germany) at ambient temperature (20−23 °C). Cantilevers where of the type MSCT, lever C, with spring constant = 0.01 N/m (Bruker Corporation). Supported membrane samples were imaged in buffer with the smallest possible contact force to minimize potential membrane deformations. Image Analysis. The domain textures were investigated in a custom-built polarization two-photon fluorescence microscope using Laurdan which aligns with the lipid acyl chains. Each texture map is based on 36 fluorescence images acquired with 10° increments in the polarization angle of the 780 nm excitation light. A Fourier decomposition of the pixel intensities enable construction of texture maps which spatially resolve the azimuth angle φc of the molecular director. Further details of the image analysis were provided previously.17

The fact that orientational textures are prevalent in gel-phase domains represents a new level of complexity in membrane organization, and the possible biological implications of this fact are yet to be explored. A recent study by Hirst et al. has demonstrated long-range orientational order in free-standing membranes of giant unilamellar vesicles (GUVs) composed of the lipid DPPC.30 This work highlights the coupling between membrane shape and topological texture defects. Interestingly, the paper suggests that the crumbled 3D shape of GUVs in the gel phase may be influenced by nucleation of topological defects. In texture studies involving supported membranes, the membrane curvature is constrained, but the planar geometry facilitates easy visualization of the orientational texture field using polarized fluorescence microscopy. This is opposed to curved membranes where the lipid orientation is superimposed on the membrane shape, presenting a considerable challenge for an experimental visualization of texture patterns. The full molecular conformation of lipids in the membrane gel phase is difficult to measure, but molecular dynamics simulations have provided some insight. In the context of the present work it is relevant to briefly review these results. Simulations of the gel phase have been reported both at the atomistic and coarse-grained level giving access to different length and time scales. Early atomistic simulations of the gel phase were restricted to one lipid and one-phase only but provided basic predictions on the lipid packing.31 In some of these studies a so-called cross-tilted (pleated) conformation of the gel phase lipids was predicted.32−34 Newer studies do not reproduce this particular feature of the gel phase and instead report a parallel lipid conformation across leaflets.35 An attempt has been made to model more experimentally relevant binary membranes and demonstrated lipid demixing during formation of the gel phase.36 Simulations of the formation of spatially defined gel domains in a liquid disordered matrix requires a larger simulation system and longer time scale. For this purpose, coarse-grained simulations have shown nucleation and growth of gel domains following a thermal quench of the membrane.10 This study, however, did not display any lipid tilt in the gel phase, obviating the possibility of orientational texture in the simulated gel domains. At present, simulations do not exist which reproduces the nucleation and growth of gel domains with tilted lipids and orientational texture. Experiments therefore provide the main source of information. In this work we systematically explore the variations in gel domain texture that arise due to differences in the acyl chain structure of the lipid components of the membrane. The aim is a more comprehensive picture of the mechanisms that generate specific texture patterns and give rise to subtle variations in the texture defects as a function of the lipid composition. To simplify data interpretation and minimize confounding factors, we use the same physical growth conditions as well as binary membranes having identical phosphatidylcholine (PC) head groups but varying acyl tail length and/or the presence of double bonds. This provides a way to selectively modulate the hydrophobic mismatch between the gel and fluid phases while retaining the headgroup chemistry.

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RESULTS AND DISCUSSION

For this study, three different lipids with a low melting temperature Tm were chosenDOPC, POPC, and DLPC combined with three lipids with a high melting temperature DMPC, DPPC, and DSPC. The lipids with low Tm constitute the main component of the ld phase surrounding domains while the lipids with high Tm will make up the main component of gel domains. In the following we examine systematic variations in the orientational texture of gel domains with respect to the lipid composition. Variation in Texture with Lipid Composition. Prior to measuring the texture patterns, we optimized the experimental conditions for the growth of domains that are suitable for

MATERIALS AND METHODS

Materials. Binary membranes were composed of a lipid with high melting temperature (Tm) and a low-Tm lipid characterized by phase diagrams with a gel−ld coexistence regions covering a wide temperature range. The high-Tm lipids were 1,2-dimyristoyl-sn10679

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of a domain as well. This could possibly point to a partial coupling between the domain boundary and the director orientation in these cases. For the binary membranes in Figure 1G,H containing DMPC, the texture is of a different type. For DOPC, DMPC we find few domains with a completely uniform texture while most domains have a large uniform region connected to smaller areas with other orientations. For POPC, DMPC we find only uniform domain textures where each domain has one single orientation. Differently oriented domains are observed in Figure 1H, which suggests that the uniform texture orientations are not aligned with the crystal structure of the underlying mica substrate. However, some degree of interdomain coupling between uniform domains due to electrostatic dipolar interactions is likely to exist. The shape of the uniformly textured domains in Figure 1G,H is significantly more compact than for domains containing a topological vortex defect. There is some indication that these domains have polygonal shapes and furthermore that the orientation of the uniform director is coupled to the orientation of the polygons. This suggests a link between domain shape and orientational texture in the case of uniform alignment. We note that similar rhombus-shaped gel domains were recently observed in giant vesicles by Petrov et al. in the system of diphytanoyl-PC/DPPC.41 As suggested by these authors, rhombus-shaped domains may reflect a particular molecular organization of lipids. Structure of the Central Defect and Correlation with Hydrophobic Mismatch. The detailed structure of the central defect is accessible from the measured texture maps, and we find that systematic variations in the vortex structure can be linked to the membrane lipid composition. For the domains in Figure 1 which display a central defect, this defect has in all cases the structure of a pair of half-integer point disclinations. We analyze this defect type using the concepts described in reference.17 Briefly, we model a pair of m = 1/2 point disclinations by an expression of the form

texture characterization. The goal was to reproducibly create sufficiently large domains having a size of about 10−20 μm and stabilized as close to ambient (room) temperature as possible. All domains where stabilized at room temperature during texture measurements due to the use of a high numerical aperture (NA) immersion objective in thermal contact with the sample, which cannot be temperature regulated. Air objectives, which are well thermally isolated from the sample, are possible alternatives, but these have an insufficient numerical aperture (NA) and light gathering power for our measurements. There are two main experimental parameters which allow us to modulate domain sizes for a given the temperature: One is the stoichiometric ratio of the lipids which sets the horizontal position in the binary lipid phase diagram, and thereby the relative proportion of the two membrane phases. The second parameter is the cooling rate during domain nucleation which affects the area density of domain nucleation sites14 and hence also the size of the domains at any given temperature. Using these two control parameters, we optimized the growth conditions for each of the different mixtures shown in Table 1. Table 1. Measured Texture Parameters for Gel Domains in the Series of Binary Membranesa lipid composition

ratio

DOPC:DPPC POPC:DPPC DLPC:DPPC DOPC:DSPC DLPC:DSPC POPC:DSPC DOPC:DMPC POPC:DMPC

1:1 1:1 1:1 2:1 2:1 2:1 1:3 1:2

AFM height h (nm)

defect angle φs0 (deg)

± ± ± ± ± ± ± ±

60.5 ± 6.5 73.9 ± 6.6 63.7 ± 6.6 62.8 ± 10.8 48.5 ± 7.8 36.8 ± 12.3 (uniform) (uniform)

1.18 1.32 1.41 1.47 2.13 2.33 0.9 0.6

0.15 0.27 0.15 0.15 0.22 0.26 0.15 0.15

N 10 9 16 13 25 8

a The AFM height represents the height difference h (in Figure 1I) between the gel phase and the surrounding fluid phase, as obtained from >20 domains in each case. The uncertainty given is ±1 standard deviation of the measured values. N is the number of domains analyzed for obtaining φs0.

φs(x , y) =

An overview of typical domain textures for each of the binary phospholipid compositions is shown in Figure 1. Several qualitative features of these patterns can immediately be noted. The membrane samples containing either DSPC (1A-1C) or DPPC (1D-1F) as the high melting temperature lipid all have a qualitatively similar appearance which corresponds well to our observations for the composition DOPC,DPPC.17,18 These domains are all characterized by branched and soft shapes, but with a slightly varying compactness among the different compositions. The domain texture for these samples is characterized by a topological defect in the center of domains, and this defect has recently been shown to coincide with the position of the nucleation site.19 The central defect is a vortex with an |index| = 1 in the far field, but this is split into two closely spaced defects of |index| = 1/2. We discuss the detailed variation in the vortex structure below. For all domains containing a vortex, there is a continuous deformation of the director field close to the center of the vortex. For the membranes containing DPPC, the peripheral regions of the domains are split into distinct subdomains each with a well-defined and nearly uniform orientation of the director. But for the systems containing DSPC there is a significant variation in the director orientation inside each arm

⎛ y − y2 ⎞ ⎛ y − y1 ⎞ 1 1 tan−1⎜ ⎟ + φs0 ⎟ + tan−1⎜ 2 2 ⎝ x − x2 ⎠ ⎝ x − x1 ⎠ (1)

where φs(x,y) is the modeled azimuth angle of the molecular director in the vicinity of the disclination pair. The two defects are located at positions (x1, y1) and (x2, y2). When modeling the experimentally measured textures, the two defect positions are obtained by fitting 2D Gaussian functions to the gradient norm |∇φc| of the measured director angles φc (defined in Figure 1I). The defect positions are identified as local maxima in |∇φc|. We denote the angle φs0 in eq 1 as the defect angle, and it is graphically illustrated in Figure 2A (inset). The value (0°− 90°) of the defect angle determines whether the vortex has primarily bend or splay character, corresponding to φs0 values of 90° and 0°, respectively. The value of φs0 is obtained experimentally from the measured texture maps by fitting to eq 1. The defect angle is a simple quantitative parameter which allows us to describe numerically the state of the vortex defect and its correlation to other membrane properties. Figure 2A,B shows examples of the spatial variation of the director in the vicinity of the disclination pair in the domain center. The domain texture in these images is for membranes with compositions: POPC:DPPC, 1:1 (2A) and POPC:DSPC, 2:1 (2B) and was chosen to illustrate cases where the texture 10680

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Figure 1. Overview of typical domain shapes and textures for gel domains in a series of binary phospholipid membranes (A−H). The specific lipids used for each bilayer system are given as the combination of the column and row names. The lipid stoichiometry used in each case is indicated as (column:row) for each frame. The in-plane orientation of lipids is indicated with black lines in the domains. A schematic illustration of tilted lipids in a gel domain and the geometry of the measurement setup (I). The Laurdan probe in the gel phase (blue lines) aligns with the ordered lipid acyl chains in the gel phase. Laurdan in the fluid phase (green) is randomly oriented. The angle of the molecular director (φc) is experimentally obtained by rotating the polarization direction of the excitation light. The color wheel in (I) gives the color code used to represent textures in (A−H).

pattern has primarily bend (2A, φs0 = 73.9 ± 6.6°) or splay character (2B, φs0 = 36.8 ± 12.3°). AFM imaging of the gel/fluid domain boundary is illustrated in Figure 2C,D for the same two membrane compositions as shown in Figure 2A,B. It is first noted that the orientational texture present in the domains is not revealed by the AFM topography images. The reason being that, to a first approximation, variations in the azimuth angle φc do not couple to the membrane thickness and are therefore not detectable in AFM. Instead, AFM was used to measure the difference in height h (Figure 2C,D) between the gel and ld phases, as a quantitative measure of the hydrophobic mismatch between the phases. The value of h was obtained as an average over parallel lines drawn across the domain boundary. We previously studied the relationship between the total membrane thickness and the AFM topography using imaging ellipsometry.38 Here it was found that the total thickness difference for domains amounts to twice the height difference (i.e., 2h) measured by AFM. This implies that for our supported membranes the protrusion of the gel relative to the fluid phase is symmetrically distributed on both membrane surfaces. The values of h measured by us compare well with previously reported values for binary membranes.37,42 Figure 3 shows the experimental correlation between the hydrophobic mismatch h and the texture defect angle φs0 corresponding to the domain textures in Figure 1. For our set of binary membrane compositions we find that the texture

belongs to one of two types: It is either having a double-vortex type defect or it is uniform. The transition between the two regimes correlates with the hydrophobic mismatch h and the transition occurs for a mismatch around h = 1.0 ± 0.2 nm.19 In the vortex regime where h > 1.0 ± 0.2 nm, the defect angle correlates with the hydrophobic mismatch such that φs0 decreases for increasing values of h. For vortex domains with h close to the threshold of 1 nm, the defect angle φs0 is close to 90° and thus corresponding to pure bend. On the other hand, for large hydrophobic mismatch values the value of φs0 is decreasing toward zero and approaching a splay-type texture for the largest values of h. For reference, the measured values of h and φs0 are given in Table 1. To interpret the correlation between h and φs0, it is relevant to consider the structure of the nucleation core or of the early domain present close to the time of nucleation. We have previously argued19 that the vortex defect is likely to be present in the early domain and that its formation is associated with the process of domain nucleation. On experimental time scales, the texture in late (i.e., micrometer large) domains will not reach equilibrium due to slow lipid mobility in the gel phase. But for a nucleation core with nanometer dimensions, the time scale for diffusion and lipid reorganization across the entire domain becomes sufficiently short that equilibration of the texture is possible. The boundary between a textured domain and the surrounding fluid membrane is characterized by an anisotropic 10681

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Ei =

∮ d s · γ (β )

(2)

As illustrated schematically in Figure 4, the lowest value of the boundary energy will occur if the texture at the domain border

Figure 4. Schematic illustration of the concept of a textured nucleation core (A). The anisotropic line tension of the domain boundary γ depends on the angle β between the director (ĉ) and the boundary normal n̂ (B). A global minimum in γ occurs for the angle βmin(h) and this orientation defines the equilibrium texture of the core. The schematic plot in (C) illustrates the concept behind a variation in βmin with respect to the hydrophobic mismatch.

is everywhere oriented with an optimum angle βmin which produces the lowest value of the line tension. Assuming that the texture of the nucleation core is equilibrated to the boundary, our experimentally measured defect angle φs0 is suggesting the value of βmin. The data for the variation of defect angle in Figure 3 are implying that the minimum in the anisotropic line tension βmin is linked to the hydrophobic mismatch h such that βmin(h). The observation of a correlation between the hydrophobic mismatch and the texture orientation at the boundary of a nucleation core points to an underlying molecular mechanism for the effect. It could be associated with steric constraints for membrane lipids that are close to the gel/fluid domain border in a manner that depends on the thickness mismatch. We remark that it is not realistic to devise a pure experiment where only the hydrophobic mismatch is varied while all other relevant parameters are constant. It is possible that the hydrophobic mismatch is linked to other properties of the lipid bilayer which are ultimately responsible for the variations in texture that we observe. But this is beyond what can be concluded from the present experimental data. The structure of the nucleation core could possibly be investigated with targeted molecular dynamics simulations designed to address the questions above, but as described in the Introduction, simulations of this kind are presently not available. A detailed characterization of nucleation phenomena is an important, but challenging, task in many condensed matter systems. This is primarily due to the short length and time scales associated with the existence of the nucleus. Test of Director Orientation at the Boundary of Large Domains. The results described above reveal a correlation between the defect angle of the vortex and the hydrophobic mismatch at the domain border. This indicates that there is a preferred orientation of the molecular director at the border of a small and compact domain nucleus. One obvious question to ask is whether or not such a relationship between texture and domain shape also persists for domains grown to micrometer dimension as those shown in Figure 1. A quantitative test of this hypothesis was performed and an example of the analysis is shown in Figure 5 for domains in a membrane with composition POPC:DPPC (1:1). Briefly, the domain boundary was identified using a brightness threshold as indicated by the black line of Figure 5A. The normal to the boundary n̂(rb) was then constructed for each pixel on the boundary (Figure 5B),

Figure 2. Examples of measured vortex patterns and corresponding AFM scans for binary membranes with the two compositions (POPC:DPPC, 1:1) (A, C) and (POPC:DSPC, 2:1) (B, D). The double defect texture pattern found in the center of gel domains is shown in (A, B). The two lipid compositions used represent cases where the vortex pattern in the domain center has mainly bend character (A) and splay character (B). The inset in (A) defines the texture defect angle φs0 with respect to the positions of the two point disclinations. The AFM scans in (C, D) include height profiles across the domain boundary from which the step height is obtained as h = h1 − h2. The AFM profiles are constructed as averages over parallel lines inside the white box. The scale bars are 1 μm (A, B) and 2 μm (C, D).

Figure 3. Plot of the texture defect angle φs0 versus the AFM height h. The transition between uniform and vortex texture occurs approximately for h = 1.0 ± 0.2 nm, as indicated by the vertical dashed line. In the vortex regime (right side of dashed line) the defect angle φs0 decreases with increasing h. The solid line is a linear fit to the experimental data in the vortex regime.

line tension γ(β(rb)). We assume that for early domains the lipid orientation at the boundary is equilibrated according to minimization of this line tension. Here β(rb) is the angle between the molecular director c and the boundary normal while rb is a position on the boundary line. The total boundary contributes to the free energy of the nucleation core with a term Ei: 10682

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the present study, domains are more compact than classical DLA clusters pointing to a significant influence of line tension on the domain shapes. A hypothetical possibility for equilibration of the texture relative to the boundary is a fast collective reorientation of the lipids in a domain. This could potentially lead to a more favorable final state of the domain. But we have never observed fast collective reorganization of texture patterns in any of our samples. This suggest that the activation barrier for such a process is quite high, making it unlikely to occur on experimental time scales. We finally remark that for the uniformly textured domains in Figure 1H the histogram of the angle Δb has a broad peak implying a preferred orientation of the director at the boundary with |Δb| = 45 ± 10° (see Figure 6). As previously discussed,

Figure 5. An examination of the orientation of the molecular director relative to the boundary of domains. Panel A shows an example of domain texture in a membrane with composition POPC:DPPC, 1:1. The orientation of the boundary normal (green) in a section of the domain (B), as indicated by the rectangle in (A). The orientation of the director (red) at the boundary (C). The peaked histogram in (D) shows the distribution of the azimuth angle φc, providing the orientation of the director, along the domain boundary for the entire domain in (A). The histogram in (E) shows the difference Δb between the orientation of the boundary normal and the director. Scale bar in (A) is 5 μm.

Figure 6. Histogram of the difference Δb between the orientation of the boundary normal and the director for a domain (inset) in a membrane with composition POPC:DMPC (1:2). Same nomenclature as used in Figure 5.

and the texture azimuth angle φc(rb) was identified for the same boundary pixels (Figure 5C). A histogram of the texture angle φc(rb) in Figure 5D shows three peaks corresponding to six dominating orientations of the molecular director c at the boundary. In contrast, the difference Δb between the texture angle and the orientation of the boundary normal yields a quite uniform histogram as shown in Figure 5E. The analysis of texture at the domain boundary was also performed for the other compositions in this study. All domains containing a vortex defect (Figure 1A−F) showed a uniform distribution of Δb indicating no correlation between the domain shape and the texture. This indicates that although the texture inside a domain may have preferred orientations this is not reflected in a particular domain shape since the boundary orientation is effectively decoupled from the molecular director field in this case. A possible explanation for this effect can be that the boundary shape is dominated by the kinetics of domain growth and that equilibration of the domain shape to the texture (or the opposite) happens on much longer time scales than the phase transformation to the gel state. It is well-known from the theory of aggregation and crystal growth that an advancing solidification front where growth is dominated by diffusion is inherently unstable toward shape perturbations. This was modeled by Mullins and Sekerka43 for diffusion of heat away from the growth front, but the concept applies also to solidification limited by diffusion of chemical components as in the present systems.44 The diffusion-limited-aggregation (DLA) algorithm provides an alternative and conceptually simple realization of the phenomenon of cluster growth.45 In

this is also reflected in the rhombus shape of this class of domains, as seen in Figure 1H. The reason why uniformly textured domains do not develop shape instabilities to the same extent as vortex domains is at present not clear. It may be a consequence of the slower growth rate employed for these domains or their uniform texture which may couple more strongly to the domain shape.

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CONCLUSION In this work we have examined variations in gel domain shape and orientational texture arising due to differences in the acyl chain structure of the lipid components of membranes. We have specifically investigated eight binary phospholipid membranes with gel−fluid phase coexistence. The different lipid compositions result in variations in the hydrophobic mismatch between the gel and fluid phases while the headgroup chemistry is maintained. Using polarized two-photon microscopy, we measured the in-plane orientational texture in the tilted gel phase. The domains were furthermore characterized with AFM providing an accurate quantification of the hydrophobic mismatch h at the domain border. Domains with a mismatch h above ≈1 nm have a vortex defect while domains with mismatch below ≈1 nm display uniform orientational texture. The vortex defect is characterized by a defect angle indicating the bend/splay nature of the defect. 10683

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There is a systematic correlation between the hydrophobic mismatch and the defect angle such that large h correlate with splay while small h correlate with bend. The results gives an indication that the texture of the nucleation core or the early domain is either directly or indirectly coupled to the mismatch value h. It implies that the orientation of the molecular director at the domain border is linked to the mismatch. We also examined the correlation between the domain shape and texture at the border for large domains. We found no systematic relationship for vortex domainsan observation indicating that the domain shape for large domains is primarily set by diffusion-limited growth kinetics and not equilibrated to the texture. For uniformly textured domains there is a trend toward rhombus-shaped domains having texture alignment of 45° relative to the boundary. In summary, our study reveals systematic variations in the domain texture for simple binary membranes. This variability is explained by a possible coupling to the hydrophobic mismatch. The study establishes a useful framework for studying texture phenomena in membranes with higher compositional complexity.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.C.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Experiments were carried out on the facilities of DaMBIC Danish Molecular Biomedical Imaging Center.

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REFERENCES

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dx.doi.org/10.1021/la5023054 | Langmuir 2014, 30, 10678−10685