Polka-Dotted Vesicles: Lipid Bilayer Dynamics and Cross-Linking

(1-7) Veatch and Keller have studied lipid bilayer phase shifts and spinodal decomposition, which depict a membrane where small perturbations lead to ...
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Polka-Dotted Vesicles: Lipid Bilayer Dynamics and Cross-Linking Effects Michael S. Kessler,† Robin L. Samuel,† and Susan D. Gillmor*,† †

Department of Chemistry, 107 Corcoran Hall, George Washington University, 725 21st Street, N.W., Washington, D.C. 20052, United States S Supporting Information *

ABSTRACT: We have investigated the effects of cross-linking perturbations on lipid phase-domain coalescence. Our model system explores cross-linking in the fluid-disordered phase of two-phase vesicles. Here, we quantify the vesicle population shift from the expected predominance of twodomain, two-phase configuration to a multidomain vesicle majority. We have found that the increase in multidomain vesicles is a distinct outcome from the cross-linking of biotinylated lipids and avidin. Analysis of our cross-linking data suggests that avidin forms clusters on the surface of the fluid-disordered domains, resulting in a large immobile fraction and restricted diffusion. In cellular membranes, receptor concentrations are similar to our experimental model, and we expect similar cluster formations, leading to nonideal mixing and lateral heterogeneity. We have induced and quantified a global response by cross-linking only a small percentage of lipids in our system, similar to receptor−ligand interactions on the cell membrane. Common activities, such as ligand−receptor coupling, contribute to lateral heterogeneity and membrane protein clustering, adding to cell membrane complexity. Fundamental studies into subtle shifts such as cross-linking events, which induce global cellular response, are pertinent to understanding membrane activities and effects of external stimuli.

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INTRODUCTION Cell membranes act both as a barrier between the cytosol and the extracellular space and as the site for protein−ligand interactions, signal transduction, and endo- and exocytosis. In exploring these roles, recent debates have centered on the lateral heterogeneity of lipids and proteins that allow for these various actions.1−7 Veatch and Keller have studied lipid bilayer phase shifts and spinodal decomposition, which depict a membrane where small perturbations lead to phase transitions.8−11 Dynamic changes in membranes fuel the discussion on the form and consequence of nanoscale composition fluctuations known as cholesterol-enriched transient zones or “rafts”.3−5,8 When cross-linking occurs at lipid compositions close to a phase transition, biomembrane constructs, or vesicles, have been observed to transition from one phase into two phases.12,13 While previous work on cross-linking-induced phase separation has focused on induced phase separation in vesicle examples,12−16 we analyze cross-linking behavior as a shift in vesicle population. Our study reveals that cross-linking induces an increase of multidomain vesicles as compared with the expected two-phase two-domain outcome. These findings indicate that the cross-linking agent, avidin, perturbs phasedomain coalescence. In addition to inducing phase separation,12,13,17 these results indicate that cross-linking in a twophase system also stabilizes islands of differing compositions, analogous to the shift from a transient raft to a stable receptor platform. In a study focusing on plasma membrane spheres, Lingwood, Simons, and co-workers have analyzed the response to © 2013 American Chemical Society

ganglioside GM1 and cholera toxin B (CTB) cross-linking, which reorganizes the membrane into liquid-disordered and liquid-ordered (Ld and Lo) domains globally. Membrane proteins then segregate into these phases according to their packing preferences.13 Hammond et al. have shown similar findings in model membranes, where GM1-CTB cross-linking induces phase separation and drives transmembrane peptide redistribution into Lo or Ld phases.12 These results are relevant to many biological membrane systems. However, they are individual examples instead of finding the overall response to an external stimulus. By analyzing the population shift in a twophase system, we investigate the general response of a specific stimulus, avidin cross-linking of lipids, and study the shift in behavior. The population response allows us to draw parallels from our model to biological examples, instead of highlighting one vesicle transformation. In this report, we examine multidomain vesicle configurations stabilized through avidin−biotin cross-linking. We have investigated two-phase vesicles 24 and 48 h after crosslinking. For our system (DOPC:DPPC:cholesterol, 1:1:1 (mol/ mol); dioleoylphosphocholine, DOPC; dipalmitoylphosphocholine, DPPC), two fluid phases, Lo and Ld, exist at room temperature (23−25 °C).10 We introduce 3.3 mol % biotinDOPE (1,2 dioleoyl-sn-glycero-3-phosophethanolamine-N-biotinyl) into the lipid composition and expose vesicles to avidin to mimic cross-linking in biological systems. For our avidin− Received: October 23, 2012 Revised: January 4, 2013 Published: January 29, 2013 2982

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Figure 1. Quenching rates and categorizing vesicles. (A) Quenching two-phase (Ld/Lo) vesicles. After cross-linking and incubating at 45 °C for 60 min (above the Tm of the Ld/Lo vesicle system), the vesicles are quenched. The rate of temperature change is compared between room temperature (24.6 ± 0.2 °C, - • -) and cold (12.7 ± 0.1 °C, - - -) water baths and air quenching (25.7 ± 0.1 °C, ). (B) A cross section of a wide-angle image is used to count and categorize the vesicles. (C) In a single-phase vesicle the dye (DiD) is evenly dispersed in membrane. (D) In a two-phase vesicle both Ld and Lo are present in two domains. (E) A two-phase vesicle with multiple domains represents incomplete domain coarsening. (F) A polkadotted vesicle is shown in a three-dimensional rendering. The dye (DiD) partitions in to the Ld phase, allowing us to image the vesicles and record their phase configuration. solvent ratio of 9:1 (v:v). For the cross-linking experiments, we used a lipid ratio of 0.1:1:1:1 for biotin-DOPE:DOPC:DPPC:cholesterol. The fluorescent dye (DiD) was incorporated at an overall 0.08 mol % compared to the lipids. The solvated lipids and dye were mixed thoroughly to obtain homogeneity. Small droplets of the mixed sample (∼2 μL) were applied to two Pt wire electrodes, each with a diameter of 1.2 mm. The lipid cake was applied as discrete droplets and allowed to evaporate. Once both electrodes were coated with ∼10 μL of sample, the Pt wires were placed under vacuum for 2 h to ensure complete solvent evaporation. A nonelectrolyte buffer solution of freshly prepared 2% sucrose was heated to 80 °C and combined with the Pt wires in a plastic cell. Multiple electrodes were then connected in parallel to a Hewlett-Packard waveform generator. The sample temperature was maintained at 80 °C during the entire process. The electroformation procedure began at 0.7 V with a frequency of 10 Hz. In a stepwise fashion, the voltage was increased 0.05 V every 5 min up to 1.4 V, where it was maintained for 3 h. A final step of 0.6 V and 4 Hz was used to separate the vesicles from the wires. Sample cells were then removed from the oven and allowed to cool slowly (overnight for +12 h) to room temperature. We have evaluated the effect of 4.5 h at 80 °C on DOPC vesicles via mass spectrometry and see no evidence of oxidation. The high temperature does give us a higher proportion of two-phase vesicles (data not shown). Cross-Linking and Quenching. Three aliquots of 300 μL of fully cooled vesicle suspensions in 2% sucrose solution were heated to 45 °C in a constant temperature water bath. Avidin from 1.0 mg/mL stock solution was added into two of the aliquots (0.50 μL or 0.50 μg (1.67 μg/mL, 25 nM) and 0.75 μL or 0.75 μg (2.50 μg/mL, 38 nM)). The third aliquot acted as the control, and no avidin was added. The vesicle suspensions were maintained at 45 °C for 1 h to allow ample time for the avidin−biotin interaction. The aliquots were quenched at varying rates and allowed to equilibrate to room temperature before assembly onto microscope slides for imaging. For the cold (12.7 ± 0.1 °C) quench, samples were put directly into a water bath maintained at 12−14 °C for 30 min. The room temperature (24.6 ± 0.2 °C) and nonspecific binding samples were placed into a water bath of 22−25 °C. The air-quenched samples were not placed in a water bath but

biotin−DOPE system, the association between the ligand and protein is exceptionally strong (KD = 10−15 M) with a slow offrate (koff = 9 × 10−8 s−1),18 which allows us to assume a stable linkage. Further, the geometry of avidin and the location of its biotin binding sites limit its cross-linking to two sites on one vesicle.19 By limiting the avidin in our system (2.50 μg/mL, 38 nM), we ensure a subsaturating amount of bound avidin within a given vesicle and minimal cross-linking between them. In contrast to the two-phase two-domain outcome in the controls, cross-linking results in a significant fraction of multidomain vesicles, which we examine and quantify. We find that the shift from majority two-phase two-domain to multidomain vesicles is due to the specific binding of avidin to biotinylated lipids. Further analysis of our data shows that cross-linking inhibits coalescence and mobility, leading to incomplete domain coalescence in the vesicle population. These results suggest that protein-induced cross-linking may regulate and stabilize domain sizes in biomembranes that lead to lateral heterogeneity and to increased membrane complexity.

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MATERIALS AND METHODS

Materials. The following lipids were obtained from Avanti Polar Lipids (Alabaster, AL) and used without modification: 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC), cholesterol, and 1,2-dioleoyl-sn-glycero-3phosophethanolamine-N-biotinyl (biotin-DOPE). From Invitrogen (Carlsbad, CA), the following dyes were used without modification: 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD), 3,3′-dilinoleyloxacarbocyanine perchlorate (DiO), and avidin Alexa Fluor 488 (avidin-488). Vesicle Preparation. Formation of giant unilamellar vesicles (GUVs) via an electrochemical cell was described previously.20 GUVs were synthesized using electroformation with modified procedures adapted from earlier works.21,22 The lipids were combined in a 1:1:1 molar ratio of DPPC:DOPC:cholesterol in a chloroform and methanol 2983

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taken out of the 45 °C cross-linking bath and allowed to equilibrate to room temperature (24−27 °C, see Figure 1a). Air quenching gave inconsistent data, and all results presented in this report have been prepared using water bath quenching. Microscope Slide Preparation. Slides were prepared using 150 μL of the GUV suspension that was placed into a spacer (0.25 mm depth, 2.5 cm diameter) on a standard microscope slide and sealed with a coverslip (micro cover square glass No. 1 VWR). Vesicles in the prepared slides were allowed to settle and reach thermodynamic equilibrium for 24 h prior to imaging and were again imaged 48 h after cross-linking. All samples were stored at 22−25 °C between viewings. Supported Lipid Bilayer Preparation. To form supported lipid bilayers, we modified previously published protocols23−25 to fuse small unilamellar vesicles (SUVs) onto clean glass surfaces. For SUV preparation, the appropriate lipids, in predetermined molar ratios (DOPC alone or DOPC:biotin-DOPE (95:5)), were mixed and dried under nitrogen in a test tube previously cleaned with base bath (ethanol−potassium hydroxide saturated solution) and rinsed several times with distilled water. When measuring the diffusion rate of the lipids, DiO was mixed in with the lipids at 0.136 mol %. The lipid cake was further dried under vacuum for 2 h. Next, the vesicles were swelled by adding 1 mL of heated (∼80 °C) 2% sucrose solution for a total lipid concentration of 2.5 mg/mL. The samples were incubated at 80 °C for 15 min, followed by 30 s of vortexing. These steps were repeated until the sample was vortexed a total of three times. After sonicating for 15 min in an ultrasonic water bath cleaner (Fisher Scientific, Pittsburgh, PA), the sample was passed through a nitrogen gas-powered extruder (Northern Lipids LIPEX Extruder, Burnaby, British Columbia) with a membrane pore size of 100 nm. To obtain SUVs (with a radius below 25 nm), the sample required a minimum of five extrusions. This process produced SUV’s with an average radius of 5.5 ± 0.81 nm as measured by dynamic light scattering (DLS, Wyatt Technologies, Santa Barbara, CA). The SUVs were fused into supported lipid bilayers with 1 M calcium chloride solution on a glass bottomed well plate that had been cleaned with a dilute base solution (1% KOH). After 1 h of fusion and equilibration, excess SUVs and calcium solution were removed by washing a mimimum of five times with ∼150 μL (750 μL total) of 2% sucrose solution. Unlabeled or labeled avidin was added at room temperature and was allowed to cross-link overnight for a final concentration of 1.0 μg/mL or 15 nM. Microscopy. An inverted Zeiss LSM 510 confocal miscroscope was used with a 63× 1.2 NA water objective to image the vesicles. The dye (DiD) was excited using a HeNe 633 nm laser, and the image was obtained by collecting the emissions from 650 to 750 nm. For FRAP experiments, imaging was conducted using 3% power of a 30 mW argon 488 nm laser line, and bleaching was conducted using 100% power of the same laser. Vesicle Categorization and Chi-Squared (χ2) Analysis. To characterize the vesicle population phase configurations, vesicles are imaged in the maximum window views and large z-stacks to document as many vesicles as possible in one image stack. Figure 1B shows a single cross section. To catalogue our vesicles, we separate them into three different types and record the numbers of each. Although we have chosen lipid ratios (1:1:1 DOPC:DPPC:cholesterol) that partition into two phases at room temperature, the vesicle population displays a variety of phase configurations. We observe single-phase vesicles, two-phase two domain, and two-phase multidomain vesicles, as illustrated in Figure 1B−E. These vesicle data give rise to categorical data for populations. Chisquared (χ2) is used to determine the statistical significance of the experimental findings. Comparing the two-domain population to the multidomain vesicle population is the purpose of the categorization. Only the two-domain and multidomain vesicles are used for the χ2 calculations, allowing for a straightforward analysis.26 A 4-fold table (shown in Table S1) compares the frequencies of multiple domain and two-domain vesicles between different experiments. We have analyzed the effects of avidin concentration, time, and quenching temperature on the distributions of phases in the vesicles. To determine χ2 in the 4fold table, we calculate the following:

X2 =

N (ad − bc)2 (a + b)(c + d)(a + c)(b + d)

(1)

where a, b, c, and d are the different vesicles population values for twophase and multidomain vesicles between control and variable experiments and N is the sum of a, b, c, and d. From the χ2 calculations, we then determine the p-value from distribution tables. For (χ2) values above the given 5% probability level (3.841) the null hypothesis is rejected. For the chi-squared test, the null hypothesis stipulates that the data are independent from the differing variable between the two groups tested. The p-values or probabilities (from 0 to 1) infer that for two data sets a random sampling will give similar or different results. High p-values indicate that the data sets are similar, while low (p < 0.05) indicate independence between data sets.26 Calculated p-values are shown in Table S2. Fluorescence Recovery After Photobleaching (FRAP). Using FRAP (fluorescence recovery after photobleaching), we have determined the mobility and diffusion properties of lipids in the Ld phase and the cross-linking protein, avidin. We have recorded the recovery of fluorescence signal after bleaching a small area on DOPC only or DOPC:biotin-DOPE (95:5) bilayers both with and without avidin. We have conducted the same procedure for DOPC:biotinDOPE (95:5) bilayers that have been cross-linked with labeled avidin488. The dye (DiO or avidin-488) is excited, imaged, and bleached with a 488 nm argon laser. Time and intensity data are collected for two coconcentric regions of interest (ROIs): (1) the bleach area (radius of 5 pixels) and (2) the surrounding area (r = 75 pixels). ImageJ (NIH Bethesda, MD) and MatLab (Mathworks Natick, MA) are used to analyze the data. A full description of the FRAP analysis is available in the Supporting Information.

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RESULTS

Many studies on liquid-ordered membranes and plasma membranes have suggested nanoscale heterogeneities.4−8 As such, we test cross-linking behavior to introduce lateral composition variations in the form of multiple phase domains. A two-phase system leads to a straightforward outcome of twophase two-domain or two-phase multidomain populations. We analyze the population shift to understand the overall phase configuration behavior instead of focusing on individual vesicle transformations. To test the effects of cross-linking on a two-phase vesicle composition, we form vesicles of DOPC:DPPC:cholesterol (1:1:1 mol/mol) and incorporate 3.3 mol % biotin-DOPE for a final composition ratio of biotin-DOPE:DOPC:DPPC:cholesterol (0.1:1:1:1). To maximize consistency, we gently heat the vesicles to 45 °C, which is well above the transition temperature for the Lo to Ld phase transition (Tm = 29 °C,10), introduce the cross-linking agent (avidin), and maintain it for 60 min. The temperature is rapidly reduced by placing the samples in a water bath. When we compare the cooling curves in Figure 1A, the water baths reach temperature equilibrium within ∼10 min. This difference in quenching temperature is statically insignificant although the step of quenching is key to consistent data, when the vesicles are categorized and counted (see Materials and Methods section for categorization details). All imaging is conducted at room temperature. When we incorporate biotin-DOPE into the bilayer, the avidin−biotin association occurs in the Ld phase of Ld−Lo vesicles, which is confirmed in Figure S1. Cross-linking in the Ld phase results in a significant increase in multidomain vesicles, shown in Figures 2 and 3. To analyze the population shift from two-phase two-domain to multidomain vesicles, we calculate chi-squared (χ2, eq 1) and determine the overall probability or p-values. High p-values (p > 0.05) indicate that 2984

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Figure 2. Comparison of cross-linked vesicles quenched to 25 °C. We characterized two-phase vesicles (Ld/Lo) with biotinylated lipids (biotin-DOPE) exposed to two avidin concentrations as well as no avidin present (controls). As an additional control, we also compare our results to nonspecific-binding (NSB, no biotin-DOPE, Figure 5) experiments. All samples are quenched from 45 to 24.6 ± 0.2 °C in a water bath. We show the distribution of each phase configuration (single phase (white), two-phase two-domain (gray), and multidomain vesicles (black)) as a percentage of the vesicle population. The insets show examples of each vesicle category. From these data, we observe that (A) at 24 h avidin cross-linking results in a significant increase of multidomain vesicles over non-cross-linked (no avidin, control) samples. In comparing the cross-linked two-phase vesicles to the controls, we calculate p-values of 0.0001 (∗) for both 1.67 and 2.50 μg/mL avidin, confirming that the cross-linked samples are quite different from the controls. In contrast, when we add 2.50 μg/mL of avidin to vesicles without biotin-DOPE (nonspecific binding, NSB samples) and compare them to the controls (biotinylated lipids, no avidin), the p-value increases to 0.42 (∗∗), indicating the two vesicle populations are quite similar. See the text for a detailed discussion. (B) After 48 h, there are slight changes for all samples compared to the 24 h data. The cross-linked samples maintain their high portion of multidomain vesicles. The p-values for the cross-linked samples compared to the controls are unchanged (0.0001, ∗) from the 24 h analysis. For the NSB vesicles compared to the control, the p-value decreases to 0.046 (∗∗).

Figure 3. Comparison of cross-linked vesicles quenched to 13 °C. We characterized two-phase vesicles (Ld/Lo) with biotinylated lipids (biotin-DOPE) exposed to two avidin concentrations as well as no avidin present (controls). All samples are quenched from 45 to 12.7 ± 0.1 °C in a water bath, and we report the distribution of each phase configuration (single phase (white), two-phase two-domain (gray), and multidomain vesicles (black)) as a percentage of the vesicle population. (A) Similar to Figure 2a, at 24 h, avidin cross-linking results in a significant increase of multidomain vesicles over non-crosslinked (control) samples. In comparing the cross-linked two-phase vesicles to the controls, we calculate p-values of 0.0093 (∗) for 1.67 μg/mL of avidin and 0.0001 (∗∗) for 2.50 μg/mL of avidin. (B) After 48 h, there is a slight change for all samples, indicating that the majority of vesicles have reached a stable plateau. We calculate p-values of 0.0001 (∗) for both cross-linked samples compared to the controls. See the text for details.

24 h; 64%, 48 h) and single-phase vesicles decreases (6 and 9% at 24 and 48 h, respectively) over the 1.67 μg/mL case. Clearly, the specific binding of avidin to biotinylated lipids results in a measurable shift to phase-partitioning vesicles, at both 24 and 48 h, between the controls and all cross-linked samples. When we examine the cross-linking case with the cold bath samples (quenching at 12.7 ± 0.1 °C and imaging at room temperature) in Figure 3, we see a similar picture to room temperature (24.6 ± 0.2 °C) quenching. We observe a significant shift from non-cross-linked to cross-linked samples (27% multidomain vesicles after 24 h). For both 1.67 and 2.50 μg/mL avidin concentrations, the number of multidomain vesicles (49% and 63%, respectively, at 24 h) is close to the room temperature quenching. Furthermore, there is little change between 24 and 48 h, indicating that the multidomain state is stable. The difference in temperature (25 or 13 °C quenching) does not appear to have large effect. In Figures 2 and 3, the data at 24 and 48 h reveal that the cross-linked system has reached a stable plateau after 24 h. There are no significant shifts between the three general categories of vesicles, indicating that cross-linking has stabilized

the data are similar, while low values reveal significant statistical differences. All p-values are shown in Table S2. When 0.50 μg of avidin (1.67 μg/mL, 25 nM) is added and allowed to incubate at 45 °C for 1 h before quenching to 24.6 °C (Figure 2), multidomain configurations are the majority in the vesicle population, 54% at both 24 and 48 h, which is not the case for the controls (no avidin, identical quenching conditions17% multidomain vesicles after 24 h; 7% after 48 h). When we compare the control population to those incubated with 1.67 μg/mL avidin at 24 and 48 h, we note a significant difference. The number of single-phase vesicles decreases compared to the control population, which confirms previous findings.12,17,27,28 With 0.75 μg of avidin (2.50 μg/mL, 38 nM), the number of multidomain vesicles increases (68%, 2985

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Figure 4. Comparison of multidomain vesicles. The increase of multidomain, two-phase vesicles (Ld/Lo) in the presence of a cross-linking pair (avidin−biotin) is a distinct outcome. We have analyzed temperature quenching, time, and avidin concentrations. Data sets correspond to 24.6 °C quenching (gray, 24 h; white, 48 h); 12.7 °C quenching (green, 24 h; blue, 48 h), and nonspecific binding samples (NSB; red, 24 h; orange, 48 h) quenched to 24.6 °C. When comparing these factors to non-cross-linked (no avidin, controls) and NSB data, we conclude that cross-linking is the greatest factor to increasing the fraction multidomain vesicles.

avidin there is a slight increase of multidomain frequency at both 24 and 48 h. After 48 h, avidin appears to have an effect on the percent of multidomain vesicles in the overall population, although not to the scale seen with the biotinylated vesicles. Possible electrostatic interactions between avidin and the zwitterionic PC lipids would affect the final phase state.29 To understand the multidomain outcome, we use fluorescence recovery after photobleaching (FRAP) to investigate the effects of cross-linking on mobility and diffusion in supported lipid bilayers. The cross-linking takes place in the Ld phase, and so for simplicity, we examine bilayers composed of only DOPC and DOPC with 5 mol % biotin-DOPE. Upon cross-linking using 1.0 μg/mL of avidin, both diffusion and the mobile fraction drop from 0.31 ± 0.15 to 0.14 ± 0.07 μm2/s and 0.88 ± 0.03 to 0.57 ± 0.07, respectively. As well, lipid diffusion in the cross-linked and control samples fit Brownian, random walk motion. The diffusion values are lower than the expected 1−5 μm2/s from previous findings.7,25,30 However, the high mobile fraction indicates that these bilayers are freely diffusing and represent an accurate snapshot of our system. We have performed the same experiments using fluorescently labeled avidin on DOPC:biotin-DOPE (95:5 mol ratio) bilayers to measure the mobility of the cross-linked protein. In this case, we find that the mobile fraction drops to 0.44 ± 0.18 and diffusion is no longer Brownian. The large standard deviation (±0.18) along with the low avidin mobility infers greater heterogeneity of avidin mobility than that of the lipids. From these data (summarized in Table S4), we conclude that crosslinking has an effect on the diffusion and mobility of tethered lipids and of avidin, which leads to potential lateral heterogeneity.

the multidomain configuration. When we compare multidomain vesicle populations from each experiment in Figure 4, the specific cross-linking of avidin to biotinylated lipids results in a distinct outcome. The controls (no avidin) and nonspecific-binding experiments display a majority of twophase two-domain vesicles whereas the cross-linked cases exhibit a majority of multidomain vesicles in all cases. Table S3 shows the vesicle distributions in all categories. To characterize the non-cross-linked populations, we compare control vesicles (biotinylated lipids without avidin) at different quenching conditions in Figure 5A, B. In these data, we observe that 54% of vesicles show two-phase two-domain and only 17% exhibit multidomain outcomes after quenching to 24.6 °C at 24 h. The numbers shift slightly after 48 h (56% twodomain, 7% multidomain), although the difference is not significant. The lower temperature quench, 12.7 °C, yields a population of 61% two-domain and 27% multidomain vesicles at 24 h and after 48 h, 74% and 19%, respectively. The differences between 24 and 48 h are not significant, suggesting that the first 24 h after phase separation is sufficient for most vesicles to reach their final phase configuration. When we compare the different quenching conditions (24.6 °C vs 12.7 °C), the colder temperature does produce a greater fraction of multidomain vesicle (27% vs 17% after 24 h; 19% vs 7% after 48 h). However, these populations are statically indistinguishable. We also compare the cross-linking and nonspecific binding (NSB) behavior of avidin. When avidin is added to vesicles without biotinylated lipids (Figure 5C, D), we observe behavior similar to the controls, where multidomain vesicles represent less than 20% of the total population. With the addition of 2986

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Figure 5. Non-cross-linked vesicles. We characterize the distribution of two-phase vesicles (Ld/Lo) with and without biotinylated lipids (biotinDOPE). All samples are quenched from 45 to 24.6 °C or 12.7 °C, and we report the distribution of each phase configuration (single-phase, white; two-phase two-domain, gray; and multidomain vesicles, black) as a percentage of the vesicle population. The non-cross-linked (no avidin) experiments show similar results at both (A) 24 h and (B) 48 h. The p-values in (A) compare the two-phase vesicles of 24.6 °C to the 12.7 °C and the nonspecific binding (NSB, no biotin-DOPE) experiments. The high p-values of 0.317 for the comparison to 12.7 °C (∗) and 0.075 (∗∗) for NSB vesicles indicate a high degree of similarity in all three vesicle populations. In (B), the p-values have shifted to 0.24 (∗) for 12.7 °C and 0.99 (∗∗) for NSB after 48 h. These p-values confirm that the non-cross-linked samples show a high degree of similarity. In the NSB (no biotin-DOPE) cases (C, D), vesicles are quenched to room temperature and exposed to avidin (2.50 μg/mL). (C) There is only a slight difference with the exposure to avidin (p = 0.31, ∗) after 24 h. (D) For 48 h, p = 0.025 (∗∗). See text for details.

sitol phosphate 2 (PIP2)35 initiate signaling cascades, leading to global cellular responses. In our model system, we have induced a shift from a two-phase two-domain configuration to a multidomain one when we cross-link a small percentage of lipids. We have analyzed the effect of avidin cross-linking on biotinylated vesicles using population statistics to capture the overall result on the system instead of investigating single events. The specific cross-linking binding of avidin to biotinylated lipids produces a population shift from two-phase two-domain majority to multidomain majority. It reveals a decrease in domain coalescence, which is stable in the majority of vesicles. Avidin to biotin cross-linking occurs above the plane of the bilayer and perturbs the bilayer behavior. To understand the overall process in our model system, we examine pertinent factors that induce multidomain behavior. We evaluate contributors such as phase nucleations, domain growth, and the lipid specific factors for phase dynamics to explain multidomain stability. To understand the observed multidomain vesicle outcome, let us first analyze the cross-linking system. Our study has evaluated the effects of temperature quenching, coalescence time, and cross-linking concentration. Of these three, crosslinking concentration is the dominant factor.

To rule out nonspecific binding as a factor in the decreased mobility and diffusion rate, we add avidin to non-biotinylated bilayers. While the lipid diffusion coefficients are similar to cross-linked bilayers (∼0.14 μm2/s), the mobile fraction (0.85 ± 0.05) is nearly identical to the controls (no avidin, 0.88 ± 0.03), which suggests transient associations between the avidin and lipids. We see this trend as well in the vesicle NSB data (Figure 5C,D) where the 48 h population displays a minor increase in multidomain vesicles over 24 h (24 h, 14%; 48 h, 19%). The nonspecific adhesion to the lipids results in minor effects on the mobile fraction, indicating possible electrostatic interactions between avidin and zwitterionic PC lipids.29 Crosslinking induces large differences in lipid and avidin mobility due to specific cross-linking on the bilayer surface. We are currently investigating diffusion effects on this system in more detail.

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DISCUSSION Lipid composition in the cell membrane is in constant flux due to mixing dynamics. Its lateral heterogeneity is subject to intense debate.1−5,7,31 A small percentage of ligand−receptor binding events on the plasma membrane can trigger a systemic response, corresponding to molecular rearrangements, cofactor recruitment, receptor platform formation, and other events. For example, cross-linking and oligomerization from antigens− immunoglobulin in B cells32−34 and calcium−phosphatidylino2987

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the highest coverage of avidin molecules on a vesicle of r = 8.9 μm if the entire surface is available. However, the biotin binding sites are confined to Ld phase domains (see Figure S1), reducing the available surface and limiting the avidin to ∼1.5 × 106 molecules. Overall, the increase in avidins available for binding results in an increase of surface coverage of the avidins on the vesicle bilayer. However, how does avidin binding and surface coverage perturb domain coalescence, which results in the population shift from two-phase two-domain majority to multidomain majority? First, let us approach this query by examining phase dynamics from the seeding and growth of the second phase (Lo) from the first (Ld). Phase nucleation and growth has been studied in crystals and thin films for ∼100 years. Nucleation, growth, and coarsening of the second phase lead to a stable two-phase system during phase separation. If nucleation occurs uniformly, then the kinetics of the growth and coarsening stages will lead to uniform crystal grains, which are useful in ceramics, thin films, and, most recently, quantum dot applications.43,44 When we apply these concepts to lipid phase dynamics, we find that they provide a useful framework.36,45−49 For example, in polka-dotted vesicles, if we consider a uniform bilayer that undergoes a phase transition from a single phase (Ld) to Ld and Lo phases, we expect that nucleation events will be uniformly distributed on the bilayer. As the nucleation seed grows to a critical size, it becomes a stable domain with the composition of a second (Lo) phase. Since the second phase is rich in cholesterol and saturated lipids, the growth of the small domain from the nucleation event depletes the surrounding area of cholesterol and saturated lipids, which will inhibit nucleation events directly adjacent to the newly formed domain. The diffusion rate of lipids determines the size of the depletion zone and the growth size restriction of the phase domain. In a uniform nucleation field and a diffusion-limited model, we expect multiple domains of similar sizes. Unlike crystals and thin films, however, lipid domains in a Ld/Lo system are mobile and will eventually coalesce. Given our avidin−biotin cross-linking system, the difference between cross-linked and un-cross-linked vesicle populations appears to be a classic diffusion-limited outcome; however, there are many other factors at play. In particular, phase coalescence depends on line tension to minimize the energetic cost of a phase boundary. Avidin perturbation to line tension potentially alters the balance to favor multidomain vesicles. Our heterogeneous model membrane consists of two phases (Lo and Ld): lipids and lipid−avidin complexes. In this study, we have examined the effects of temperature quenching, coalescence time, and cross-linking concentration. Crosslinking appears to be the dominant factor. In addition to avidin concentration and surface coverage effects, avidin binding on the bilayer surface may contribute to lowering the line tension between the phase domains. Marsh and Swamy have measured a vertical displacement of avidin binding to biotinylated lipids and have found ∼8 Å height difference. Additionally the increase in curvature about avidin bound to biotinylated lipids affects up to 50 lipids per binding event.29 Since line tension, the driving force of coalescence, depends on height differences between phases,50 such a displacement along a phase boundary has the potential to decrease line tension. At sufficiently low line tensions, the entropic gain of many nanodomains overcomes the energetic penalty of phase boundaries.51 In this system, we have documented micrometer-size multiple domains, indicating

In this study, we have considered temperature and quenching effects as possible contributors to multidomain outcomes. However, from our statistical analysis, it is clear that they are not the primary factors in the observed results. Quenching enables repeatable, consistent data, and rapidly cooling from 45 °C to 25 or 13 °C increases the number of nucleation sites.36 The effect of quenching on diffusion and mobility is less evident. Diffusion and viscosity are directly related to temperature (ηD/ς ∝ kBT, where η is the viscosity coefficient, ζ is the drag coefficient, kB is Boltzmann’s constant, and T is temperature37). Therefore, the lower temperature leads to a slower diffusion rate. When the temperature is decreased from incubation (45 °C) to 24.6 or 12.7 °C, the theoretical diffusion decreases by a factor of 1.11 from the ratio of the two final temperatures (318 K to 298 or 286 K). Previous studies indicate that once the temperature has dropped below the transition point, demixing occurs in a short time frame between seconds and hours.10,38,39 Since we document vesicle domain configurations after 24 and 48 h, the effect of temperatureinduced diffusion delay is small. From our calculated p-values, the difference between quenching temperatures is statically insignificant for the relevant populations. We have collected data at two time points, 24 and 48 h, to evaluate end points, since un-cross-linked two-phase vesicles coalesce into two-phase two-domain configurations rapidly.10,38,39 In all cases, the difference between 24 and 48 h not is statistically significant. It demonstrates that population shift from two-phase two-domain to multidomain configurations is stable and has reached a local minimum or a kinetic trap. From our data analysis, different quenching temperatures (25 and 13 °C) and coalescence time (24 and 48 h) produce statistically indistinguishable data. When we combine Figures 2 and 3 into a single data set, we find that their values are within standard deviations (see Table S5). For the condensed data of 1.67 μg/mL avidin, we see 10 ± 4% of vesicles in onephase configuration, 39 ± 7% in two-phase two-domain, and 52 ± 3% multidomain vesicles for all conditions. As well, when 2.50 μg/mL is present, we observe 7 ± 1% in one-phase configuration, 28 ± 3% in two-phase two-domain, and 65 ± 2% multidomain vesicles for all conditions. However, the controls (no avidin) have the greatest variance. For one phase, we observe 21 ± 14%; for two-phase two-domain, 61 ± 9%; and for multidomain vesicles, 18 ± 8% (all values in Table S5). In particular, there is large range in values for the one-phase configurations of the controls, and budding and trapped coarsening may play a role (see Supporting Information). From combining the data sets of the differing conditions, it is evident that avidin concentration plays the determining role in the population shift. Indeed, if we consider a vesicle with a radius of 8.9 μm and a surface area of 1000 μm2, we calculate a minimum of 2.7 × 10−16 mol of lipid, using a lipid head-to-head distance of 35 Å.40,41 Given our 3.3 mol % of biotin-DOPE, we estimate ∼2.68 × 106 biotin molecules available for avidin binding on the outer leaflet. In a 5 μm distance beyond vesicle, we calculate that 1.67 μg/mL of avidin gives a minimum of 7700 avidins circulating in solution free to bind with the vesicle surface. For 2.50 μg/mL of avidin, 11 600 avidins occupy the same volume. From the ratio of biotin to avidin, we expect that the increased number of avidins available would result in increased avidins bound on the vesicle surface until it reaches a maximum. For an avidin size of 6.0 × 5.5 × 4.0 nm,42 we expect ∼3.0 × 106 to be 2988

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interactions on the surface of the bilayer, decreasing domain coalescence and giving rise to lateral heterogeneities. We have found that avidin cross-linking produces a population shift from majority two-phase two-domain to majority multidomain vesicles in our Ld/Lo system. To understand this effect, we have considered line tension and diffusion perturbations due to avidin binding on the bilayer surface, leading to clustering and surface crowding. Our outcome of stable multidomain vesicles results from a combination of these factors. A decrease in line tension from bound avidin decreases the energetic cost of the Ld/Lo phase boundary. From our FRAP data, we observe a decrease in lipid diffusion and subanomalous avidin behavior, indicating surface crowding and avidin−avidin clustering. These data imply that biomembrane shifts and local variations rely on the interplay between protein and lipid dynamics to add complexity and lateral heterogeneity.

that line tension dominates over entropy. However, we expect that the rearrangement of the bilayer and domain coalescence will differ according to diffusion factors and potential avidin− biotin obstacles. Avidin−biotin is a stable linkage, leading to a rearrangement time scale far longer than most cell membrane components (t1/2 = 200 days18,52), suggesting that any crosslinking obstacles are long-lived. If avidin perturbs the phase boundary, does it also affect the phase distribution? First, we examine the distribution of biotinylated lipids before and after cross-linking. BiotinDOPE partitions into the Ld phase due to its similarity to DOPC. Next, when we cross-link biotin-DOPE with labeled avidin, we confirm that it remains in the Ld phase (see Figure S1). When we consider the overall phase diagram, its tielines11,53 and the small potential for a two-site cross-linker to alter the phase boundary,17 we find that the ratio of Ld to Lo remains unchanged. We do not detect any difference between the phase ratios. In addition to avidin’s mechanical deformations and its effect on line tension, let us consider restricted mobility and coalescence from surface crowding. Avidin−biotin complexes form above the plane of the bilayer, and we expect avidin diffusion to be determined primarily by the lipid bilayer since the bilayer has a much higher viscosity than the surrounding aqueous environment. At low surface concentrations (0.01− 0.05 mol % biotin-lipid anchors), Horton, Rädler, and coworkers have reported normal, Brownian diffusion coefficients for avidin of (2.0−2.2) ± 0.3 μm/s2 on SOPC (stearoyloleoylphosphocholine) bilayers using fluorescence correlation spectroscopy (FCS),54 which fits with the assumption that diffusion is determined by the lipid bilayer. At 1 mol % biotinylated lipids, avidin diffusion displays anomalous behavior, characterized by a power-law relationship (D(t) ∝ tα−1, α ∼ 0.71) instead of simple Brownian motion, α = 1.00.54 When we examine lipid diffusion and mobility data from FRAP measurements, we observe a decrease in diffusion and mobility between non-cross-linked and cross-linked samples. In all cases, lipids exhibit Brownian diffusion with α = 0.92 ± 0.11, where α > 0.9 is considered Brownian (see Supporting Information). For avidin, in addition to the decreased mobility (0.44), analysis of its movement reveals anomalous diffusion (α = 0.78 ± 0.17). This decrease of cross-linked components mobility and subanomalous diffusion suggests large obstacles such as avidin−avidin clusters and lateral heterogeneities. Indeed, streptavidin forms crystals on vesicle surfaces, which requires self-association.55 Ganglioside GM1 bound to cholera toxin B (GM1-CTB) also forms complexes that exhibit clustering in several lipid combinations.15,16,56 Wagner and Tamm have reported low mobile fractions in the t-SNARE and PIP2 system.57 Our findings of low mobility and subanomalous diffusion fit the profile of these systems. As well, our calculations on a vesicle with an 8.9 μm radius are consistent to an increase in avidin surface coverage when we increase avidin concentration in solution. In all, these behaviors point to surface crowding inducing restricted mobility, clustering, and non-Brownian motion. In the cross-linked Ld domains and the Ld bilayers, we expect a variety of mobile component combinations: (a) lipids only, (b) complexes of lipid−avidin couplings affecting up to 50 lipids, and (c) clusters of lipid−avidin complexes, inducing surface crowding that inhibit mobility and domain coalescence. In these combinations, avidin plays a critical role due to its

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CONCLUSIONS In our simple two-phase (Ld/Lo) system, we have induced a population shift from majority two-phase two-domain to majority multidomain vesicles. Our population analysis quantifies the extent of the effect and pinpoints the dominant factor to be the specific avidin binding and concentration, not temperature or coalescence time. The observed multidomain vesicles and avidin subanomalous diffusion point to avidin− avidin interactions, surface crowding, and line tension perturbations. In cellular membranes, receptor concentrations are similar our experimental model (3.3 mol %), and we expect that similar effects lead to clustering of membrane and membrane-associated proteins. For example, membranebound immunoglobulins and antigens binding on the surface of B cells have been observed to form large cross-linking clusters due to multivalent antigens.32,34 Our model of this common membrane response to a simple cross-linking event demonstrates the ease of inducing lateral heterogenities and nonideal mixing into a lipid bilayer.

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ASSOCIATED CONTENT

S Supporting Information *

Further discussion of diffusion and mobility analysis, budding and trapped coarsening, tables and figures pertinent to chisquared calculations, p-values, diffusion data, and bound avidin phase preference; citation of refs 58−66. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph (202) 994-7320; Fax (202) 9945873. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The authors thank A. Lee and K. Baldwin for their efforts, J. Stolee for her help, K. Pearson for useful discussions, and E. Sheets for advice. Also, many thanks to the Keck foundation and the University Facility Fund (GW UFF) for seed money and NSF REU program #0649165. 2989

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