Engineering Lipid Structure for Recognition of the Liquid Ordered

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Engineering Lipid Structure for Recognition of the Liquid Ordered Membrane Phase Stefan S. Bordovsky, Christopher S. Wong, George D. Bachand, Jeanne C Stachowiak, and Darryl Y. Sasaki Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02636 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 29, 2016

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Table of Contents graphics

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1 2 Engineering Lipid Structures for Recognition of the Liquid Ordered Membrane Phase 3 4 Stefan S. Bordovsky, Christopher S. Wong, George D. Bachand, Jeanne C. Stachowiak, and Darryl Y. 5 Sasaki 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 In the design of lipids to selectively partition into the liquid ordered membrane phase 37 38 lipid tail packing is the main driving force, however, a hydrophobic headgroup can 39 redirect the phase partitioning to the disordered membrane phase. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 ACS Paragon Plus Environment 56 57 58

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Engineering Lipid Structure for Recognition of the Liquid Ordered Membrane Phase Stefan S. Bordovsky†£, Christopher S. Wong†, George D. Bachand§, Jeanne C. Stachowiak£, and Darryl Y. Sasaki†* Sandia National Laboratories, †Biotechnology and Bioengineering Dept., Livermore, CA and §Nanosystems Synthesis/Analysis Dept., Albuquerque, NM £

The University of Texas at Austin, Department of Biomedical Engineering

Abstract Selective partitioning of lipid components in phase separated membranes is essential for domain formation involved with cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Here, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (Lo) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails we found that although the lipid tails can direct selective partitioning to the Lo phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (Ld). The PEG spacer can serve as a buffer to mute headgroup-membrane interactions and thus improve Lo phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescent-labeled lipids with selective affinity for the Lo phase.

ACS Paragon Plus Environment

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Introduction Recognition events at the membrane surface initiate important cellular processes, such as endocytosis,1 immunological synapse formation,2 and antigen binding.3 Interactions of signaling molecules,4 viral particles,5 and other biomolecules to membrane receptors reorganize membrane components to form supramolecular complexes that perform a specific mechanical or chemical activity, such as membrane invagination and enzymatic processes. Aspects of molecular recognition on lipid films,6 pioneered by the Kunitake research group, and recognition-induced mechanical processes have been studied in minimal systems in an effort to understand how binding events affect membrane behavior. Examples include chemical recognition on functionalized lipid films causing molecular aggregation via multivalent binding interactions7,8 or dispersion through electrostatic repulsive forces,9,10 or curvature induced on membrane domains through steric interactions of proteins bound to the interface.11 While gains have been made in understanding these interfacial interactions, far less is understood about lateral interactions between lipids within the plane of the membrane. It would be instructive to understand the forces that drive in-plane organization of molecular complexes and the extent to which these phenomena can be used to generate supramolecular membrane complexes. The study of lateral formation of molecular complexes in the lipid membrane is inspired by the lipid raft hypothesis, which attributes a range of cellular activities to the presence of phase-separated domains of membrane components.12 The presence of rafts in live cells, however, is difficult to detect due to their small size and purportedly dynamic assembly and disassembly.13 Minimal systems employing common lipids from


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cellular membranes have revealed the existence of phase separated lipid domains in cellsized giant unilamellar vesicles (GUVs).14 Of particular relevance is the coexistence of liquid ordered (Lo) and liquid disordered (Ld) membrane phases that mimic the raft and non-raft phases thought to exist in cellular membranes. A common system employed incorporates a PC lipid with long saturated tails yielding a high phase transition temperature (Tm), such as dipalmitoylphosphatidyl choline (DPPC [Tm = 41 °C]), along with cholesterol and a low Tm lipid, such as dioleoylphosphatidyl choline (DOPC [Tm = 17 °C]).15 Favorable packing interactions between the long saturated fatty acid lipid tails with the planar ring system of cholesterol serves as the driving force that separates Lo lipid domains from the disordered structure of the Ld phase. Fluorescent lipid probes have been developed to identify membrane domains in studies of biphasic lipid films.16,17,18 However, predicting the phase partitioning behavior of these probes has been difficult due to a poor understanding of the relationship between molecular structure and phase selectivity. Many examples exist of fluorescent lipids with cis-double bonds in the tails that partition to the Ld phase due to the kinked structures in their tails that inhibit good packing interactions.19,20 Fluorescent lipids with long saturated tails, however, do not ensure partitioning to the more ordered phase.21 In fact, only a few of these lipids are known to partition well to the Lo phase. In the current work we explore the role of lipid tail, headgroup, and spacer structure for membrane phase selectivity. In particular, we focus on developing lipid designs to target the Lo phase. Previously, we found that the spacer chemistry and length play a deciding factor in the phase partitioning behavior of biotinylated lipids.22 This tuning of selectivity of the biotinylated lipids to either the Lo or Ld phase likewise


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facilitates selective attachment of streptavidin to the respective phase. Here, we find that through favorable packing interactions the lipid tail structure can direct partitioning towards the higher ordered Lo phase, but headgroup hydrophobicity can redirect phase selectivity towards the lower ordered Ld phase. A hydrophilic polyethylene glycol spacer, however, can be used to buffer headgroup interaction with the membrane and enable lipid tail structure to regain control of phase selectivity.

Experimental General Aqueous solutions were prepared from deionized water obtained through a Barnstead Type D4700 NANOpure Analytical Deionization System with ORGANICfree cartridge registering ≥ 18.0 MΩ-cm resistance. Diphytanoylphosphatidyl choline (DPhPC), dipalmitoylphosphatidyl choline (DPPC), distearoylphosphatidylethanolamine-NPEG2000-amine (DSPE-PEG2000-amine), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL) and fluorescent lipids Texas Red dihexadecanoyl phosphatidylethanolamine (DHPE-TR), Oregon Green 488 DHPE (DHPE-OG488), Fluorescein DHPE (DHPE-FITC), and NBD-PE (DHPE-NBD) were purchased from Thermo Fisher Scientific (Waltham, MA).

Lipid synthesis Procedures for the preparation and characterization data of DP-EG5-FITC, DPEG10-FITC, DP-EG15-FITC, DSPE-PEG2000-FITC, DP-EG5-RhB, DP-EG10-RhB,


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DP-EG15-RhB, DSPE-PEG2000-RhB, DP-EG15-OG488, DP-EG15-AF568, and DPEG15-Atto488 lipids can be found in the Supporting Information.

Membrane partition coefficients Giant unilamellar vesicles were prepared by electroformation following published procedures.23 All vesicles were prepared at an elevated temperature that exceeded the highest phase transition temperature of the lipid mixtures (e.g., for samples containing DPPC the GUVs were prepared at 55 – 60 °C) and contained 0.3 mol% of a red fluorescent-labeled lipid (DHPE-TR) to identify the Ld phase. Vesicles were initially prepared in sucrose solution (350 mOsm) and following electroformation the vesicles were diluted in glucose solution (350 mOsm). The mixture was then placed in a channel structure constructed of a glass coverslip and slide sandwiching parallel strips of doublesided tape (3 layers thick) spaced 2 – 3 mm apart. The ends of the fluidic structure were then sealed with wax to minimize evaporation and imaged within five minutes to ensure sample freshness. Images were captured in epifluorescence (Figures 4 A and B, S3, S4, S6, S7, S9, and S10) using a Zeiss Axiovert 200M microscope with a Sutter Lambda XL broadband light source under green (filter set of λex = 472 nm, 30 nm bandpass; λem = 520 nm, 35 nm bandpass) and red channels (filter set of λex = 543 nm, 22 nm bandpass; λem = 593 nm, 40 nm bandpass). Partitioning coefficients were determined using the pixel intensities of the captured images. The concentration of lipid label used (0.3 mole%) facilitated good imaging conditions for evaluating phase partitioning while maintaining a reasonably low FRET-based quenching. However, to eliminate losses of fluorescence intensity to FRET


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between the green and red fluorescent dyes, Kp (ILo/ILd) values were obtained using only one dye present in the membrane. Using the Plot Profile feature on ImageJ, the average pixel intensity along the Lo and Ld domains were determined for each vesicle. No fewer than 20 measurements were taken for each averaged value reported in Table 1.

Octanol-water partition coefficients The partition coefficients for the fluorescent dyes were determined using a published procedure.24 In brief, fluorescent dye was dissolved in dimethylformamide (universal solvent for the dyes) at a concentration ranging between 80 – 360 µM, depending on the dye. An aliquot of the dye solution (1 mL) was then placed in a separation funnel containing octanol (20 mL) and water (20 mL), the mixture shaken, and layers separated. The aqueous layer was then diluted by 2-fold with deionized water to obtain optical clarity and the UV-vis absorption then measured. Using calibration curves determined for each dye in water (extinction coefficients reported in Table S1 (Supporting Information)) we could find the concentration of dye in the aqueous phase (Cw) and determine Kow: 𝐾"# =

𝐶&"'() − 𝐶# 𝐶#

where CTotal was obtained from the known amount of dye added to the separation funnel.

Results In this study we examined fifteen different lipids to evaluate the role of headgroup (fluorophore) structure and length of the PEG spacer between the fluorophore and lipid backbone on membrane phase selectivity. Four of the lipids, shown in Scheme 1, were


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commercially available – DHPE-FITC, DHPE-OG488, DHPE-NBD, and DHPE-TR. The other eleven were prepared in our lab using previously described procedures.22 Scheme 2 shows two sets of lipids that compare the role of spacer length with two fluorophores, fluorescein and rhodamine B. The spacer lengths were pentaethylene glycol (EG5), decaethylene glycol (EG10), pentadecaethylene glycol (EG15), or PEG2000 (i.e., EG45). Lipids with EG5 – EG15 spacers (Scheme 2 and 3) were prepared with ether linked palmityl tails at C1 and C2 of the glycerol backbone while the PEG spacer was attached to the C3 position. The PEG2000 lipids were prepared by coupling commercially available DSPE-PEG2000-amine with FITC or RhB-ITC. The partitioning behavior of the lipids in Lo/Ld phase separated membranes was measured using a membrane composition of 40:35:25:0.3 of DPhPC/DPPC/cholesterol/ fluorescent lipid. This composition corresponds to a region in the DPhPC/DPPC/ cholesterol phase diagram where separation of Lo and Ld phases is distinct and persists up to high temperature (~45 °C).25 Giant unilamellar vesicles (GUVs) of the lipid mixtures were prepared via electroformation in sucrose, diluted in glucose to allow the higher density sucrose-filled GUVs to settle onto the coverslip, and placed in a microfluidic chamber consisting of a glass coverslip and slide. The GUVs were then imaged at room temperature with an epifluorescence microscope. The first experiments examined the phase partitioning behavior of commercially available fluorescent lipids. These molecules all have the same dipalmitoylphosphatidyl ethanolamine (DHPE) lipid structure but with different fluorophores at their headgroup positions. While the literature finds that DHPE-Texas Red (DHPE-TR) partitions consistently to the Ld phase,21,26 the other lipids have reported variations in partitioning


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behavior. For example, DHPE-NBD partitions to the Lo phase for membranes consisting of DOPC/DPPC/cholesterol, but has also been found to partition to the Ld phase for membranes of similar composition (i.e., DOPC/DSPC/cholesterol).21 In our experiments we found that DHPE-NBD partitions selectively into the Lo phase (Figure 1A, Kp = 2.2). DHPE-FITC was found to partition to the Ld phase (Figure 1B, Kp = 0.28), similar to what has been observed in giant plasma membrane vesicles (GPMVs).19 On the other hand, DHPE-OG488, with an Oregon Green 488 fluorophore of similar in size and shape to fluorescein, partitions towards the Lo phase (Figure 1C, Kp = 2.1). Figure 2 plots the partition coefficients of these fluorescent-labeled DHPE lipids.

O

O O

O

O P

O

H N

O-

O

H N S

O HO 2C

O

OH

DHPE-FITC O

F

O O

O

O P

O

O

O

H N

O-

HO 2C

O

F

O

OH

DHPE-OG488

O O

O

O P

O

H N

N O N

O-

O

NO 2

O

DHPE-NBD N+

O O

O O

O P

O

O-

H N

O S O

O SO 3-

O

N

DHPE-TR

Scheme 1


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Figure 1. Fluorescent microscopic images of GUVs containing A) DHPE-NBD/DHPE-TR, B) DHPE-FITC/DHPE-TR, and C) DHPE-OG488/DHPE-TR under red (left) and green (center) filters and the merged images (right). (scale bars are 10 µm)

Figure 2. Partition coefficient Kp (Lo/Ld) of DHPE fluorescent dye-labeled lipids (green dyes).

We next examined the role of the spacer length in the phase partitioning of labeled lipids. Scheme 2 shows two series of lipids with either fluorescein or rhodamine B fluorophore at the headgroup position and increasing lengths of PEG spacer. The two fluorophores were chosen for their similar size but different hydrophobicity. We measured the octanol/water partition coefficient (Kow) of fluorescein isothiocyante (FITC)


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and found it to be 13, whereas rhodamine B isothiocyate (RhBITC) had a value of 32. Figure 3 shows a general trend between PEG length and increasing Kp value for these lipids with saturated C16-tails (GUV images in Figure S1). A plot of the Kp values against the number of ethylene glycol monomers in the spacer is also shown in Figure 3 (inset) to evaluate the structure-property relationship. It is evident that although a positive correlation exists between PEG length and Lo partitioning, Kp is also tied to the type of fluorophore. Here, we find that while fluorescein-labeled lipid goes from partitioning into the Ld phase with short spacer length (EG5) to partitioning towards the Lo phase with long spacer length (PEG2000), rhodamine B-labeled lipids remain selective towards the Ld phase even with the longest spacer of PEG2000. From this study a link between headgroup hydrophobicity and phase partitioning to the Ld phase becomes apparent.

Scheme 2


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Figure 3. Graph showing the effect of PEG spacer length on Kp (Lo/Ld) for fluorescein (FITC) and rhodamine B (RhB) labeled lipids. Inset shows the relationship between Kp and PEG length.

To test if we could now design lipids with selective partitioning to the Lo phase we prepared three additional lipids shown in Scheme 3. The fluorophores were selected using the work by Hughes, et al.,27 who measured the affinity of fluorescent probes to the lipid membrane from the bulk aqueous phase (i.e., membrane interaction factor [MIF]), as a guide. Oregon Green 488, Atto488, and Alexa Fluor 568 were selected for their low MIF values, which also corresponded with low measured Kow (Table S1). They were then attached to the 1,2-dipalmityl-glycero-3-pentadecaethylene glycol (DP-EG15) lipid body and their phase selectivity compared against those of DP-EG15-FITC and DP-EG15RhB. Figure 4 shows fluorescence microscopic images of GUVs with the five DP-EG15 lipids. Using DHPE-TR to label the Ld phase we determined that DP-EG15-FITC (Figure 4A) partitions primarily to the Ld phase (Kp = 0.41), whereas DP-EG15-OG488 (Kp = 2.7) and DP-EG15-Atto488 (Kp = 2.6) (Figure 4 B and C, respectively) partition to the Lo phase. Then, using DHPE-OG488 (Kp = 2.1) as a label for the Lo phase we found that DP-EG15-RhB (Kp = 0.13) (Figure 4D) partitions to the Ld phase whereas DP-EG15-


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AF568 (Kp = 3.6) was selective for the Lo phase. Kp values for the fluorescent lipids are shown in graphical form in Figure 5 along with the Kow for each of the headgroup fluorophores.

Scheme 3


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Figure 4. Fluorescence images of GUVs of containing DPhPC/DPPC/cholesterol in a ratio of 40:35:25 with A) 0.3% DP-EG15-FITC/0.3% DHPE-TR, B) 0.3% DP-EG15-OG488/0.3% DHPE-TR, C) 0.3% DP-EG15Atto488/0.3% DHPE-TR, D) 0.3% DP-EG15-RhB/0.3% DHPE-OG488 and E) 0.3% DP-EG15-AF568/0.3% DHPEOG488 shown in filters for (left) red emission, (middle) green emission, and the (right) merged images. Lo and Ld phases in the lipid bilayers (see text) are indicated in merged images. (scale bars are 10 µm)

Figure 5. Combined graph of fluorophore octanol/water partition coefficient (Kow) (right axis) with the Lo/Ld membrane phase partition coefficient (Kp) (left axis) of the five DP-EG15 lipids to show the relationship between fluorophore hydrophobicity and membrane phase selectivity.


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Discussion The lipids tested in this study have saturated palmityl or palmitoyl tails that are identical to lipid structures known to selectively partition into the Lo phase (e.g., DPPC28, DPIDA29). However, as we, and many others, have found the moiety at the headgroup position can dictate the partitioning behavior of the lipid. The lipids of Scheme 1 serve as an example of headgroup-driven phase selectivity. Although the lipid tails are the same, DHPE-TR and DHPE-FITC both partition to the Ld phase whereas DHPE-OG488 and DHPE-NBD partition to the Lo phase. Why do the latter two lipids partition similarly as DPPC to the Lo phase while former lipids reject the more ordered membrane state? Some argue that it is a matter of headgroup size, where the larger dye presents steric interactions that prevent partitioning to the Lo phase.19,21 Here, we find that headgroup size has little to do with partitioning behavior. For example, Oregon Green 488, in spite of being slightly larger in size compared to fluorescein with two fluorines in place of hydrogens on the hydroxyfluorone ring system, enables DHPE-OG488 to partition to the Lo phase whereas DHPE-FITC partitions to the Ld phase. So, although size is not a determining factor other interactions of the headgroup with the membrane may dictate phase selectivity. Previously, it was found that biotinylated DHPE partitions to the Ld phase and placement of a hydrophobic spacer further strengthens the preference to the Ld phase.30 We found that replacement of the hydrophobic spacer (i.e., caproyl) with a hydrophilic one (i.e., PEG) altered the phase selectivity towards the more ordered Lo phase.22 We argued that biotin, which is slightly hydrophobic (Kow = 0.39),31 prefers interaction with the membrane over the bulk aqueous solution thus driving the lipid to the more


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disordered phase of the membrane. By muting the biotin-membrane interaction through the hydrophilic cloak of the PEG spacer we were successful in enabling the palmityl tails of the biotinylated lipid to recognize the more ordered membrane phase. In that work we found that a PEG length of ten monomer units was sufficient to decouple biotin interaction with the membrane and allow highly selective attachment of streptavidin to the Lo phase. This design concept has also been shown to be effective in decoupling interaction of the large fluorescent dye KK114 from the membrane using PEG1450 to reengineer a lipid to partition from the Ld phase to the Lo phase.32 Using the lipids of Scheme 2 we examined the generality of the PEG spacer for lipid design. These lipids varied in lengths of PEG from pentaethylene glycol to PEG2000. Two fluorescent dyes, fluorescein and rhodamine B, with markedly different hydrophobicities were attached at the distal end of the spacer. Results of this study shown in Figure 3 suggest a direct relationship between PEG length and partitioning behavior to the more ordered membrane phase with these saturated lipids. However, strongly hydrophobic headgroups, such as rhodamine B, are difficult to completely decouple from membrane interaction and remain primarily in the Ld phase even with a PEG2000 spacer. While it is possible to use even longer PEG spacers to increase Kp, there is probably a point of diminishing return as higher molecular weight PEG is also known to disrupt membrane structure and phase.33,34 The trend of these results are a further demonstration that steric interactions from groups at the headgroup position, which can include the spacer, are not a direct influence on phase partitioning. Finally, the lipids of Scheme 3 were prepared to test our design concepts in optimizing selective partitioning of fluorescent labeled lipids for the Lo phase. The


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headgroups were chosen as minimally interactive with the lipid membrane and the pentadecaethylene glycol (EG15), which had been previously shown to be an effective lipid spacer for a biotin headgroup,22 was employed as the spacer. All three lipids, DPEG15-OG488, DP-EG15-Atto488, and DP-EG15-AF568, exhibited highly favorable selectivity towards the Lo phase (Table 1). From Figure 5 we can observe a direct link between headgroup hydrophobicity and selective lateral affinity for the Lo phase with these lipids. The Kp values for these engineered lipids are some of the highest reported for fluorescently-labeled lipids.16,17 Based on reported information that DPPC itself partitions 75 – 90% into the Lo phase,28 these fluorescent lipids exhibit a selectivity to the Lo phase near to that of DPPC. It should be noted that the physical characteristics (e.g., miscibility transition temperature) of the Lo phase varies with membrane composition,15,25 which suggests a change in packing order of the phase. Since packing order plays an important role in phase partitioning, phase selectivity for these fluorescent lipids may change in different bilayer systems. Nonetheless, the general concept described here provides design pathways for selectively labeling/functionalizing membrane domains.


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Table 1. Kp values for fluorescent lipids

Conclusions We have shown through these results that lipids can be successfully engineered to partition selectively to the Lo phase of Lo/Ld biphasic membranes. Through a systematic approach we evaluated the role of headgroup hydrophobicity and PEG spacer length to enable lipid tail packing interactions to drive phase selectivity. In the current lipid designs we are relying entirely upon van der Waals interactions (i.e., packing interactions) to enable phase selectivity. Future designs for phase selective lipids may benefit from concepts used in molecular recognition and supramolecular assembly, including hydrogen bond interactions, dipole-dipole interactions, and shape recognition. Further improvements in Kp values for specific membrane phases would generate unique supramolecular chemistries and architectures in engineered membrane materials.


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Acknowledgements The authors would like to thank Dr. Ryan W. Davis of the Biomass Science & Conversion Technology Dept. at Sandia National Labs for the mass spectral analyses of the lipids and Prof. Steve Boxer for insightful discussions. J. Stachowiak acknowledges support from the National Science Foundation under grant DMR1352487. This work was supported by the US Department of Energy, Office of Basic Energy Sciences, Materials Science and Engineering Division (KC0203010). Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.

*

Corresponding author, email: [email protected]

Supporting Information Available: Synthetic preparations and spectral characterization of lipids, octanol-water partition coefficients, and fluorescent images of GUVs are shown here. This material is available free of charge via the Internet at http://pubs.acs.org.


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Engineering Lipid Structure for Recognition of the Liquid Ordered

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