# Budding-driven localization of lipid domains at prescribed positions in confined giant unilamellar vesicles

Koyomi Nakazawa, Antoine Lévrier, Sergii Rudiuk, Ayako Yamada, Mathieu Morel, Damien Baigl

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Koyomi Nakazawa, Antoine Lévrier, Sergii Rudiuk, Ayako Yamada, Mathieu Morel, et al.. Budding-driven localization of lipid domains at prescribed positions in confined giant unilamellar vesicles. 2023. [hal-04237568](https://hal.science/hal-04237568v1)

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Budding-driven localization of lipid domains at prescribed
positions in confined giant unilamellar vesicles

K. Nakazawa¹, Antoine Lévrier,¹ S. Rudiuk¹, A. Yamada¹, M. Morel¹, D. Baigl¹*

¹PASTEUR, Department of Chemistry, École Normale Supérieure, PSL University,
Sorbonne Université, CNRS, 75005 Paris, France

*correspondence to: damien.baigl@ens.psl.eu

**Abstract**

Giant unilamellar vesicles (GUVs) are widely used minimal cell models where essential biological features can be reproduced, isolated and studied. Although precise spatio-temporal distribution of membrane domains is a process of crucial importance in living cells, it is still highly challenging to generate anisotropic GUVs with domains at user-defined positions. Here we describe a novel and robust method to control the spatial position of lipid domains of liquid-ordered (Lo) / liquid-disordered (Ld) phase in giant unilamellar vesicles (GUVs). Our strategy consists in confining Lo/Ld phase-separated GUVs in microfluidic channels to define free budding regions where the minority phase lipid domains localize and grow by decreasing the line tension between the co-existing phases. We show that this process is governed by the respective fraction of the two phases, and not by the chemical nature of the lipids involved. The spatial position and number of domains are controlled by the design of the confining microchannel and could result in polarized GUVs with a controllable number of poles. The developed method is versatile and user-friendly, while allowing to perform multiple single-vesicle experiments in parallel.
Introduction

Living cells are characterized by a well-defined and highly-regulated spatiotemporal
organization of their biomolecular content. In particular, non-uniformity in molecules
concentration (gradients) and distribution (e.g., polarization), is capital in many key
biological processes, from apical-basal establishment in epithelial cells[1] to asymmetric
division in stem cells,[2] or directional sensing and motility in neuronal growth cones.[3]
For most of these events, the spatial organization of membrane proteins and associated
lipids, constituting the interface with the surrounding micro-environment, contribute to
the specificity of the signaling outcome.[4] Mechanisms driving the spatial organization
of the lipid membrane are thus of particular importance to understand biological
polarization. For example, microdomains of ordered lipid phase, called “lipid rafts”, are
hypothesized to recruit specific sets of transmembrane and scaffolding proteins for
functional processes.[5] However, the understanding of the underlying mechanisms
directing ordered/disordered lipid phase segregation as well as further recruitment of
specific lipids and proteins in sub-domains, remains incomplete in particular due to the
difficulty of extracting essential parameters in the highly crowded and multi-factorial
environment of living cells.

There is thus a need for the development of reconstituted *in vitro* system, in which all
components and physico-chemical parameters could be precisely tuned, and ideally
precisely ordered in space and time. To this aim, giant unilamellar vesicles (GUVs), which
are spherical entities with a phospholipid bilayer membrane and a diameter (1-100 µm)
comparable to that of actual cells, appear as particularly suitable minimal models to
mimic living cells.[6] Various methods have been developed to control their size,[7,8] inner
content[9] and composition of their membrane (in lipids and proteins),[10,11] leading to
various relevant examples of synthetic cells, each of them mimicking, reproducing or
allowing to study one or several specific biological features.[6,12-15] Hence, the lipid
domain formation and organization has been studied using GUVs as models for lipid
phase segregation.[16,17] Indeed, mixtures of unsaturated and saturated lipids with
cholesterol in proper proportions exhibit liquid-ordered (Lo) / liquid-disordered (Ld)
phase separation.[10] Controlling domain formation in GUVs, either by lipid
stoichiometry[10] or externally by temperature[10] or light stimulus,[18] is a well-established
process but the control over the domain spatial distribution remains a highly desired
challenge that has been partially achieved in only a few notable reports. For instance,
microstructured substrates have been shown to direct the position of lipid domains at
specific locations but this involved the use of supported bilayers[19,20] and not GUVs. To
our knowledge, spatial control over lipid domain position in GUVs has been realized
either on a single vesicle under pneumatic stress[21] or on GUVs squeezed in pH-sensitive gels.[22] The first study showed that, in a GUV compressed into a pancake shape between two walls, the lipid phase in minority tended to localized away from the flat parts in contact with the walls. Such an approach was however limited to a single compressed GUV and did not allow much flexibility in terms of GUV domain localization. In contrast, the second study allowed to impose a broad variety of user-defined shapes to many constrained GUVs but required a specific pH-sensitive gel microenvironment and the control over domain distribution was not studied in detail.

Here, we report the first systematic method investigating how domains of interest can be localized in many intact GUVs in a fluid environment. The strategy was to confine vesicles in microfluidic channels to create free budding regions where the lipid phase in minority accumulated and formed a single domain minimizing line tension. GUVs of different Ld/Lo compositions were first confined in straight microchannels where we studied the spatio-temporal evolution of domain distribution. The analysis of a large number of confined GUVs allowed us to establish the probability to get the minority phase in the free budding regions of the GUVs. This resulted in the formation of polarized GUVs having one or two Lo or Ld poles at its extremities, surrounding a body mainly composed of the other phases. Finally, other confinement geometries allowed us to direct the distribution of lipid domain into 3 or 4 well-positioned poles.

## Results and Discussion

It has been theoretically and experimentally shown that the evolution of micro-scale domains in Lo/Ld phase-separated GUVs is mainly controlled by line tension, resulting in particular in domain coalescence, growth and budding[23,24] [25] (Figure 1, top). Based on that knowledge, we attempted to control the domain location, growth and coalescence by creating a “free region” where budding would favorably occur and a “confined region” at predefined position in a vesicle by confining it in a microchannel (Figure 1, bottom). At the confined region, we hypothesized that domain budding was hindered due to the close contact of the membrane with the flat walls of the microchannel. In contrast, budding was not restricted in the “free region”. As a result, this “free region” created a favorable region for the domains to coalesce, grow and bud through minimization of the contact line between co-existing phases.

We started with a microfluidic device with straight channels in parallel to capture multiple GUVs in different lanes simultaneously (Figure 2A). GUVs with a typical diameter around 60 µm were confined in the straight channels with a smaller square-
shaped constriction (*h* = *w* = 20, 30, or 40 µm where *h* and *w* where the cannel height and width, respectively). That allowed us to confine and deform GUVs into rod-shaped morphologies (Figure 2A) where the middle part of each confined GUV was in contact with the microchannel walls ("confined region"). In contrast, its extremities constituted "free regions" where the growth and coalescence of lipid domains and their budding would thus be favored. GUVs were produced by electro-formation[7] and introduced in the microfluidic device previously coated with β-casein to limit membrane adhesion. The device was heated above the mixing temperature to get an isotropic membrane[10] and render GUVs more flexible by the increase of membrane surface area as well as by the decrease of their bending rigidity.[23,26] Applying a small pressure allowed to introduce the isotropic GUVs in the confinement channels (Figure 2B). Once the GUVs were confined, the flow inside channels was stopped (Figure S1) to avoid any hydrodynamic effects on domains formation and redistribution. Phase separation was then initiated by letting the system cool down to room temperature (Figure S2). Subsequent domain formation and redistribution were observed under fluorescence microscopy and recorded by camera every 20 or 30 seconds for one hour. We chose the ternary system of DPPC/DOPC/Cholesterol for the model system of Lo/Ld phase separation. In our study, two different lipid compositions, namely *Comp.1* (DPPC/DOPC/Chol = 45/25/30 in molar ratio) and *Comp.2* (DPPC/DOPC/Chol=30/45/25), were selected to obtain Lo-rich and Ld-rich GUVs, respectively. To characterize the distribution of lipid domains, we supplemented the GUVs with fluorescent lipids, which specifically localized in Ld phase (Rhodamine-DHPE) or distributed in both phases (NBD-PE), and characterized the resulting GUVs by fluorescence microscopy. Although broadly distributed, we found that, before confinement, the Lo-rich GUVs with *Comp.1* contained a majority of GUVs with 40% of Ld phase, while the Ld-rich GUVs with *Comp.2* contained a majority of GUVs with 60% of Ld phase (Figure 2C). This phase separation was accompanied by domain coalescence and budding of the phase in minority, i.e., Ld and Lo for *Comp.1* and *Comp.2*, respectively (Figure 2D, top). Interestingly, when these phase-separated GUVs were confined, we observed that lipid domains corresponding to the minority phase were mainly localized in the "free regions" resulting in rod-like shaped GUVs with those domains at their tips (Figure 2D, bottom). Hence, with *Comp.1* (resp. *Comp.2*), Lo-rich (resp. Ld-rich) GUVs had Ld (resp. Lo) domains localized at their tips. We thus obtained the same patterning (minority phase at tips and majority phase in between) with opposite distribution of Lo and Ld phase depending on the initial lipid composition. To characterize the extent of domain localization achieved with this method, we established the distribution of Ld phase along a normalized longitudinal axis of each confined GUV
divided into 20 areas (Figure S3). We repeated this analysis on many confined GUVs and plotted the median value of Ld ratio along this axis for different compositions and geometries of confinement (Figure 2E). This shows the strong effect of domain localization for both compositions producing Lo-rich (Ld domains at the tip) and Ld-rich (Ld-rich phase in the middle) GUVs. Since switching of phases for localization was possible, the method did not depend on the chemical natures of lipids nor on the interaction between domains and walls of the passivated channels. We also obtained similar distributions regardless of the width and height of the confining channel, due to too small curvatures at tips to induce the type of domain sorting that could be observed in highly curved membranes.[27] By looking at the temporal evolution of confined GUVs during the cooling process, we observed that phase separation occurred either by nucleation or spinodal decomposition (Figure 2F, Movie S1, Movie S2). Interestingly, regardless of the mechanism of phase separation, we observed that the majority of domain coalescence and growth was localized at the “free region”. All these results show that line tension and free budding are the main driving parameters for domain localization and maturation in confined GUVs.

The domain localization at both end of the confined vesicles is reminiscent to the
polarization that can be observed in some cellular processes. To further quantify this
analogy, we determined by image analysis the probability of having domains at both ends
of GUVs in our confined system. We introduced an adimensional number referred to as
“polarization index” (PI) to classify the state of individual confined vesicles. Difference
of Ld region (area) between a “Pole” and a “Body”, A_Ld(Pole) and A_Ld(Body) respectively
as described in **Figure 3**, was obtained according to a global Ld ratio (A_Ld(Tot)) in GUV,
as PI = (A_Ld(Pole)- A_Ld(Body))/ A_Ld(Tot). Values of PI index from -1 (full polarization of
Lo domain) to 1 (full polarization of Ld domain) were plotted by two triangles,
representing the situation of each pole in the vesicle, as a function of vesicle length and
Ld/Lo ratio in **Figure 3A**. We further categorized each vesicle into “not polarized” or
“single pole” or “double poles” based on a threshold PI > 0.33 for Ld poles in Lo-rich
vesicles and PI < -0.33 for Lo poles in Ld rich-vesicles, and made a phase diagram in
**Figure 3B** (see **Supplementary Figure S4** for the details). As we see in **Figure 3B**, the
probabilities to have one and two pole(s) is nearly independent of the length of the
confined GUV. When aspect ratio of confined GUV increased, confined region increased
while ratio of free region decreased. At high aspect ratio, domains indeed localized at tips,
it however gave low PI values because of extra minority phases which stayed at middle
confined region due to poor capacity in free region (Supplementary Figure S5). In
contrast, the Ld/Lo ratio has a strong role in defining the number of poles. Many single-
and double-pole of Lo-rich and Ld-rich GUVs were found in a region where the ratio of
Ld phase was around 30-40% and 50-60%, respectively. Conversely, we found either one
pole or no poles in the region of less than 30% and more than 70% of Ld ratio. Certain
amounts of domain were thus required for the coverage of free regions.

Our understanding of domain positioning was applied to different patterning of domains using other geometries. We designed microfluidic chambers with periodically spaced 50 µm-diameter pillars distributed in a hexagonal or squared fashion (Figure 4) and applied a similar procedure of GUVs confinement as with straight channels. Vesicles being trapped between the pillars adopted the local geometry and displayed either 3 or 4 “budding” regions according to the pillar arrangement. As expected, the free budding regions defined by the area between the pillars dictated the localization of the minority phase (Figure 4 + Supplementary movie S3). This eventually led to tripolar and quadripolar GUVs. Mechanism of domain sorting in GUVs under the confinement between pillars seemed to be consistent with that in straight channel. This behavior was therefore not limited to the specific geometry, but appeared instead as a general feature in Ld/Lo phase separation GUVs under confinement. As such, it is readily extendable to other types of geometries to spatially control specific lipid domains, domain-specific membrane-associated proteins[11,30,31] and/or nucleic acids[32] in a user-defined fashion.

**Conclusion**

Spatial position of lipid domain of liquid-disordered/liquid-ordered (Ld/Lo) phase separation in a giant unilamellar vesicle (GUV) was controlled by physical confinement in microchannel. The confinement limits and determines the area where the lipid domain, minority phase of Ld/Lo phase separation, can bud and minimize line tension between co-existing two phases, resulting in a favorable growth and coalesce of domains at the prescribed area. GUVs confined in straight channels and in between pillars produce polarized GUVs from one pole to multi poles, depending on the design of microchannel. Our method is simple, user-defined, and other types of confinement with different composition/species of lipids or even domain specific membrane-associated proteins can also be controlled. Our method can be applied to a future model system for studying some biological phenomena which require polarization, such as cell division.
# Materials and Methods

## Chemicals

1,2-dipalmitoyl-*sn*-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-*sn*-glycero-3-phosphocholine (DOPC) and cholesterol (ovine wool, >98%) were purchased from Avanti Polar Lipids, Inc. N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-*sn*-Glycero-3-Phosphoethanolamine, Triethylammonium Salt (NBD-PE) and Lissamine™ Rhodamine B 1,2-Dihexadecanoyl-*sn*-Glycero-3-Phosphoethanolamine, Triethylammonium Salt (Rhodamine-DHPE) were purchased from Thermo Fisher Scientific. Sucrose, D⁺ glucose, Sodium Azide, Beta-Casein from bovine milk and PBS buffer were purchased from Sigma-Aldrich Co. LLC. Indium Tin Oxide (ITO)-coated glasses (Slide 25 x 75 x 1.1 mm, surface resistivity 15-25 Ω/sq) were from Sigma-Aldrich Co. LLC.

## Giant unilamellar Vesicles (GUVs) preparation

GUVs were prepared by the electroformation method using a custom-made protocol adapted from the historical paper[7]. DPPC, DOPC and Cholesterol with desired relative amounts were dissolved in Chloroform/Methanol 9:1 (v/v%) to a total lipid concentration of 20 mM. NBD-PE and Rhodamine-DHPE were added to the lipid mixture (0.1 mol% for each) in order to visualize Lo and Ld phases, respectively, based on the fact that Rhodamine-DHPE distributes in Ld phase while NBD-PE are in both Ld and Lo phases with strong preference in Ld phase.[33] This lipid solution was spread on the conductive side of two pre-warmed ITO-coated glass slides (70 °C, 6 µL lipid deposition for each). A lipid dry film immediately formed on the substrate and the film was further dried under vacuum for 3 hours. A chamber made by the two lipid-deposited ITO slides and a 1 mm-thick PDMS spacer was then filled by a 100 mOsm/L Sucrose, 5 mM NaN₃ aqueous solution. The chamber was sealed with a PDMS plug, inserted into a 70 °C dry oven and 600 mV AC voltage at 10 Hz was applied for 3 hours. The resulting GUV solution was pipetted to a glass tube, kept at 4 °C and used for experiments within one week.

## Microfluidic device fabrication

The microfluidic device was fabricated in poly-dimethylsiloxane (PDMS) using standard photolithography technique.[34] Briefly, the master mold was produced by spin-coating a desired thickness of negative photoresist (SU8-2025, Microchem) on a silicon wafer and exposing it to UV light through a chromium mask of the desired design (ex. Figure 2A). After development of the resist, the master mold was coated with a 100 nm layer of Parylene by vapor deposition. PDMS (Sylgard 184, Dow) was poured on the master,
degassed and then baked overnight at 72 °C. PDMS was removed from the master, the chip was cut, and inlets pierced with a 1.5 mm-diameter biopsy punch, then sealed to a glass coverslip by oxygen plasma bonding. To reduce surface interaction with vesicles, channels were passivated by flowing a PBS solution containing 0.5 wt. % Beta-Casein immediately after bonding and for at least 30 min.[35] The channels were then washed with a 100 mOsm/L sucrose solution containing 5 mM of NaN₃ as preservative.

## GUV reshaping in microfluidic device and imaging/data analysis

Experiments were conducted on an epifluorescence microscope (Observer Z1, Zeiss) equipped with a transparent heating stage (Thermo Plate, Tokai Hit), an EMCCD camera (Evolve, Photometrics) and a Xenon light source (XBO, Zeiss). GUVs solution was first loaded in a 0.5mm ID Tygon tubing (Tygon S54HL, Saint-Gobain), connected to the inlet of the microfluidic device and flowed using pressure controllers (MFCS, Fluigent). To avoid strong shear, a small pressure difference (1-2 mbars) was applied between input and output. The chip was put in a homemade humidity chamber (Supplementary Figure S1) to avoid evaporation through PDMS, and temperature was increased to 50 °C in order to melt the liquid-ordered phase (T_m= ca. 35~38 °C, depending on the composition)[10] and to increase membrane flexibility so that GUVs entered the smaller channels (20-40 µm width). The absence of residual flow was ensured by closing tubing clamps at inlet and outlet. The heating stage was then stopped, resulting in a progressive cooling from 50 °C to room temperature. Fluorescence image was acquired after 1 hour from the beginning of the cooling ramp. In order to follow the kinetics of distribution and rearrangement of domains, time lapse was applied every 20 seconds from the beginning of the cooling ramp for 1 hour with autofocus (Definite focus, Zeiss). NBD-PE was imaged through a FITC filter set (Exc: 470/40 nm Dc: 495 nm Em: 525/50 nm) and Rhodamine-DHPE through a mCherry filter set (Exc: 562/40 nm Dc: 593 nm Em: 641/75 nm). Fluorescence images were then treated and analyzed using a home-made routine (Fiji). Results of GUV detection and proportion of Rhodamine-DHPE (Ld domains) along vesicles were further analyzed using Matlab.

## Funding

This work was supported by the European Research Council (ERC) (European Community's Seventh Framework Programme (FP7/2007-2013)/ERC Grant Agreement No. 258782, D.B.), the French National Research Agency ANR contracts DYOR (ANR-18-CE06-0019, D.B.), and an Overseas Research Fellowship from the Japan Society for
the Promotion of Science (JSPS, K.N.). It has received the support of "Institut Pierre-Gilles de Gennes" (laboratoire d'excellence) and "Investissements d'avenir" program ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-31, and ANR-10-EQPX-34

**Acknowledgement**

We thank Armand Despons, Jacques Fattaccioli and Yong Chen (Ecole Normale Supérieure) for experimental support and/or discussion.

**Conflict of interest**

The authors declare no conflict of interest.

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<figure><img src="image_2.png" /><figcaption>**Figure 1.** Schematic representation of the strategy to prescribe spatial position of lipid domain (minority phase of Lo/Ld phase separation) by physical confinement of a Giant Unilamellar Vesicle (GUV). Without confinement (top), domains are randomly positioned and budding to reduce the line tension between co-existing phases. In contrast, under physical confinement in a microchannel (bottom), domains growth and coalescence are facilitated by budding at free regions, resulting in a domain localization, a process we referred to as “restricted budding”.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Microfluidic confinement of phase-separated GUVs directs the positioning and growth of minority phase lipid domains at the free regions in a composition-dependent but Lo/Ld phase-independent manner. A) A design of a microfluidic</figcaption></figure>
channel (left) and fluorescence/transmission microscopy images of confined GUV inside
straight channel (middle). Red and blue signals correspond to the region of Ld phase and
Lo phase, respectively. Right image shows schematic drawing of confined GUV from
side view. B) Experimental protocol of GUV confinement in microchannel. GUV was
heated over Tmix before the confinement and was cooled down after the confinement to
induce phase separation. C) Distributions of Ld ratio in GUVs with Comp.1
(DPPC/DOPC/Chol=45/25/30) and Comp.2 (DPPC/DOPC/Chol=30/45/25) were plotted
at the left top graph. (Number of analyzed vesicles Nv = 476 for Comp.1 and Nv = 428 for
Comp.2) D) Lo-rich GUVs with Comp.1 and Ld-rich GUVs with Comp.2 with/without
confinement in straight microchannel (h, wchannel= ca. 30 µm) were imaged using
fluorescence microscopy (left bottom images). Red and blue signals correspond to the
region of Ld phase and Lo phase, respectively. E) Statistical analysis of domain
distribution in GUVs confined in straight channel. Distribution profiles of Ld ratio across
the longitudinal axis of GUV in each 20 devided area were obtained in all the confined
vesicles, and median values of Ld ratio was plotted. Number of analyzed vesicles Nv is
indicated below the graph. Different width and height of microchannel (20, 30, 40 µm)
was tested by using Lo-rich GUVs. Profile of each individual vesicles is shown in
**Supplementary Figure S3**. F) Time lapse fluorescence microscopy images of confined
GUV in straight channel (h, wchannel= ca. 40 µm). Both Lo-rich GUV and Ld-rich GUV
were represented in top and bottom, respectively. Temperature change by time were
indicated upon each image. Red and blue signals correspond to the region of Ld phase
and Lo phase, respectively. All scale bars are 50 µm.
<figure><img src="image_4.png" /><figcaption>**Figure 3. Polarization occurrence in confined GUVs.** A) Color plot of polarization index PI = (A<sub>Ld</sub>(Pole) - A<sub>Ld</sub>(Body)) / A<sub>Ld</sub>(Tot) (A<sub>Ld</sub>: area occupied by liquid disordered (Ld) phase, A<sub>Ld</sub>(Tot): the total area of Ld domain) as a function of GUV length and global ratio of Ld phase in GUV. B) Categorization of GUVs in “single pole” (red diamond; single Ld pole, blue diamond; single Lo pole), “double poles” (red star: double Ld poles, blue star: double Lo pole) and “other situations” (black circle) based on the threshold of PI value (Figure 3A) as following; PI > 0.33 for Ld poles in Lo-rich vesicles and PI < -0.33 for Lo poles in Ld rich-vesicles. Distribution profiles of Ld phase in each category are shown in **Supplementary Figure S4**. Results are plotted and phase diagram was made as a function of GUV length and global ratio of Ld phase in GUV.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Controlled generation of multipolar GUVs using multiple free budding regions. Transmission microscopy images of pillars in microchannel (left). Schematic illustrations of confined GUVs between pillars (middle) and fluorescence microscopy images of GUVs confined between pillars (right). Lo-rich GUV with Comp. 1 and Ld-rich GUV with Comp. 2 60 min after cooling down were both imaged. Red and blue signals correspond to the region of Ld phase and Lo phase, respectively. All scale bars are 50 µm.</figcaption></figure>