Fluorescent probe partitioning in giant unilamellar vesicles of ‘lipid raft’ mixtures

Direct visualization of raft-like lo (liquid-ordered) domains in model systems and cells using microscopic techniques requires fluorescence probes with known partitioning preference for one of the phases present. However, fluorescent probes may display dissimilar partitioning preferences in different lipid sys-tems and can also affect the phase behaviour of the host lipid bilayer. Therefore a detailed understanding of the behaviour of fluorescent probes in defined lipid bilayer systems with known phase behaviour is essential before they can be used for identifying domain phase states. Using giant unilamellar vesicles composed of the ternary lipid mixture DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)/cholesterol, for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned either on the basis of quantification of the domain area fractions or by using a well-characterized ld (liquid-disordered)-phase marker. Fluorescent probes were examined both individually and using dual or triple labelling approaches. Most of the probes partitioned individually into the ld phase, whereas only NAP (naphtho[2,3-a]pyrene) and NBD-DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl] preferred the lo phase. We found that Rh-DPPE (Lissamine™ rhodamine B–1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, Tmix. Interestingly, the partitioning of DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy®-PC [2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine]. The specific use of each of the fluorescent probes is determined by its photostability, partitioning preference, ability to detect lipid phase separations and induced change in Tmix. We demonstrate the importance of testing a specific fluorescent probe in a given model membrane system, rather than assuming that it labels a particular lipid phase.

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Giant unilamellar vesicles - a perfect tool to visualize phase separation and lipid rafts in model systems.

Acta Biochimica Polonica ◽

10.18388/abp.2009_2514 ◽

2009 ◽

Vol 56 (1) ◽

Cited By ~ 68

Author(s):

Olga Wesołowska ◽

Krystyna Michalak ◽

Jadwiga Maniewska ◽

Andrzej B Hendrich

Keyword(s):

Phase Separation ◽

Lipid Membranes ◽

Average Diameter ◽

Model Systems ◽

Membrane Properties ◽

Lipid Phase ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Simple Method ◽

Wide Range

Model systems such as black lipid membranes or conventional uni- or multilamellar liposomes are commonly used to study membrane properties and structure. However, the construction and dimensions of these models excluded their direct optical microscopic observation. Since the introduction of the simple method of liposome electroformation in alternating electric field giant unilamellar vesicles (GUVs) have become an important model imitating biological membranes. Due to the average diameter of GUVs reaching up to 100 microm, they can be easily observed under a fluorescent or confocal microscope provided that the appropriate fluorescent probe was incorporated into the lipid phase during vesicle formation. GUVs can be formed from different lipid mixtures and they are stable in a wide range of physical conditions such as pH, pressure or temperature. This mini-review presents information about the methods of GUV production and their usage. Particularly, the use of GUVs in studying lipid phase separation and the appearance and behavior of lipid domains (rafts) in membranes is discussed but also other examples of GUVs use in membrane research are given. The experience of the authors in setting up the GUV-forming equipment and production of GUVs is also presented.

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Atomic force microscopy of phase separation on ruptured, giant unilamellar vesicles

10.1101/250944 ◽

2018 ◽

Cited By ~ 1

Author(s):

Yanfei Jiang ◽

Guy M. Genin ◽

Kenneth M. Pryse ◽

Elliot L. Elson

Keyword(s):

Atomic Force Microscopy ◽

Phase Separation ◽

Model Systems ◽

Study Phase ◽

Dark Phase ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Force Microscopy ◽

Atomic Force ◽

Lipid Species

AbstractGiant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) are synthetic model systems widely used in biophysical studies of lipid membranes. Phase separation behaviors of lipid species in these two model systems differ due to the lipid-substrate interactions that are present only for SLBs. Therefore, GUVs are believed to resemble natural cell membranes more closely, and a very large body of literature focuses on applying nano-characterization techniques to quantify phase separation on GUVs. However, one important technique, atomic force microscopy (AFM), has not yet been used successfully to study phase separation on GUVs. In the present study, we report that in binary systems, certain phase domains on GUVs retain their original shapes and patterns after the GUVs rupture on glass surfaces. This enabled AFM experiments on phase domains from binary GUVs containing 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). These DLPC/DSPC and DLPC/DPPC GUVs both presented two different gel phases, one of which (bright phase) included a relatively high concentration of DiI-C20 but excluded Bodipy-HPC, and the other of which (dark phase) excluded both probes. The bright phases are of interest because they seem to stabilize dark phases against coalescence. Results suggested that the gel phases labeled by DiI-C20 in the DLPC/DSPC membrane, which surround the dark gel phase, is an extra layer of membrane, indicating a highly curved structure that might stabilize the interior dark domains. This phenomenon was not found in the DLPC/DPPC membrane. These results show the utility of AFM on collapsed GUVs, and suggest a possible mechanism for stabilization of lipid domains.

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Directed tubule growth from giant unilamellar vesicles in a thermal gradient

Soft Matter ◽

10.1039/c8sm01892h ◽

2019 ◽

Vol 15 (7) ◽

pp. 1676-1683 ◽

Cited By ~ 2

Author(s):

Emma L. Talbot ◽

Jurij Kotar ◽

Lorenzo Di Michele ◽

Pietro Cicuta

Keyword(s):

Liquid Phase ◽

Thermal Gradient ◽

Phase Coexistence ◽

Lipid Phase ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Experimental Control

We demonstrate experimental control over tubule growth in giant unilamellar vesicles with liquid–liquid phase coexistence, using a thermal gradient to redistribute lipid phase domains on the membrane.

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Accurate quantification of inter-domain partition coefficients in GUVs exhibiting lipid phase coexistence

RSC Advances ◽

10.1039/c6ra13170k ◽

2016 ◽

Vol 6 (71) ◽

pp. 66641-66649 ◽

Cited By ~ 5

Author(s):

M. J. Sarmento ◽

S. N. Pinto ◽

A. Coutinho ◽

M. Prieto ◽

F. Fernandes

Keyword(s):

Partition Coefficients ◽

Phase Coexistence ◽

Lipid Phase ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Domain Partition ◽

Fluorescent Molecules ◽

Accurate Quantification

Giant unilamellar vesicles (GUVs) with phase coexistence allow for the recovery of inter-domain partition coefficients (Kp) of fluorescent molecules through comparison of fluorescence intensities in each phase.

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Quantitative optical microscopy and micromanipulation studies on the lipid bilayer membranes of giant unilamellar vesicles

Chemistry and Physics of Lipids ◽

10.1016/j.chemphyslip.2014.02.009 ◽

2014 ◽

Vol 181 ◽

pp. 99-120 ◽

Cited By ~ 30

Author(s):

Luis A. Bagatolli ◽

David Needham

Keyword(s):

Optical Microscopy ◽

Lipid Bilayer ◽

Lipid Bilayer Membranes ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Bilayer Membranes

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Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles

Nature Protocols ◽

10.1038/nprot.2017.047 ◽

2017 ◽

Vol 12 (8) ◽

pp. 1563-1575 ◽

Cited By ~ 5

Author(s):

Tripta Bhatia ◽

Flemming Cornelius ◽

John H Ipsen

Keyword(s):

Lipid Bilayer ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Free Standing ◽

Dynamic Structures

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Microfluidic Deformability Study of an Innovative Blood Analogue Fluid Based on Giant Unilamellar Vesicles

Journal of Functional Biomaterials ◽

10.3390/jfb9040070 ◽

2018 ◽

Vol 9 (4) ◽

pp. 70 ◽

Cited By ~ 4

Author(s):

Denise Carvalho ◽

Ana Rodrigues ◽

Vera Faustino ◽

Diana Pinho ◽

Elisabete Castanheira ◽

...

Keyword(s):

Ethical Issues ◽

Soybean Lecithin ◽

Flow Behavior ◽

Extensional Flow ◽

Point Of View ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Lipid Mixture ◽

Further Development ◽

Good Agreement

Blood analogues have long been a topic of interest in biofluid mechanics due to the safety and ethical issues involved in the collection and handling of blood samples. Although the current blood analogue fluids can adequately mimic the rheological properties of blood from a macroscopic point of view, at the microscopic level blood analogues need further development and improvement. In this work, an innovative blood analogue containing giant unilamellar vesicles (GUVs) was developed to mimic the flow behavior of red blood cells (RBCs). A natural lipid mixture, soybean lecithin, was used for the GUVs preparation, and three different lipid concentrations were tested (1 × 10−3 M, 2 × 10−3 M and 4 × 10−3 M). GUV solutions were prepared by thin film hydration with a buffer, followed by extrusion. It was found that GUVs present diameters between 5 and 7 µm which are close to the size of human RBCs. Experimental flow studies of three different GUV solutions were performed in a hyperbolic-shaped microchannel in order to measure the GUVs deformability when subjected to a homogeneous extensional flow. The result of the deformation index (DI) of the GUVs was about 0.5, which is in good agreement with the human RBC’s DI. Hence, the GUVs developed in this study are a promising way to mimic the mechanical properties of the RBCs and to further develop particulate blood analogues with flow properties closer to those of real blood.

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