Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

Cellular Functions ◽

Unilamellar Vesicles ◽

Synthetic Cells ◽

Buffer Composition ◽

Protein Incorporation ◽

Functional Investigation

Giant unilamellar vesicles (GUVs) are micrometer-sized model membrane systems that can be viewed directly under the microscope. They serve as scaffolds for the bottom-up creation of synthetic cells, targeted drug delivery and have been used in many in vitro studies of membrane related phenomena. GUVs are also of interest for the functional investigation of membrane proteins that carry out many key cellular functions. A major hurdle to a wider application of GUVs in this field is the diversity of existing protocols that are optimized for individual proteins. Here, we compare PVA assisted and electroformation techniques for GUV formation under physiologically relevant conditions, and analyze the effect of immobilization on vesicle structure and membrane tightness towards small substrates and protons. There, differences in terms of yield, size, and leakage of GUVs produced by PVA assisted swelling and electroformation were found, dependent on salt and buffer composition. Using fusion of oppositely charged membranes to reconstitute a model membrane protein, we find that empty vesicles and proteoliposomes show similar fusion behavior, which allows for a rapid estimation of protein incorporation using fluorescent lipids.

Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion

FEBS Letters ◽

10.1002/1873-3468.12233 ◽

2016 ◽

Vol 590 (14) ◽

pp. 2051-2062 ◽

Cited By ~ 19

Author(s):

Olivier Biner ◽

Thomas Schick ◽

Yannic Müller ◽

Christoph von Ballmoos

Keyword(s):

Membrane Proteins ◽

Imaging analysis to quantitate the Interplay of membrane and cytoplasm protein dynamics

10.1101/2021.12.01.470244 ◽

2021 ◽

Author(s):

N Kislev ◽

M Egozi ◽

D Benayahu

Keyword(s):

Membrane Proteins ◽

Protein Expression ◽

Glucose Transporter ◽

Imaging Method ◽

Protein Distribution ◽

Cellular Functions ◽

Membrane Expression ◽

Cellular Processes ◽

Imaging Tool

AbstractPlasma membrane proteins are extremely important in cell signaling and cellular functions. Protein expression and localization alter in response to various signals in a way that is dependent on cell type and niche. Compartmental quantification of the expression of particular proteins is a very useful means of understanding their role in cellular processes. Immunofluorescence staining is frequently used to investigate the distribution of proteins of interest. Here, we present an imaging method for quantifying the membrane to cytoplasm ratio (MCR) of proteins analyzed at single-cell resolution. This technique provides a robust quantification of membrane proteins and contributes new insights into membrane expression dynamics. We have developed a protocol that uses immunostaining to assess protein expression according to the fluorescent cellular distribution and to compute the MCR. The method was applied to measure the MCR of glucose transporter 4 (GLUT4) in response to insulin in 3T3-L1 cells, an in-vitro model for adipocyte function and adipogenesis. The results revealed informative changes in the subcellular localization of GLUT4 following insulin induction. MCR analysis is a powerful imaging tool that can be generally applied to membrane proteins to provide a rapid and efficient quantitative analysis of protein distribution and sub-cellular processes in cells.

Surfactant-free production of biomimetic giant unilamellar vesicles using PDMS-based microfluidics

Communications Chemistry ◽

10.1038/s42004-021-00530-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Naresh Yandrapalli ◽

Julien Petit ◽

Oliver Bäumchen ◽

Tom Robinson

Keyword(s):

High Throughput ◽

Paradigm Shift ◽

Mammalian Cells ◽

Lipid Vesicles ◽

Unilamellar Vesicles ◽

Artificial Cells ◽

Membrane Function ◽

Lipid Diffusion ◽

Protein Incorporation

AbstractMicrofluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.

Distribution, Lateral Mobility and Function of Membrane Proteins Incorporated into Giant Unilamellar Vesicles

Biophysical Journal ◽

10.1529/biophysj.104.053413 ◽

2005 ◽

Vol 88 (2) ◽

pp. 1134-1142 ◽

Cited By ~ 107

Author(s):

Mark K. Doeven ◽

Joost H.A. Folgering ◽

Victor Krasnikov ◽

Eric R. Geertsma ◽

Geert van den Bogaart ◽

...

Keyword(s):

Membrane Proteins ◽

Lateral Mobility ◽

Unilamellar Vesicles ◽

And Function

Determining Unitary Water Permeability of Membrane Proteins Reconstituted into Giant Unilamellar Vesicles

Biophysical Journal ◽

10.1016/j.bpj.2015.11.1195 ◽

2016 ◽

Vol 110 (3) ◽

pp. 215a ◽

Cited By ~ 1

Author(s):

Danila Boytsov ◽

Christof Hannesschlaeger ◽

Andreas Horner ◽

Peter Pohl

Keyword(s):

Membrane Proteins ◽

Water Permeability ◽

A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

Biophysical Journal ◽

10.1529/biophysj.104.040360 ◽

2004 ◽

Vol 87 (1) ◽

pp. 419-429 ◽

Cited By ~ 161

Author(s):

Philippe Girard ◽

Jacques Pécréaux ◽

Guillaume Lenoir ◽

Pierre Falson ◽

Jean-Louis Rigaud ◽

...

Keyword(s):

Membrane Proteins ◽

New Method ◽

Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles via Peptide-Induced Fusion

Biophysical Journal ◽

10.1016/s0006-3495(01)75801-8 ◽

2001 ◽

Vol 81 (3) ◽

pp. 1464-1474 ◽

Cited By ~ 112

Author(s):

Nicoletta Kahya ◽

Eve-Isabelle Pécheur ◽

Wim P. de Boeij ◽

Douwe A. Wiersma ◽

Dick Hoekstra

Keyword(s):

Membrane Proteins ◽

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

10.1101/2021.02.24.432456 ◽

2021 ◽

Cited By ~ 1

Author(s):

Lori Van de Cauter ◽

Federico Fanalista ◽

Lennard van Buren ◽

Nicola De Franceschi ◽

Elisa Godino ◽

...

Keyword(s):

Encapsulation Efficiency ◽

Double Emulsion ◽

Dna Nanostructures ◽

Unilamellar Vesicles ◽

High Quality ◽

Emulsion Method ◽

Pure Protein ◽

Biochemical Systems ◽

Synthetic Cells

AbstractGiant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.