Development of Giant Unilamellar Vesicles for the Study of Crowded Protein Environments David D. Gooray, Sandeep Dhall, and Victor G. J. Rodgers Bioengineering Department, University of California, Riverside Giant unilamellar vesicles (GUVs) are supramolecular structures consisting of amphiphiles that range in sizes from 10-100 µm. [1] They provide an easy method to: (1) observe environment for in-vitro studies of compartmentalized reactions and (2) model particular cell behavior. By studying the effects of crowded protein environments within GUVs, we will achieve a greater understanding of processes and properties such as mitochondrial swelling and osmotic pressure. Phosphatidylcholine lipids are the only lipids that can form GUVs under electroformation. [2] GUVs with a diameter ranging between 50-100 µm will withstand micromanipulation techniques and allow us to study the effects of crowded protein environments. We prepared GUVs using the 3 types of phosphatidylcholine lipids; 1- palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), L-α- phosphatidylcholine (Egg PC) and 1-2- dioleoyl-sn-glyeo-3- phosphocholine(DPC). o A square chamber was cut from teflon and molded to fit the microscope slide. o Two cylindrical platinum wires (Sigma-Aldrich) of 1.0 mm diameter were housed approximately parallel to each other with a 3.0 mm separation o Each wire was 1.5 mm from transparent viewing slide o All drilled holes and platinum-teflon interfaces were sealed with silicone o Preparation of GUVs experiments o Obtain GUVs with 50-100 µm diameter o Develop a crowded protein environment Research Objectives Conclusion & Future Work Results (Continued) Results GUVs that were obtained were found attached to the electrode. They will be micromanipulated for studying crowded protein environments. 50 microns 25 microns 25 microns POPC GUVs 25 microns Egg PC GUVs 25 microns DPC GUVs Acknowledgements oB2K Group, oDr. Vullev, Dr. Anvari, and Dr. Park oJun Wang and Bioengineering department. Electroform ation Chamber Function Generator Inverted Microscop e Digital Imaging Camera Oscillosc ope Pt Terminals Silicone Sealant Chamber designed by B2K Teflon Housing The GUVs selected for micromanipulation had an average size of 66.9 µm and were observed using a light microscope with a phase contrast filter. Average diameter of GUVs were determined using ImageJ. The average diameters of the GUVs obtained are: o POPC GUVs - 52.5 µm, o EPC GUVs - 67.6 µm, and o DOPC GUVs - 70.4 µm We successfully setup the experiments for preparing GUVs and obtained the required sizes. GUVs with individual distribution on the wire were dependent on the precision of lipid application on the wire. The future direction includes loading of the GUVs with proteins while surrounding their environments with a similar concentration of the protein. This is to reduce the osmotic pressure within the GUVs and their respective loading characteristics. We will investigate better microinjection apparatus to enhance the precision of our piercings and load delivery into the GUVs. Materials & MethodsYa o Stock solutions of 1-palmitoyl-2- oleoyl-sn-glycero-3- phosphocholine (POPC), L-α- lysophosphatidylcholine (EPC) and 1-2-dioleoyl-sn-glyeo-3- phosphocholine (DPC) were made. (Avanti Polar Lipids) o Frequency: 10 Hz o A-C Voltage Ramp: 0.3 V increased to 2.3 V in 15 min o 2.3 V applied for 105 min [1] Luisi, P. and Walde, P. Giant Vesicles. New York: John Wiley & Sons Ltd. 2000 [2] Angelova,M.I., S.Soleau, P.Melead,J.- Faucon, and P.Bothorel. Preperation of giant vesicles by external ac electric fields: kinetics and applications.Prog. Colloid Polym. Sci. 89:127- 131(1992) Experimental Setup References Single GUV Experimental set up Size distribution of GUVs Introduction

Development of Giant Unilamellar Vesicles for the Study of Crowded Protein Environments David D. Gooray, Sandeep Dhall, and Victor G. J. Rodgers Bioengineering

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Development of Giant Unilamellar Vesicles for the Study of Crowded Protein Environments

David D. Gooray, Sandeep Dhall, and Victor G. J. Rodgers

Bioengineering Department, University of California, Riverside

Giant unilamellar vesicles (GUVs) are supramolecular structures consisting of amphiphiles that range in sizes from 10-100 µm.[1] They provide an easy method to: (1) observe environment for in-vitro studies of compartmentalized reactions and (2) model particular cell behavior. By studying the effects of crowded protein environments within GUVs, we will achieve a greater understanding of processes and properties such as mitochondrial swelling and osmotic pressure.

Phosphatidylcholine lipids are the only lipids that can form GUVs under electroformation.[2] GUVs with a diameter ranging between 50-100 µm will withstand micromanipulation techniques and allow us to study the effects of crowded protein environments.

We prepared GUVs using the 3 types of phosphatidylcholine lipids; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), L-α-phosphatidylcholine (Egg PC) and 1-2-dioleoyl-sn-glyeo-3-phosphocholine(DPC).

o A square chamber was cut from teflon and molded to fit the microscope slide.o Two cylindrical platinum wires (Sigma-Aldrich) of 1.0 mm diameter were housed

approximately parallel to each other with a 3.0 mm separationo Each wire was 1.5 mm from transparent viewing slideo All drilled holes and platinum-teflon interfaces were sealed with silicone

o Preparation of GUVs experimentso Obtain GUVs with 50-100 µm diametero Develop a crowded protein environment

Research Objectives

Conclusion & Future Work

Results (Continued)

Results

GUVs that were obtained were found attached to the electrode. They will be micromanipulated for studying crowded protein environments.

50 microns

25 microns

POPC GUVs

25 microns

Egg PC GUVs

25 microns

DPC GUVs

Acknowledgements

oB2K Group, oDr. Vullev, Dr. Anvari, and Dr. ParkoJun Wang and Bioengineering department.

Electroformation Chamber

Function Generator

Inverted Microscope

Digital Imaging Camera

Oscilloscope

Pt Terminals

Silicone Sealant

Chamber designed by B2K

Teflon Housing

The GUVs selected for micromanipulation had an average size of 66.9 µm and were observed using a light microscope with a phase contrast filter. Average diameter of GUVs were determined using ImageJ. The average diameters of the GUVs obtained are:o POPC GUVs - 52.5 µm,o EPC GUVs - 67.6 µm, and o DOPC GUVs - 70.4 µm

We successfully setup the experiments for preparing GUVs and obtained the required sizes. GUVs with individual distribution on the wire were dependent on the precision of lipid application on the wire.

The future direction includes loading of the GUVs with proteins while surrounding their environments with a similar concentration of the protein. This is to reduce the osmotic pressure within the GUVs and their respective loading characteristics.

We will investigate better microinjection apparatus to enhance the precision of our piercings and load delivery into the GUVs.

Materials & MethodsYa

o Stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), L-α-lysophosphatidylcholine (EPC) and 1-2-dioleoyl-sn-glyeo-3-phosphocholine (DPC) were made. (Avanti Polar Lipids)

o Frequency: 10 Hzo A-C Voltage Ramp: 0.3 V increased to 2.3 V

in 15 mino 2.3 V applied for 105 min

[1]Luisi, P. and Walde, P. Giant Vesicles. New York: John Wiley & Sons Ltd. 2000

[2]Angelova,M.I., S.Soleau, P.Melead,J.- Faucon, and P.Bothorel. Preperation of giant vesicles by external ac electric fields: kinetics and applications.Prog. Colloid Polym. Sci. 89:127-131(1992)

Experimental Setup

References

Single GUV

Experimental set up Size distribution of GUVs

Introduction