Periodic Reporting for period 1 - BiLamVesicles (Novel bi-lamellar lipid vesicles for studying double-membrane transenvelope proteins)
Période du rapport: 2020-08-01 au 2022-07-31
Bi-lamellar membranes, composed of two lipid bi-layers, are ubiquitous in cellular organisms with examples ranging from the double membrane envelope of Gram-negative bacteria to the two lipid-bilayers of eukaryotic subcellular organelles like the cell nucleus and mitochondrion. These double-membranes host sophisticated protein systems that perform many key activities which are crucial for the survival of the cell. For instance, bacterial efflux pumps such as AcrAB-TolC in Escherichia coli are perhaps the most important component in the bacterial line of defense against antibiotic treatment; and while molecularly detailed structures of many types of multidrug transporters are now available, the complex nature of double membranes renders the study of these proteins in live cells highly challenging. In addition, contemporary artificial vesicles like liposomes and other artificial membrane models are inappropriate for hosting and studying this important class of proteins as they either composed of a single or multiple lipid bilayers. My postdoctoral research, as a Marie-Curie fellow, aims to develop a novel artificial double-membrane vesicle for studying the permeation and transport of various molecules through a membranous model that mimics the membrane architecture of various bacteria and organelles.
BiLamVesicles focuses on the synthesis of novel bi-lamellar (or double membrane) giant unilamellar vesicles for studying the transport activity of membrane proteins that naturally span across two membranes. The implementation of BiLamVesicles involved the use of various optical techniques and development of novel microfluidic approaches to set up a framework for synthesizing giant vesicle models and studying mass transport across their membrane. To enable the controlled production of artificial giant vesicle models, the first part of the project was mainly focused on design and development of a novel integrated microfluidic device capable of producing, manipulating and purifying giant unilamellar vesicles (GUVs). Examination of the synthesized vesicles and their properties was mainly attained using various optical techniques, including atomic force microscopy as well as epifluorescence and confocal microscopy. Then, through exploiting the developed microfluidic platforms, two different approaches for generating double membrane giant vesicles were explored and attempts to incorporate the bacterial efflux pump AcrAB-TolC were made. The last part of the project was mainly devoted for studying ion transport across GUV membranes and elucidating the association between ionic fluxes and generated electrochemical gradient across the lipid bilayer. For that purpose, a transport assay with a suitable image analysis script was designed.
The layer-by-layer synthesis of bi-lamellar vesicles was performed in a stepwise manner. In the first stage, GUVs with a narrow size distribution were produced using octanol-assisted liposome assembly (OLA), a novel droplet-based microfluidic technique. The intermembrane space was then successfully formed by incubating the GUVs with poly-L-lysine (PLL), a positively charged polymer. In the last stage of constructing the outer membrane through SUVs rupture, it was crucial to remove excess of free PLL from the GUVs solution. For this purpose, I designed a microfluidic device capable of separating GUVs from a broad range of residual components, including dye molecules, polymers and oil droplets. Furthermore, by combining the purification device with a microfluidic production module (OLA), I developed the first integrated microfluidic chip capable of simultaneously producing, manipulating and purifying GUVs. The results, design and operation of the integrated microfluidic device were summarized and published in a peer-reviewed journal (ACS Synthetic Biology).Utilizing the microfluidic-based purification platform I managed to purify the poly-L-lysine (PLL) coated GUVs and create giant vesicles with an intermembrane spacer the mimics the periplasm of gram negative bacteria. Subsequently, I verified that negatively charged SUVs rupture on the positively charged intermembrane space and decorate the GUVs. However, attempts to create an intact outer lipid-bilayer were unsuccessful. Therefore, an alternative synthesis approach was taken through growing a GUV within an existing mother GUV and positive preliminary results were obtained. Owing to setbacks in construction of the double-membrane, ongoing efforts to reconstitute the full efflux pump, AcrAB-TolC, in a single phospholipid-bilayer are currently taking place. Taking into consideration that proton electrochemical gradients power the function of many membrane transporters, including AcrAB-TolC, I proceeded with studying the correlation between proton (H+) transport and electrochemical gradient evolution across the GUV membrane. By directly measuring the permeability rate of protons across the membrane of single GUVs the resultant electrochemical gradient, that is generated in response to proton flux, could be quantified. The obtained results open the way for correlating energy input to efflux pumps activity towards elucidating their underlying operational mechanism. These findings were summarized and published in a peer-reviewed journal (Biophysical Journal). To broaden the understanding of protein-based transport we explored the transport of potassium (K+) across two archetypical cation-selective channels, gramicidin A (gA) and the bacterial porin OmpF (currently under review). Analysis of potassium fluxes across ion channels reconstituted in the membrane of GUVs, trapped in microfluidic chip, provided a useful insight into their cation selectivity and role in determining the rate of charge accumulation in the vesicle interior, at the single-vesicle-level.
The layer-by-layer synthesis of bi-lamellar vesicles was performed in a stepwise manner. In the first stage, GUVs with a narrow size distribution were produced using octanol-assisted liposome assembly (OLA), a novel droplet-based microfluidic technique. The intermembrane space was then successfully formed by incubating the GUVs with poly-L-lysine (PLL), a positively charged polymer. In the last stage of constructing the outer membrane through SUVs rupture, it was crucial to remove excess of free PLL from the GUVs solution. For this purpose, I designed a microfluidic device capable of separating GUVs from a broad range of residual components, including dye molecules, polymers and oil droplets. Furthermore, by combining the purification device with a microfluidic production module (OLA), I developed the first integrated microfluidic chip capable of simultaneously producing, manipulating and purifying GUVs. The results, design and operation of the integrated microfluidic device were summarized and published in a peer-reviewed journal (ACS Synthetic Biology).Utilizing the microfluidic-based purification platform I managed to purify the poly-L-lysine (PLL) coated GUVs and create giant vesicles with an intermembrane spacer the mimics the periplasm of gram negative bacteria. Subsequently, I verified that negatively charged SUVs rupture on the positively charged intermembrane space and decorate the GUVs. However, attempts to create an intact outer lipid-bilayer were unsuccessful. Therefore, an alternative synthesis approach was taken through growing a GUV within an existing mother GUV and positive preliminary results were obtained. Owing to setbacks in construction of the double-membrane, ongoing efforts to reconstitute the full efflux pump, AcrAB-TolC, in a single phospholipid-bilayer are currently taking place. Taking into consideration that proton electrochemical gradients power the function of many membrane transporters, including AcrAB-TolC, I proceeded with studying the correlation between proton (H+) transport and electrochemical gradient evolution across the GUV membrane. By directly measuring the permeability rate of protons across the membrane of single GUVs the resultant electrochemical gradient, that is generated in response to proton flux, could be quantified. The obtained results open the way for correlating energy input to efflux pumps activity towards elucidating their underlying operational mechanism. These findings were summarized and published in a peer-reviewed journal (Biophysical Journal). To broaden the understanding of protein-based transport we explored the transport of potassium (K+) across two archetypical cation-selective channels, gramicidin A (gA) and the bacterial porin OmpF (currently under review). Analysis of potassium fluxes across ion channels reconstituted in the membrane of GUVs, trapped in microfluidic chip, provided a useful insight into their cation selectivity and role in determining the rate of charge accumulation in the vesicle interior, at the single-vesicle-level.
The expected results of the projects are the synthesis of a novel double-membrane giant vesicle capable of hosting a range of transmembrane proteins, such the bacterial efflux pump AcrAB-TolC.
The results of the projects will deepen our understanding of how efflux pumps operate and transport powered by the electrochemical proton gradient that is generated by cells and, thus, provide a and what is the corresponding transport activity of the pump. Since efflux pumps and ion transport are crucial to the function of cells in health and disease, I anticipate that the results of this project will impact researchers from various scientific communities, including biophysics, biology and medicine.
The results of the projects will deepen our understanding of how efflux pumps operate and transport powered by the electrochemical proton gradient that is generated by cells and, thus, provide a and what is the corresponding transport activity of the pump. Since efflux pumps and ion transport are crucial to the function of cells in health and disease, I anticipate that the results of this project will impact researchers from various scientific communities, including biophysics, biology and medicine.