Integrin Reconstitution

Due to the difficulty in reconstituting membrane proteins directly into preformed GUVs we found that the best, most reproducible approach was to firstly create smaller proteoliposomes before later “growing” these to GUVs. To create these smaller αIIbβ3-reconstituted proteoliposomes, two different methods were regularly utilised. The first method utilised a protocol based on Müller et al.³⁴ whereby lipid was fully solubilised by the detergent triton x-100 and the surfactant later removed by the addition of bio-beads. Alternatively a protocol based on Dennison et al.²⁷ was used whereby sonicated SUVs were brought to the onset of solubilsation with β-OG

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and the surfactant removed later removed via dialysis. Because of these different protocols for integrin αIIbβ3 reconstitution, two different methods were used to detect the presence of αIIbβ3 in the liposomes.

Detection of integrin in Triton X-100 created proteo-liposomes

While the extruded vesicles used for integrin reconstitution were of uniform dimater, DLS measurements showed that the use of triton x-100 and biobeads to insert αIIbβ3, as per the Müller protocol, had lead to a shift in the dispersity of the resultant proteoliposomes. Proteoliposomes produced via Müller’s full solubilisation method were found to have a diameter in the micrometer range. This change in vesicle size can be seen in Figure 44. Due to this increased vesicle diameter it was decided to use flow cytometry in order to detect the presence of αIIbβ3 in the proteoliposomes. Control liposomes were prepared via the same detergent solubilisation method but in the absence of integrin αIIbβ3 in order to yield protein-free liposomes of equivalent size. In order to identify integrinin the lipid membrane the anti-αIIbβ3 antibody CD41a was used. This antibody has general affinity for αIIbβ3

and will bind the protein’s extracellular domain regardless of integrin activation state. To detect the presence of αIIbβ3 in the reconstituted proteoliposomes, flow cytometry was first performed on control liposomes using FITC labelled CD41a. To act as a negative control, αIIbβ3 free vesicles were passed through the flow cytometer and counts for the fluorescence data channel were piled on the lowest values of the logarithmic x-axis of the histogram. Using a gate 5 % of these counts were located within this region. Therefore, any CD41a binding leading to a shift in the histogram to the right can be quantified by the percentage gated value increasing within the region. After the control liposomes, αIIbβ3-reconstituted liposomes were passed through the flow cytometer and the counts were overlayed on the same histogram.

A typical histogram for CD41a-FITC featuring both control and αIIbβ3-reconstituted liposomes is given in Figure 45. A shift in the histogram to the right can clearly be seen in the αIIbβ3-reconstituted liposomes due to the CD41a binding. Using this method, it was determined that the percentage gated value increased from a baseline of 5 % to 35.47 % ± 5.74 % in αIIbβ3-reconstituted liposomes showing the

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presence of the integrin protein within the vesicle lipid membrane in proteoliposomes produced via the Müller method.

Figure 44: DLS measurements of vesicles after extrusion but before integrin insertion (blue), and after triton x-100-mediated integrin reconstitution. After extrusion vesicles were mono-disperse, around the 100 nm diameter mark. Due to detergent solubilisation and then detergent removal, reconstituted vesicles were poly-disperse with many proteoliposomes in the micrometer range.

Figure 45: Representative histogram of FITC labelled CD41a binding in control liposomes (yellow fill, black outline) and reconstituted liposomes (red outline). The gate displayed is set so that 5 % of control liposome counts fall within the region. A shift in the histogram to the right can clearly be seen for αIIbβ3 liposomes showing the reconstitution protocol was successful.

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Detection of integrin in β-OG created proteoliposomes

When compared to proteoliposomes produced via the Müller method, proteoliposomes produced via the Dennison method were relatively small. In fact, no change in vesicle diameter was detected by DLS before and after detergent treatment where the mean vesicle diameter was 31 nm ± 5 nm throughout. This was likely due to the fact that sonicated SUVs were only brought to the onset of solubilisation in this protocol, rather than being fully solubilised. Due to their SUV size it was not possible to use flow cytometry to detect the presence of αIIbβ3 within the vesicle membrane. Instead an alternative assay had to be used. To do so, newly reconstituted proteoliposomes that had been separated from detergent via dialysis were run on a size exclusion column to remove free integrin. The fractions were collected and analysed for lipid concentration and the presence of protein. The lipid concentration of each fraction was quantified by the presence of radiolabelled lipid.

The protein (integrin αIIbβ3) concentration of each fraction was quantified by running each fraction on a polyacrylamide gel. Densitometry was subsequently used on each lane of the gel. Combined these two assays showed that the lipid and αIIbβ3 were present in the same fractions and that the concentration of each component in the fractions was roughly correlated. This is shown in Figure 46. Due to the fact that free integrin had previously been removed through a size exclusion column and only the vesicle-containing fractions collected, this co-association of lipid and protein in the same fractions showed that αIIbβ3 had been reconstituted in SUVs created using the Dennison protocol.

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Figure 46: Lipid and protein levels in recovered fractions. A) A graph showing detected disintegrations per minute (DPM) for each fraction in black and relative density of each fraction (as determined by densitometry) in red. To determine densitometry values, each fraction was run on a polyacrylamide gel and later stained with coomasssie blue to highlight any protein present. ImageJ was used to analyse each lane and the lane with the highest density value was set to 1 (lane 4). From this value the relative density of the other lanes could be determined. It is evident that there is a correlation between the lipid concentration in each fraction (black) and the protein concentration in each fraction (red). B) A coomassie stained polyacrylamide gel from which the densitometry values were obtained. A molecular weight marker is also included (leftmost lane). αIIbβ3 is reduced into three subunits during the PAGE step. The alpha subunit is divided into a heavy (125 kDa, top band) and light chain (25 kDa, run off gel). The beta subunit is 105 kDa (lower band).

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