A microfluidic approach for studying integrin αIIbβ3 activation and clustering

“Formation and adhesion of protocells containing integrin and THD. a) Schematic depiction of the formation of functional cell-sized liposomes that contain integrin and THD by droplet-based microfluidics. Scale bar: 50 μm. I) Encapsulation of bare liposomes (≈100 nm diameter), integrin-reconstituted proteoliposomes, and THD at defined concentration into microfluidic water-in-oil droplets of 30 μm diameter. II) Liposomes rupture at the droplets’ inner interface, fuse, and form a continuous lipid bilayer, the dsGUV. III) After the addition of destabilizing surfactant, the GUV is released from the stabilizing oil phase into an aqueous phase, i.e., an appropriate buffer solution. IV) Free-standing GUV that contains integrin and THD of defined concentrations, after its release. V) Released GUV adhering to a fibrinogen-coated surface as a result of integrin-activation by THD. Depiction not to scale. b) 3D-reconstitutions of integrin-containing protocells (lipid: ATTO488 (green), integrin: Alexa568 (red)). The images show increasing adhesion to fibrinogen-coated surfaces in correlation to an initially provided THD concentration of: I) 2 nM, II) 10 nM, III) 30 nM, IV) 80 nM, V) 200 nM at a constant integrin concentration. The spreading value of each vesicle shown is close to the mean value obtained from a corresponding sample of vesicles. For correlating data refer to Table 1. Scale bar: 10 μm. c) Integrin-clustering in the contact area of protocells adhering to fibrinogen-coated surfaces. The lipid composition of the protocells is 18:72:10 DOTAP:DOPC:Egg PC with reconstituted integrin (Alexa568) activated by: I) 1 mM Mn²⁺/1 mM Mg²⁺ (vesicle diameter: 12 μm) and II) 200 nM THD (vesicle diameter: 14 μm). The difference in the overall signal and concentration of clusters can be due to bleaching effects. Scale bar: 3 μm”. Reproduced under Creative Commons Attribution 4.0 International License from Benk, L.T., Benk, A.S., Lira, R.B., Cavalcanti-Adam, E.A., Dimova, R., Lipowsky, R., Geiger, B. and Spatz, J.P. (2022), Integrin α_IIbβ₃ Activation and Clustering in Minimal Synthetic Cells. Adv. NanoBiomed Res. 2100094.