Cis and Trans Cooperativity of E-Cadherin Mediates Adhesion in Biomimetic Lipid Droplets  Lea-Laetitia Pontani, Ivane Jorjadze, Jasna Brujic  Biophysical Journal  Volume 110, Issue 2, Pages 391-399 (January 2016) DOI: 10.1016/j.bpj.2015.11.3514 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 (A) Schematic representation of functionalized droplets. The oil/water interface is stabilized with a mixture of phospholipids and SDS. (B) A portion of the lipids exhibit a NTA-Ni group on the surface, allowing for the grafting of histidine-tagged GFP, as revealed by the fluorescent signal on the surface of the droplets observed through a confocal microscope. (C) The droplets are also labeled with a hydrophobic dye (Nile Red) and index matched with the aqueous phase, which allows for the imaging of transparent 3D samples through confocal microscopy. Biophysical Journal 2016 110, 391-399DOI: (10.1016/j.bpj.2015.11.3514) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 (A) In a low salt buffer the droplets are repulsive and cream under gravity to form a random close packing with an average number of contacts per particle 〈z〉 = 5.5 (N = 551). (B) The droplets are functionalized with cadherins in a low salt buffer. The adhesive interactions between the droplets are strong enough to stabilize voids in looser packings with 〈z〉 = 4.12 (N = 668). Biophysical Journal 2016 110, 391-399DOI: (10.1016/j.bpj.2015.11.3514) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 (A) Calcium is added at t0 to cadherin-coated droplets. (B) The addition of salt triggers rapid fusion of the droplets. Biophysical Journal 2016 110, 391-399DOI: (10.1016/j.bpj.2015.11.3514) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 (A) Labeling the cadherins reveals the presence of protein clusters in the adhesion area between contacting droplets after fusion. (B) Surface tension measured from a pendant drop experiment. The silicone oil contains various amounts of phospholipids and is dropped in an aqueous solution containing 1mM SDS and 2 mM Tris, pH = 7.5. Biophysical Journal 2016 110, 391-399DOI: (10.1016/j.bpj.2015.11.3514) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 (A) The average radius of the droplets at steady state is measured as a function of cadherin concentration, in the presence of 1.67 mM Ca2+ (N ≈ 200 for each point). (B) Average radius of functionalized (red squares, 228 nM cadherin) or control droplets (blue circles, no cadherin) as a function of calcium concentration. The average size of functionalized droplets increases with salt but remains constant in the absence of cadherins (N ≈ 300 for each condition). Biophysical Journal 2016 110, 391-399DOI: (10.1016/j.bpj.2015.11.3514) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 (A) Immiscible lipid mixtures on the droplets drive domain formation. The disordered (Ld) domains contain DSPE-PEG-biotin lipids and are labeled with Texas Red streptavidin (red), while the ordered phase (Lo) enriched in cholesterol contains the DGS-NTA-Ni lipids labeled with GFP-His (green). (B) Clustering is enhanced by the addition of 10% cholesterol in the lipid mixture (black diamonds, N ≈ 200 for each point) or by the addition of 100 nM anti-human antibody (purple triangles, N ≈ 200 for each point). (C) The use of fluorescent antibodies reveals large aggregation patterns on the fused droplets at steady state. (D) Higher fluorescence in the contact areas reveals higher concentration of the cadherins in the adhesion patch. Biophysical Journal 2016 110, 391-399DOI: (10.1016/j.bpj.2015.11.3514) Copyright © 2016 Biophysical Society Terms and Conditions