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. 2021 Apr 15;10:e61401. doi: 10.7554/eLife.61401

Figure 2. Real-time monitoring of protein–oil interaction in a microfluidic system shows irreversible adsorption of Plin4 12mer-A488 on triolein.

(A) Scheme of the microfluidics experimental set-up. (B) Top row: confocal images of the triolein-buffer interface as formed in the microfluidic system after adsorption of Alexa488-labeled Plin4 12mer on the triolein surface and after rinsing with buffer. Bottom row: control experiment with the free fluorophore Alexa488. The intensity profile along the channel center is shown below each confocal image. The protein adsorbs irreversibly at the oil surface, whereas Alexa488 conjugated to free cysteine (A488) does not. See also Videos 1–4. A representative of three independent experiments is shown. (C) Time course of the signal of Alexa488-labeled Plin4 12mer or of free Alexa-488 in the side channel as quantified from the experiment shown in B. A representative of three independent experiments is shown.

Figure 2.

Figure 2—figure supplement 1. Microfluidic experiments demonstrate that Plin4 12mer lowers the surface tension of oil.

Figure 2—figure supplement 1.

(A) Plin4 12mer readily adsorbed to both oil and glass surfaces and even intercalated into the oil-glass interface, making the glass surface visibly heterogeneous (Videos 1 and 2). In this way, it induced a marked hysteresis in the oil-glass contact angle. The range of equilibrium contact angles spanned from 69° to 75° for buffer, and from 58° to 84° for Plin4 12mer. This hysteresis can result from chemical heterogeneities on a flat glass substrate (Joanny and de Gennes, 1984). (B) Equilibrium pressure difference vs curvature for buffer (black points) and Plin4 12mer (blue points). The slope of the lines represents the surface tension of the interface. In the case of oil-buffer interface the surface tension was 36 ± 3 mN/m (N = 5), and for PLIN4-12mer, the surface tension decreased to 25 ± 1 mN/m (N = 5).
Figure 2—figure supplement 2. Plin4 mutant 4T > S-A488 also adsorbs to the oil surface, but is outcompeted by Plin4 12mer-A488.

Figure 2—figure supplement 2.

(A) Confocal images of the oil-buffer interface in the microfluidic system after adsorption of Plin4 4mer mutant 4T > S-A488 (0.1 mg/ml) on the oil surface and after rinsing with buffer. The intensity profile along the channel center is shown below each confocal image. (B) Time evolution of signal intensity on the oil surface and in the bulk solution during adsorption and rinsing. A representative of three independent experiments is shown. (C) Competition between Plin4 12mer and 4T > S mutant. The ratio between the intensity on the oil surface and in the bulk solution for a mixture of unlabeled Plin4 12mer: Plin4 12mer-A488 (50:1) and a mixture of unlabeled 4T > S: Plin4 12mer-A488 (50:1). Mean ± SD of the fluorescence intensity of surface/bulk solution is shown.