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. 2022 Jan 20;11:e72588. doi: 10.7554/eLife.72588

Figure 2. FAK and paxillin undergo liquid-liquid phase separation.

(A) Molecular interactions of IAC proteins, KD values indicated where known. Details and references in Supplementary file 1. (B) Solution turbidity measurements with increasing concentrations of FAK. (C) Spinning disk confocal fluorescence microscopy images of droplets formed from 1 μM FAK (1% Alexa647 labeled). The same image is displayed with two different contrast settings for comparison to panel E. (D) Solution turbidity measurements. 250 nM FAK was combined with increasing concentrations of talinH (magenta), kindlin (black), or paxillin (green). (E) Spinning disk confocal fluorescence microscopy images of droplets formed from 1 μM FAK (1% Alexa647 labeled) and 1 μM FAK paxillin (5% Alexa546 labeled). For comparison, contrast settings of FAK image are identical to those in 2 C, low contrast. (F) Quantification of constituent partitioning into droplets. Each condition contains at least 750 measurements from four or more independent experiments. Significance tested with student’s t-test. (G) Fluorescence Recovery After Photobleaching (FRAP) measurements of droplets. Droplets formed from 1 μM of FAK (1% Alexa647 labeled, cyan) or 1 μM each of FAK (1% Alexa647 labeled, dark blue) and paxillin (5% Alexa546 labeled, magenta). Each point represents the mean ± SEM of at least 15 independent measurements. Recovery curves were fit with a biexponential model and the fits are overlayed on the graph (black line). Detailed fit information in Supplementary file 4. In (B) and (D), each point represents the mean ± SEM of three independent measurements. In (F), each gray point represents an individual measurement, and the mean is indicated by black line. All scale bars = 10 μm.

Figure 2.

Figure 2—figure supplement 1. Solution turbidity measurements.

Figure 2—figure supplement 1.

(A) Increasing concentrations of Kindlin, Talin Head or Paxillin were added to solution. (B) 250 nM FAK and 250 nM Paxillin were combined with increasing concentrations of Talin Head or Kindlin. In A-B, each point represents the mean ± SEM of three independent measurements.
Figure 2—figure supplement 2. Droplets form with physiological protein concentrations.

Figure 2—figure supplement 2.

(A) Spinning disk fluorescence microscopy images of 5 nM or 40 nM FAK (1% Alexa647). (B) Quantification of number of droplets per mm2. (C) Spinning disk fluorescence microscopy images of 5 nM FAK (1% Alexa647) ± 5 nM paxillin. (D) Quantification of number of droplets per mm2. All images are displayed with matched contrast settings. Scale bar = 10 μ m. In (B) and (D), each gray point represents an individual measurement of a single, randomly selected field of view, and the mean is written and indicated by black line. In (D), significance tested with Student’s t-test.
Figure 2—figure supplement 3. Representative fluorescence recovery after photobleaching (FRAP) data.

Figure 2—figure supplement 3.

(A) Top: Representative spinning disk fluorescence images of FAK fluorescence recovery after photobleaching (FRAP) experiment. Droplets were formed with 1 μ M FAK (1% Alexa-647), and a single droplet is bleached at t = 0 s. Botton: Quantification of fluorescence intensity of the bleached droplet over time. (B) TOP: Representative spinning disk fluorescence images of FAK FRAP experiment. Droplets were formed with 1 μ M FAK (1% Alexa-647) and 1 μ M paxillin, and a single droplet is bleached at t = 0 s. Bottom: Quantification of fluorescence intensity of the bleached droplet over time. (C) TOP: Representative spinning disk fluorescence images of paxillin FRAP experiment. Droplets were formed with 1 μ M FAK and 1 μ M paxillin (5% Alexa-561), and a single droplet is bleached at t = 0 s. Bottom: Quantification of fluorescence intensity of the bleached droplet over time. All scale bars = 5 μm.