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

Figure 6. Integrin adhesion complexes are sensitive to genetic perturbations that alter Cas-dependent phase separation.

(A) In vitro solution turbidity measurements. 500 nM Nck and 1 µM N-WASP were combined with increasing concentrations of phosphorylated Cas (pCas, magenta) or Y15F Cas (black). Each point represents the mean ± SEM of three independent measurements. (B) Representation of in vitro experiments in C-D. (C) Spinning disk confocal fluorescence microscopy images of droplets formed with the Cas + FAK mix. TOP: 1 μM each of pCas, Nck, N-WASP, Kin, FAK, and paxillin (15% Alexa546); BOTTOM: 1 μM each of Y15F Cas, Nck, N-WASP, Kin, FAK, and paxillin (15% Alexa546). (D) Quantification of paxillin partitioning into droplets. Each condition contains at least 40 measurements from two independent experiments. (E) Spinning disk fluorescence microscopy images of MEFs with immunostaining for endogenous paxillin. (F) Quantification of number of adhesions. (G) Quantification of paxillin partitioning into adhesions (partition coefficient). Significance tested by one-way ANOVA followed by a Tukey multiple comparison test. In (C) scalebar = 5 μm. In (E) scalebar = 10 μm. Note that the pCas protein used in Figure 6 is a different batch than protein used in Figures 1—5 (See Figure 1—figure supplement 3).

Figure 6.

Figure 6—figure supplement 1. Cas Y15F does not form droplets.

Figure 6—figure supplement 1.

Spinning disk fluorescence microscopy images of solution containing 1 μM Cas Y15F, 1 μM N-WASP and 1 μM Nck (15% Alexa-647). Scale bar = 10 μm.
Figure 6—figure supplement 2. Measuring number of integrin adhesions during cell spreading.

Figure 6—figure supplement 2.

(A) Experimental workflow (B) Example of image analysis workflow. Scale bar = 10 μm.
Figure 6—figure supplement 3. Westernblot analysis of MEF cell lines.

Figure 6—figure supplement 3.

Cell lysates were blotted with antibodies to p130Cas (Cas), FAK, and actin. (A) Cas MEF cell lines were assessed (WT MEF, Cas knockout (KO)+ Control vector, Cas KO+ WT Cas vector, Cas KO+ Y15 F Cas vector). (B) FAK MEF cell lines were assessed (WT MEF, FAK KO, FAK KO+ WT-FAK-GFP).
Figure 6—figure supplement 3—source data 1. Uncropped gel images and unprocessed gel.tif files from Figure 6—figure supplement 3.