Figure 3. Talin mediates interactions between full-length vinculin and actin.

(A) Actin bundling co-sedimentation assay with filamentous actin (2.5 µM) and Tn2 with various vinculin fragments (2.5 µM, see Figure 3—figure supplement 1), supernatant and pellet samples were analyzed using SDS-PAGE. Graph shows individual data points and mean densitometry for three independent samples. (B) Actin bundling co-sedimentation assay with filamentous actin (2.5 µM) and various talin2 mutants and fragments (see Figure 3—figure supplement 1), with and without Vn^2A (2.5 µM), supernatant and pellet samples were analyzed using SDS-PAGE. Graph shows individual data points and mean densitometry for talin2 proteins alone (dark blue) and with Vn^2A (aqua) for three independent samples. Cosedimentation experiments performed in 20 mM HEPES, pH 7.5, 75 mM KCl, 2 mM MgCl₂, and 0.2 mM ATP. Controls for (A,B) found in Figure 3—figure supplement 2.(C) Representative images of two-color TIRFm with 1 µM actin (5% ATTO488-actin) and Vn^2A (1.5 µM) with different SNAP-647-labeled Tn2 proteins (1.5 µM). Schematics above images indicate domain locations of mutations and truncations. Quantification can be found in Figure 3—figure supplement 4. Scale bar = 5 µm. TIRFm experiments were carried out in TIRFm buffer with 15 mM glucose, 20 µg/mL catalase, 100 µg/mL glucose oxidase, 1 mM DTT and 0.25% methyl-cellulose (4000 cp).

Figure 3—figure supplement 1. Domain schematics of talin mutants and vinculin truncations.

Domain schematic of talin mutants, and truncations used in this study (top). Domain schematic of vinculin full-length and truncations used in this study (bottom).

Figure 3—figure supplement 2. Cosedimentation controls corresponding to Figure 3.

(A) SDS-PAGE from Figure 3A, adjusted to clearly show Vn^D1 and Vn^Tail (top) and actin-negative control samples (bottom) (B) Actin bundling co-sedimentation assay with actin (2.5 µM), Vn^2A (2.5 µM), and increasing amounts of talin VBS peptide (10 µM to 100 µM). Representative SDS-PAGE of supernatant and pellet samples. (C) Cosedimentation control samples without actin for Figure 3B. Cosedimentation experiments were performed in 20 mM HEPES, pH 7.5, 75 mM KCl, 2 mM MgCl₂, and 0.2 mM ATP.

Figure 3—figure supplement 3. Mass spectrometry of Tn2^ΔHead and quantification of Vn cosedimentation with Tn2 mutants.

(A) Coverage map of Tn2^ΔHead compared to Tn2, from mass spectrometry of the purified proteins. (B) Quantification of Vn^2A co-sedimented with actin and Tn in the actin bundling assay shown in Figure 3B. n.s. >0.5, *p<0.5, by one-way ANOVA.

Figure 3—figure supplement 4. Quantification of Tn2 mutant TIRFm data.

(A) Violin plot shows the distribution of estimated number of actin filaments per bundle for three independent experiments. From left to right, n = 35, 53, 63, 63, 87, 62. (B) Graph shows individual data points for Tn2 enrichment at actin filaments, binned according to number of filaments within an individual bundle, based on average filament fluorescence in the actin alone control, from three independent experiments. A value of 1 indicates no enrichment of Tn2 at the actin filament or bundle. From left to right, total measurements, n = 53, 63, 102, 87, 62. n.s. >0.5, *p<0.5 **p<0.05, ***p<0.005.