Figure 4.

Partitioning increases the local protein concentration in coacervates. (A and B) Confocal fluorescence micrographs are given of polypeptide coacervates containing variable concentrations of TMR-actin on nonadherent substrates, focused at the interface of the coacervates and the substrate (surface, A), or near the droplet midplane (B). Contrast is adjusted individually for each concentration of TMR-actin, but is consistent between confocal slices for each condition. (C) Magnified images are given of the yellow boxed regions outlined in (A) and (B). Scale bars, 5 μm (A and B) and 2 μm (C). (D) Fluorescence intensity is given along the colored dashed lines in (B). (E) Mean fluorescence intensity is given of interior (circle, black), periphery (triangle, cyan), and exterior (square, red) of coacervates droplets, obtained from the black-, cyan-, and red-shaded regions in (D). (F) Auxiliary RAM values, showing ratios of peripheral to exterior fluorescence (triangles, cyan) and interior to exterior fluorescence (circles, black), are given for the data in (D) and (E). Red line indicates where product of the auxiliary RAM value and the total actin concentration equals the critical concentration for actin assembly of ∼0.1 μM. Filaments are expected to the right of the red line, but not to the left. Conditions are 47% TMR-labeled Mg-ATP-actin at a range of concentrations, incubated with 5 mM pLK either alone (1.5- and 0.5-μM actin) or with 0.25 μM 91% Alexa-647-labeled BSA (0.25-, 0.1-, 0.05-, 0.01-μM actin) before addition of 5 mM pRE in 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM imidazole (pH 7.0), and 72 μM ATP (all concentrations final). To see this figure in color, go online.