Panel A shows Commassie stained SDS gel images and a bar graph from a low speed pelleting assay, demonstrating that combined fascin and espin produce greater filamentous actin bundling than would be expected from the sum of their individual effects. Panel B shows a line graph and a bar graph, indicating that fascin plus espin causes a larger increase in normalized light scattering than individual proteins (and more than would be expected from the sum of their individual effects), reflecting enhanced bundle formation. Panel C shows fluorescence microscopy images comparing fascin, espin, and combined conditions, revealing longer and more prominent actin bundles when both bundling proteins are present together. Panel D shows a bar graph of bundle length in micrometers, demonstrating that fascin plus espin generates substantially longer actin bundles than fascin or espin alone (and more than would be expected from the sum of individual effects). Panel E shows microscopy images of actin bundles formed with fascin, espin, or both proteins, illustrating thicker and more densely packed bundles in the combined condition. Panel F shows a bar graph of bundle width in nanometers, indicating that fascin plus espin produces the widest actin bundles compared with bundles formed by either protein individually (or the sum of their individual effects).
Fascin and espin synergize in actin bundles formation. (A) Low-speed sedimentation assays show that together fascin and espin bundle more actin filaments than would be expected from the sum of their individual effects. A representative SDS polyacrylamide gel shows supernatant (S) and pellet (P) fractions of actin in the low-speed sedimentation experiments (1). The corresponding percentage of actin in the pellets (mean values ± SEM) is shown in 2. Dashed line indicates the sum of fascin (blue) and espin (pink) values. [F-actin] = 2 μM; [Fascin] and/or [Espin] are indicated in the figure. n = 5 independent experiments/conditions. (B) Individual or combined effects of the bundling proteins on actin, as followed by light-scattering changes at λ = 325 nm. Black trace corresponds to polymerized unbundled actin (F-actin), and the arrow indicates the addition of bundling protein(s). Results of a representative experiment are shown in 1, and the corresponding percentage increase of light scattering (mean ± SEM) from n = 3 independent experiments/conditions is shown in 2. Dashed line indicates the sum of fascin (blue) and espin (pink) values. [F-actin] = 5 μM; [Fascin] = 0.25 μM; [Espin] = 0.75 μM; n = 3 independent experiments/conditions. (C) TIRFM images of actin bundles taken 5 min after addition of bundling proteins to the on-slide polymerized actin filaments. [Actin] = 1 µM, 20% Alexa488-labeled; [Fascin] = 0.05 μM and/or [Espin] = 0.2 μM. Scale bars: 10 μm. (D) Average bundles length (±SEM) determined from TIRFM images taken in three independent experiments. n = 27, 55, and 71 bundles analyzed for fascin only, espin only, and fascin + espin conditions, respectively. Dashed line indicates the expected sum of fascin (blue) and espin (pink) values. (E) EM images of actin bundles in the presence of fascin or espin or both. In the presence of both fascin and espin, much thicker bundles are formed than would be expected from the sum of their concentrations (shown as dashed line in F). Scale bars: 50 nm. (F) Average bundles width (±SEM) determined from EM images taken in three independent experiments. n = 3, 7, and 8 bundles analyzed for fascin only, espin only, and fascin + espin conditions, respectively. [F-actin] = 1 μM, [Fascin] = 0.05 μM, and/or [Espin] = 0.2 μM. Dashed line indicates the expected sum of fascin (blue) and espin (pink) values. Source data are available for this figure: SourceData F2.