Figure 5. Fimbrin and espin segregate to filaments bundled by fascin.

Three-color TIRFM of two-filament bundles formed by fascin and α-actinin with (A–C) fimbrin, (D–F) espin, or (G and H) muscle tropomyosin. Red and white lines indicate fascin and α-actinin domains. White arrowheads and arrows indicate leading and trailing barbed ends.

(A) Schematic of bundles formed by fimbrin, espin, fascin and α-actinin [8, 14].

(B–C) 1.5 μM actin (15% Oregon green-actin), 400 nM α-actinin dimers, 75 nM Cy5-fascin (red) and 15 nM TMR-fimbrin (Cyan). See also Movie S4

(B) Micrographs (left) and corresponding kymographs (right) of a parallel two-filament bundle, with Oregon green-actin (top), TMR-fascin (middle) and Cy5-fimbrin (bottom). Scale bars=2 μm.

(C) Cy5-fimbrin molecules on single filaments (n=6), two-filament parallel α-actinin bundles (n=23) and two-filament fascin bundles (n=16). Error bars indicate SEM.

(D–F) 1.5 μM actin (15% OG-labeled), 100 nM Cy5-fascin (red), 400 nM α-actinin dimers and a range of concentrations of SNAP-647-espin-2B (Cyan). See also Movie S5

(D) Micrographs (left) and corresponding kymographs (right) of a parallel two-filament bundle, with Oregon green-actin (top), TMR-fascin (middle) and SNAP-647-espin-2B (bottom). Scale bars=2 μm.

(E) Number of SNAP-647-espin-2B molecules per μm of single filament (n=15), parallel α-actinin bundles (n=15) or fascin bundles (n=17). Error bars indicate SEM.

(F) Ratio of the total domain length of TMR-fascin two-filament parallel bundles over the total length of two-filament parallel bundles as a function of SNAP-647-espin-2B concentration. Error bars indicate SEM; n=2 movies.

(G–H) 1.5 μM actin (10% Alexa-488-labeled), 100 nM Cy5-fascin (red), 150 nM α-actinin and 100 nM skeletal muscle α/β TMR-tropomyosin (Cyan).

(G) Micrographs (left) and corresponding kymographs (right) of a parallel two-filament bundle, with Alexa-488-actin (top), Cy5-fascin (middle) and TMR-tropomyosin (bottom). Scale bars = 2 μm.

(H) Normalized Tropomyosin fluorescence on control actin filament, α-actinin and Fascin parallel bundles. Error bars indicate SEM.

(I) High speed co-sedimentation assays with 5 μM Mg-ATP actin, 600 nM fascin (blue) or α-actinin (red), and an increasing concentration of skeletal muscle α/β tropomyosin. Gels (inset) show fascin or α-actinin depletion from the pellet with increasing tropomyosin. Graph is the fraction of total fascin or α-actinin in the pellet as a function of tropomyosin concentration. Normalized curves were fit to a single exponential decay function.

See also Figure S6