Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

We examine the interaction between cytoskeletal $F$-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in $F$-actin as well as facilitates bundle formation. Mutations in one of the two $F$-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

Figure 1

(a) Circularly averaged diffraction from rE3A-actin mixtures at different $\rho$ showing hexagonally indexed peaks and layer line peaks. (b) Partially aligned 2D diffraction pattern of rE3A-actin bundles at $\rho =0.2$. (c) Proposed espin-actin bundle structure; espin linkers are omitted for clarity. Angularly averaged wedges along the radial ($q_r$) and axial ($q_z$) directions of (b) are shown in (d) and (e). Labeled layer line diffraction peaks are (d) 0.120, (e) 0.135, and (f) $0.146{\AA }^{-1}$. Inset in (a) shows coexisting bundled and unbundled $F$-actin layer line peaks (i) 0.114 and (j) $0.124{\AA }^{-1}$ for $\rho =0.02$.

Figure 2

(a) Model 2D diffraction pattern of a near-crystalline hexagonally ordered rE3A-actin bundle aligned parallel to $q_z$. Horizonal layer lines are only visible at $q_r$ values where $S(q)$ is nonzero. The peaks labeled d, e, and f correspond to the peaks in Fig. 1e. (b) Fit to the intrahelical diffraction peaks from Fig. 1e using the 4-sphere $G$-actin model multiplied by $S(q)$. Arrows indicate corresponding peak positions of $-13/6$ native actin twist symmetry.

Figure 3

(a) 2D diffraction pattern from a NN suspension of actin-hE3AdelK at $\rho =0.05$. (b) Proposed actin-hE3AdelK NN structure oriented with (a); espin linkers are omitted for clarity. Circularly averaged diffraction intensities for suspensions of $F$-actin mixed with hE3A, hE3AdelK, and hE3AdelCt are shown in (c), (d) and (e), respectively. Dashed curve in (f) is nematic $F$-actin at $70\mathrm{mg}/\mathrm{ml}$. Labeled diffraction peaks d, e, f, i, and j are the same as those in Fig. 1. The broad feature at h is the nematic correlation, $q_n=0.024–0.026{\AA }^{-1}$.

Figure 4

Fluorescence microscopy images of $0.5\mathrm{g}/\mathrm{L}$, $10\mu \mathrm{m}$ $F$-actin (a) bundled with hE3A at $\rho =0.2$, (b) condensed with hE3AdelK at $\rho =0.06$, (c) coexisting with bundles of hE3A-actin at $\rho =0.03$. Alexa546-phalloidin dyed $F$-actin concentration is fixed at $0.03\mathrm{g}/\mathrm{L}$. Coexisting isotropic $F$-actin in (b) and (c) are visible as faint gray lines. Scale bar is $10\mu \mathrm{m}$. (d) Phase diagram of espin-actin complexes determined from the SAXS data showing predominant phases as a function of $\rho$ and different ratios of hE3A and hE3AdelK. On the far right, the hE3AdelCt-actin phase behavior is included. The location of images (a), (b), and (c) are also indicated in (d). All phases include some isotropic $F$-actin. Dashed lines estimate the phase boundaries.