The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function

Abstract

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

FIGURE 1:

(A) Domain structure of Tmod1 and Lmod2 based on biochemical and structural analysis of Tmod1 and sequence homology of Lmod2 to Tmod1. A1, A2, and WH2 are actin-binding sites; Tpm1 and Tpm2 are Tpm-binding sites; LRR is the leucine-rich repeat domain; and P is the proline-rich domain. Truncated fragments are indicated by lines. (B) Amino acid sequence alignment of N-terminal fragments for Lmod2 and Tmod1 made using MultAlin. Identical amino acids in the alignment are shaded. The prediction of the secondary structure in Lmod2 was made using Jpred. α-Helical regions are shown in bold italic. Arrowheads and vertical dashed lines show the end of Lmod2 fragment 1–94 and Tmod1 fragment 1–92. Arrows indicate the residues mutated in Lmod2. Lmod2 residues 45–51 are shown in bold underlined.

FIGURE 2:

Both Lmod2 [1-94] and Lmod2 [1-201] inhibit pointed-end polymerization. Effect of 0.2 μM Lmod2 fragments in the absence (A) and presence (B) of a saturating concentration of Tpm1.1 (1 μM) on the polymerization of 1.1 μM G-actin at the pointed end of gelsolin-capped actin filaments. Control, actin alone in the absence of Tpm1.1 and Lmod2 fragments. (C) Dependence of inhibition of actin polymerization on the concentration of Lmod2 fragments in the presence of 1 μM Tpm1.1. (D) The inhibition of actin polymerization by 0.2 μM Lmod2 fragments calculated as R_exp/R_control. Initial rates (R) were determined as the first derivatives at time 0 after fitting. Asterisks indicate statistically significant groups determined using one-way ANOVA with Tukey–Kramer post hoc test. All values are mean ± SD (n = 3–6); *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3:

Formation of a complex between the fragment Lmod2 [45-94] and G-actin. NMR proton spectra of 0.2 mM Lmod2 [45-94] in the absence (red) and presence (blue) of 10 μM G-actin in the aromatic/amide (top) and side-chain (bottom) chemical shift regions in 2 mM potassium phosphate, 0.1 mM ATP, 0.1 mM CaCl₂, and 0.01% NaN₃ at pH 6.8. In the presence of actin, a large number of resonance peaks manifested differential broadening/shifts (regions with broadened peaks are boxed). The NMR spectra were recorded at 25°C.

FIGURE 4:

Complex formation between αTM1bzip and Lmod2 [1-201] or Lmod2 [1-201(L30E)] monitored by native (nondenaturing) PAGE. αTM1bzip/Lmod2 molar ratio was 0:1 (lanes 1 and 5), 1:1 (lanes 2 and 6), 2:1 (lanes 3 and 7), and 1:0 (lane 4). Concentration of Lmod2 fragments was 5 μM. αTM1bzip is positively charged and does not enter the gel. Arrows indicate Lmod2 fragments and the complex.

FIGURE 5:

Titration of Lmod2 [1-201] by αTM1bzip. A stock solution of Lmod2 [1-201] was diluted to a final concentration of 5 μM with different concentrations of Tpm peptide in 20 mM Tris-HCl, pH 7.4. The decrease of free Lmod2 [1-201] and the increase of the complex were monitored by scanning and quantifying the Lmod2 [1-201] band in native polyacrylamide gels. (A) 9% native gel. (B) Dependence of the amount of free Lmod2 [1-201] (○) and density of the complex band (●) on the amount of αTM1bZip added. Lanes on the gel correspond to points on the graph. Lanes 1–11 contain αTM1bzip/Lmod2 in ratios of 0, 0.125, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, and 2.5, respectively.

FIGURE 6:

Mutations L30E and W73D reduce the capping ability of the Lmod2 [1-201] truncated fragment. Polymerization of 1.1 μM G-actin at the pointed end of gelsolin-capped actin filaments was measured in the presence of a saturating concentration of Tpm (1 μM) and various concentrations of Lmod2 [1-201(L30E)] (A) and Lmod2 [1-201(W73D)] (B). Control, actin alone in the absence of Tpm1.1 and Lmod2 fragments. (C) Dependence of inhibition of actin polymerization on concentration of Lmod2 [1-201] fragments, wild type, and with the mutations. Initial rates (R) were calculated as the first derivatives at time 0 after fitting. The inhibition of polymerization was calculated as R_exp/R_control.

FIGURE 7:

The N-terminal TM- and actin-binding domains of Lmod2 contribute to its localization and thin filament elongation function. (A) Representative images of rat neonatal cardiomyocytes expressing GFP alone, GFP-Lmod2, GFP-Lmod2 (L30E), or GFP-Lmod2 (W73D) stained with phalloidin to label F-actin and for sarcomeric α-actinin to label the Z-disks. Arrows mark pointed-end assembly. Scale bar, 10 μm. Insets in GFP/α-actinin merged images represent twofold magnification to highlight pointed-end assembly of each construct. Scale bar, 2 μm. (B) Pointed-end assembly of cytosol-extracted rat neonatal cardiomyocytes transduced with GFP, GFP-Lmod2, GFP-Lmod2 (L30E), and GFP-Lmod2 (W73D). Graph shows the percentage of cardiomyocytes demonstrating pointed-end assembly (mean ± SEM); 300 total counts from ∼100 cells/culture, three cultures; **p < 0.01, ***p < 0.001, one-way ANOVA. (C) Thin filament lengths of rat neonatal cardiomyocytes transduced with GFP, GFP-Lmod2, GFP-Lmod2 (L30E), and GFP-Lmod2 (W73D). All values are mean ± SEM; 54–66 total measurements from ∼10 cells/culture, 3-4 cultures; ***p < 0.001, one-way ANOVA.

FIGURE 8:

Fluorescence recovery after photobleaching of GFP-Lmod2 and GFP-Lmod2 (W73D). (A) Mean relative recovery after photobleaching over time for GFP-Lmod2 (blue triangles) and GFP-Lmod2 (W73D) (red squares). (B) Recovery data fit using nonlinear regression curves with the single-exponential association equation R = M[1 − exp(−kt)] for each individual cell, where R is the relative recovery of fluorescence at time t. Mean mobile fraction (M), rate (k), and half-time of recovery (t_1/2) are indicated ± SEM. Bold indicates values that are statistically significantly different between GFPLmod2 and GFPLmod2 (W73D). n = 34–36 from three independent cultures; *p < 0.05, Student’s t test.