Molecular basis of F-actin regulation and sarcomere assembly via myotilin

Figures

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  Fig 1. Myotilin displays conformational ensemble in solution. (A) Schematic representation of striated muscle sarcomere with its major thick (myosin based) and thin (actin based) filaments, titin, and the Z-disc anchoring cross-linker α-actinin. Extending from one Z-disc to the next, sarcomere represents the fundamental unit of muscle contraction. (B) Schematic presentation of myotilin and its predicted compactness as a function of residue position. The N-terminal part of myotilin contains the SRR, which comprise hydrophobic residues stretch (H, yellow) and represents “mutational hotspot” of the protein (gray). Known disease-causing mutations are shown. Ig domains 1 and 2 are colored blue and red, respectively, followed by the carboxyl-terminal tail. Within this study, full-length myotilin construct fused to N-terminal Trx (violet) was used. Large meta-structure derived compactness values indicate residue positions typically buried in the interior of the 3D structure, whereas small values are found for residues exposed to the solvent. Dashed line depicts the average compactness value (about 300) of the well-folded protein. Significantly smaller values (200) are found for structurally…

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  Fig 2. Myotilin influences F-actin dynamics by binding via its Ig domains and disordered flanking regions. (A) Summarized results of the actin co-sedimentation assays. The asterisk denotes values obtained in assays performed under B1 conditions (for details, see Materials and methods). (B) Surface electrostatic potential mapped on the Ig1Ig2 structural model [23]. Surfaces are colored on a red white blue gradient, as calculated by Adaptive Poisson-Boltzmann Solver [28]. Residues forming a basic patch (blue) on both Ig domains are labeled. The residue (R405) involved in muscular dystrophy–causing mutation R405K is shown in green [27]. (C) Binding of Ig1Ig2250–444 to F-actin at increasing salt conditions. Graph data are presented as a percentage of theoretical total binding (100%) corresponding to the total amount of protein (8 μM) used in each experiment. Data represent mean values ± SEM of 3 independent experiments. The mean binding of Ig1Ig2250–444 to F-actin in 50 mM KCl was significantly different to the means for all other tested concentrations of KCl. Significance was assessed using 1-way ANOVA with Holm–Sidak test, p < 0.001 for salt concentration of 50 mM vs. all other tested…

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  Fig 3. Integrative structural model of myotilin:F-actin complex. Summary of experimental data on myotilin:F-actin complex, obtained from XL-MS (orange), mutagenesis/co-sedimentation analysis (medium slate blue), and NMR (green). K354 was found to be involved in the interaction by both XL-MS and mutagenesis/co-sedimentation analysis (magenta). Thick, gray dashed lines represent N-terminal and carboxyl-terminal flanking regions of myotilin Ig domains. Selected residues found in cross-links between actin and myotilin are shown in orange. NMR, nuclear magnetic resonance; XL-MS, cross-linking coupled to mass spectrometry.

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  Fig 4. Myotilin regulates binding of tropomyosin to F-actin and interacts with α-actinin-2. (A) Model of tandem Ig domains of myotilin bound to F-actin superimposed with the cryo-EM structures of cardiac tropomyosin bound to F-actin in the blocked (magenta ribbons, PDB: 5NOG), closed (cyan ribbons, PDB: 5NOL), and open (green ribbons, PDB: 5NOJ) structural states [35]. Residues of actin found in cross-links with N-terminal and carboxyl-terminal regions flanking myotilin Ig domains are shown in orange (see Fig 3). (B) Effects of myotilin on tropomyosin:F-actin interaction. Myotilin (Trx-MYOT) at increasing concentrations was added to F-actin before (Trx-MYOT before tropomyosin) or after (tropomyosin before Trx-MYOT) incubation of F-actin with the fixed amount of tropomyosin. Mean values (± SEM) of 3 independent experiments are shown. For “Trx-MYOT before tropomyosin,” the mean binding of tropomyosin to F-actin at all concentrations of Trx-MYOT was significantly different from the means in the absence of Trx-MYOT. Significance was assessed using 1-way ANOVA with Holm–Sidak test, p < 0.01 for 0 vs. 1.2 μM Trx-MYOT and p < 0.001 for 0 vs. all other concentrations of Trx-MYOT. For…

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  Fig 5. Binding of myotilin with F-actin affects its mobility and dynamics. (A) FRAP analysis of the dynamics of myotilin and its mutant variants in Z-discs. Localization of EGFP fusion proteins of WT myotilin and its variants with the indicated mutations in Ig2 before bleaching (pre-bleaching), directly after bleaching (post-bleaching), and after the indicated time points after bleaching. White rectangles show ROIs, used for bleaching during FRAP experiments. Scale bars, 5 μm. (B) Representative FRAP recovery curves of the myotilin variants. (C) Half-times of myotilin mutants compared to WT myotilin. Quantification of data from FRAP studies shown in (A and B). Statistical data are depicted in box and whisker plots. Calculated median half-times are shown as a line surrounded by a box, representing the interquartile range comprising the median ± 25% of the data. Whiskers extend at most 2 standard deviations from the median. (D) Mobile fractions of mutants compared to WT myotilin. Graph reports mean ± SEM, n = 8 myotubes for each variant. A total of 1–3 Z-discs were bleached per myotube. Significance was assessed using 2-tailed Student t test, *p < 0.1, **p < 0.05, and ***p < 0.01. Data…

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  Fig 6. Model illustrating the role of myotilin in the organization of sarcomeric Z-disc assembly. Myotilin is recruited to the Z-disc by binding to F-actin, α-actinin-2, and/or other Z-disc components e.g., filamin C, ZASP. As a part of the recruitment process, myotilin can further stabilize (in its propose antiparallel dimeric form) [11] 2 α-actinin-2 dimers on F-actin through interaction of its N-terminal region with CAMD of α-actinin-2. Consequently, together with α-actinin-2 and/or other Z-disc components, myotilin can help to displace tropomyosin from the Z-disc. For simplicity, only some of the Z-disc components are shown. α-Actinin-2 dimers are schematically shown in an open conformation bound to myotilin, with ABD highlighted as red spheres and EF12 and EF34 of CAMD as violet and purple spheres, respectively. Myotilin and tropomyosin are shown in color code as used in Figs 1B and 4A. The 3D model presented in this panel is available in the following link: https://skfb.ly/6YGQF. ABD, actin binding domain; CAMD, calmodulin-like domain.