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

Abstract

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs—the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin’s structural role in Z-discs.

Order from disorder in the sarcomere: FATZ forms a fuzzy but tight complex and phase-separated condensates with α-actinin

“…Most of these interactions were identified on a binary level, but it is not clear which higher-order complexes can actually coexist in Z-disks. While titin Z-repeats, myotilin, myopodin, and myopalladin are known to bind to α-actinin-2 EF3-4 via their 1-4-5-8 motif, the ZASP PDZ domain binds to the C terminus of EF3-4 ( 61 – 64 ). The titin Zq motif was shown to bind to the α-actinin-2 rod (SR2-SR3) and modeled as a dimer of α helices running perpendicular to the centre of the rod ( 59 , 65 ).…”

Unbiased gene expression analysis of the delayed fracture healing observed in Zucker diabetic fatty rats

“…The significance of this is unclear but could reflect inappropriate mesenchymal differentiation away from osteogenic lineages (chondroblast-osteoblast) towards myoblast, as the gene product myotilin is best characterized as a protein involved in striated muscle contraction. 33 Further evidence for aberrant cell differentiation was obtained when GSEA of the entire gene set was performed ( Table II ). Of the top 13 hallmark gene pathway sets identified, epithelial-mesenchymal transition and myogenesis were positively associated with the tissue response in ZDF callus.…”

Myofilament-associated proteins with intrinsic disorder (MAPIDs) and their resolution by computational modeling

“… Michie et al (2016) used a similar strategy to identify a linker in the M-domain of MyBPC3 for binding calmodulin via SAXS scattering and computational modeling. Another excellent MAPID study that utilized both experiment and simulation was performed by Kostan et al (2021) . Conformations comprising myotilin’s ensemble were selected from a pool of structures generated by the ‘EOM’ approach ( Bernadó et al, 2007 ) to match experimental SAXS profiles.…”

“…Conformations comprising myotilin’s ensemble were selected from a pool of structures generated by the ‘EOM’ approach ( Bernadó et al, 2007 ) to match experimental SAXS profiles. These conformations, together with biochemical binding assays, provide an integrative structural model that rationalizes the mechanism, by which myotilin’s IDR binds to F-actin ( Kostan et al, 2021 ).…”