Electron Microscopy and Persistence Length Analysis of Semi-Rigid Smooth Muscle Tropomyosin Strands

Abstract

The structural mechanics of tropomyosin are essential determinants of its affinity and positioning on F-actin. Thus, tissue-specific differences among tropomyosin isoforms may influence both access of actin-binding proteins along the actin filaments and the cooperativity of actin-myosin interactions. Here, 40 nm long smooth and striated muscle tropomyosin molecules were rotary-shadowed and compared by means of electron microscopy. Electron microscopy shows that striated muscle tropomyosin primarily consists of single molecules or paired molecules linked end-to-end. In contrast, smooth muscle tropomyosin is more a mixture of varying-length chains of end-to-end polymers. Both isoforms are characterized by gradually bending molecular contours that lack obvious signs of kinking. The flexural stiffness of the tropomyosins was quantified and evaluated. The persistence lengths along the shaft of rotary-shadowed smooth and striated muscle tropomyosin molecules are equivalent to each other (approximately 100 nm) and to values obtained from molecular-dynamics simulations of the tropomyosins; however, the persistence length surrounding the end-to-end linkage is almost twofold higher for smooth compared to cardiac muscle tropomyosin. The tendency of smooth muscle tropomyosin to form semi-rigid polymers with continuous and undampened rigidity may compensate for the lack of troponin-based structural support in smooth muscles and ensure positional fidelity on smooth muscle thin filaments.

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation

“…For the WT Tpm, we obtained a PL of 877 Å which is a little less than the value obtained for actin-free Tpm [*1000 Å , see Li et al (2010b) and Sousa et al (2010)], suggesting the Tpm is more curved upon binding to F-actin. For both WT and Tpm mutants, we found moderate correlation between PL and RMSF h i Tpm or RMSF h i FÀactin (with Pearson correlation = -0.5), which is consistent with the idea that the PL not only depends on flexibility but also on the end-to-end curvedness of Tpm (Li et al 2010b).…”

“…To complement the RMSF analysis and compare with other studies of Tpm flexibility (Li et al 2010a(Li et al , b, c, 2012Loong et al 2012a, b;Sousa et al 2010), we have calculated the apparent persistent length (PL) with the commonly used tangent correlation method (also used in previous studies, see (Li et al 2010b;Loong et al 2012b)) for the WT and Tpm mutants (see Table 1). For the WT Tpm, we obtained a PL of 877 Å which is a little less than the value obtained for actin-free Tpm [*1000 Å , see Li et al (2010b) and Sousa et al (2010)], suggesting the Tpm is more curved upon binding to F-actin.…”

Thin Filament Structure and the Steric Blocking Model

“…Li et al found that the tropomyosin coiled-coil is rather stiff with a persistence length about 12 times its own length (108,(134)(135)(136)(137)199). Thus, local troponin-and myosininduced shifts of semirigid tropomyosin on the actin filament can be expected to be propagated along the tropomyosin cable with limited decrement, producing expected filament cooperative effects.…”

Polymer physics of the cytoskeleton

“…The very small persistence length of the 10 nm diameter IF containing many coiled-coil dimer subunits per cross-section is surprising since the persistence length of a single coiled-coil dimer such as tropomyosin is itself approximately 100 nm. [20]. The thinner, semiflexible F-actin is an order of magnitude stiffer than IFs and has a persistence length ranging from 3–17 um [21], which is comparable to the typical filament length.…”