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.

Figure 1

EM of isolated tropomyosin molecules. (a and b) Rotary-shadowed bovine cardiac tropomyosin molecules. (c) Rotary-shadowed cardiac tropomyosin complexed with TnT. (d and e) Chicken gizzard smooth muscle tropomyosin molecules. (a and d) Survey fields showing monomers (single arrows), dimers (double arrows), and polymers (open arrows). (b, c, and e) Montages showing examples of monomers, dimers, and polymers. (b) Top row: cardiac tropomyosin monomers; bottom two rows: dimers. (c) Top row: cardiac tropomyosin monomers complexed with TnT; bottom row: dimers with TnT (arrow indicates possible contribution of TnT at the center region of the dimers). (e) Top row: gizzard tropomyosin monomers; middle two rows: dimers; bottom row: polymers. Scale bar: 100 nm for each set of images. Molecules were examined from two or more different protein preparations each.

Figure 2

Apparent persistence length measurements of rotary-shadowed tropomyosin dimers. The tangent correlation method used to calculate the persistence length (ξ) plots ln 〈cos θ〉 as a function of arc length along tropomyosin (where θ is the deviation angle from a straight rod); the inverse slope of the regression line yields 2ξ. Persistence length measurements of (a) cardiac and (b) smooth muscle tropomyosin; (♦) plots for 25 nm distal end (labeled ends) sections of tropomyosin; (x) plots for 25 nm central (labeled middle) sections of tropomyosin. Regression lines through the respective points are represented by tick marks. The tangent angles determined for the arc lengths measured were normally distributed.