Myosin assembly of smooth muscle: from ribbons and side polarity to a row polar helical model

Figures

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  Fig. 1. Low power magnification of cross sections of taenia coli muscle. At this magnification, the myosin filaments (some of them exhibiting ribbon-like features) are clearly visible even at 1.5-fold magnification. These shorter ribbons, and not the not recognizable regular arrangements of the actin filaments, later described as actin lattices, are clearly seen in Fig. 3. The applied preparation protocol was as the one used for the molluscan smooth muscle (Fig. 2; see also (Sobieszek 1973)). For more details see “Materials and methods” and “Discussion” sections. Magnification: ×24,000

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  Fig. 2. Low power cross section of an EM image of ABRM smooth muscle of Mytilus edulis. The black dots of variable diameter correspond to the myosin filaments, which additionally contain paramyosin and myorod. For more details, see “Discussion” section. Magnification: ×19,500

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  Fig. 3. Smooth muscle cell of taenia coli at high magnification. Note, the clear presence of the actin filament lattices. In contrast, most of the myosin filaments are not round but in the form of a ribbon-like shape of variable width. Magnification: ×107,500

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  Fig. 4. Cross section of molluscan smooth muscle. At this magnification, the myosin cross-bridges are often recognizable as projecting away from the thick filament’ surfaces or connections between the two kinds of actin filaments. The dense areas are structures analogous to the Z-line of skeletal muscle, also present in the vertebrate smooth muscles. Magnification:×63,000

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  Fig. 5. Paracrystals formed from purified light meromyosin fragment (LMM) of chicken gizzard myosin at low acidic conditions. Most commonly they were of the size shown in (B), but often they could be relatively broad (C) or rather long (D). Those in form of a short filament were very rare (A). Note the presence of the 7.15 nm striation and the corresponding 14.3 nm so-called leucine repeat resulting from the coiled-coil structure of the LMM and their regular longitudinal assembly. This periodicity was at 90° to the paracrystals axis, which is clearly visible on the large and flat paracrystal shown in (B). Magnifications: A × 103,000; B ×82,200; C ×94,700; D ×45,000

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  Fig. 6. Short filaments or assemblies formed from purified myosin rod fragments at low ionic strength (BW pH 5.6). In these rod filament preparations, there were many assemblies present, in which the filaments were splitting at one end forming arrowhead-like structures (labelled by letter “V”). Note also the presence of the 7.15 nm coiled-coil repeats as observed for the LMM paracrystals. As it is clear from the optical diffraction analysis, this repeat appears to be at about 6°–7° to the longitudinal filament axis. Magnification: ×72,000

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  Fig. 7. Filaments assembled from purified myosin rod fragment by slow dialysis against low ionic strength buffer at pH 5.6. Under these conditions, javelin-like filaments were formed, exhibiting the same 7.15 nm coiled-coil repeat. Noticeable in their appearance is the somewhat flattened shape, which sometimes resulted in a 90° rotation of the two ends of the javelins in opposite directions. This is clearly seen on the horizontal upper insert (H) and the one labeled with an asterisk. The vertical insert (V) on the left shows an interesting and unique flattened javelin in which three rotated threads are recognizable of the kind shown in Fig. 8. For more details, see “Discussion” section. Magnification: ×72,000

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  Fig. 8. Threads formed from myosin rods after their solubilization at 0.6 M KCl under low acidic conditions (pH about 4.0) with subsequent neutralization (pH 6.8). Note the formation of extremely long thread-like structures, often in form of triple ribbons, which might be related to the coiled-coil structure of the proposed helical thread (see “Discussion” section). Magnification: ×72,000

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  Fig. 9. Myosin filaments assembled from purified pig stomach myosin by slow overnight dialysis. These were exceptionally fixed with 1% glutaraldehyde fixation on the EM grid to check whether this would result in their elongation. Apparently, this was not the case because their length was comparable to those of gizzard myosin. Magnification: ×22,000

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  Fig. 10. Filaments assembled from pig stomach smooth muscle myosin purified by our improved procedure described in “Materials and methods” section. Note the excellent preservation of the myosin cross-bridges. All the previously described features of such long filaments are clearly seen (see “Introduction” section). This particular EM image was selected because it included an about twofold thinner assembly (at the lower right part of the image) which may correspond to one of the three strands of the supra-coiled-coil assembly proposed (see “Discussion” section). Magnification: ×92,500

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  Fig. 11. Optical diffraction analysis of LMM paracrystals (A), javelin rod filaments (B) and myosin filaments isolated from vertebrate (C) as well as those of invertebrate smooth muscle (D). All were from purified chicken gizzard, except the last one (D), which was of Mytilus edulis, and the corresponding EM image used is shown in (E). The 7.15 nm meridional reflection is more intense than the 14.3 nm, both characteristic of the coiled-coil structure of the myosin rod. Significantly, in the case of javelins, the 7.15 nm reflection was tilted about 6°–7° relative to the meridian, indicating that the rods were packed at this angle to the javelins’ axis. Note also that in the pattern obtained from intact myosin in C there are contributions of the other side of the helix, which results in more symmetrical patterns or individual LLs. The LLs at about 20 and 34 nm are consistent with the 172 nm repeat but not necessarily the 43 nm one. The pattern of (D) was an FFT obtained from a scan of the EM image shown below (E), using the ImageJ software and not our diffractometer. The FFTs are comparable to those obtained by optical diffraction, except that there were differences due to the large…

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  Fig. 12. Optical diffraction patterns from myosin filaments (A) and (C). The patterns (A) and (D) are perhaps characteristic of the smooth muscle with numerous LLs, which makes their interpretation rather difficult. Nevertheless, the most intense ones are consistent with the proposed repeat. In (C) the symmetrical LL spacing was about 72 nm and the spacing of the other one (connected) to a meridional reflection was close to the 43 nm. This pattern was obtained from myosin filament isolated cells of taenia coli. All LLs at about 86 nm can also be identified in this pattern. In the (C) pattern, the 34–36 nm LLs and even the nearer 24 nm LLs are also connected in an interesting doublet