Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle

Abstract

The Z-band in vertebrate striated muscle crosslinks actin filaments of opposite polarity from adjoining sarcomeres and transmits tension along myofibrils during muscular contraction. It is also the location of a number of proteins involved in signalling and myofibrillogenesis; mutations in these proteins lead to myopathies. Understanding the high-resolution structure of the Z-band will help us understand its role in muscle contraction and the role of these proteins in the function of muscle. The appearance of the Z-band in transverse-section electron micrographs typically resembles a small-square lattice or a basketweave appearance. In longitudinal sections, the Z-band width varies more with muscle type than species: slow skeletal and cardiac muscles have wider Z-bands than fast skeletal muscles. As the Z-band is periodic, Fourier methods have previously been used for three-dimensional structural analysis. To cope with variations in the periodic structure of the Z-band, we have used subtomogram averaging of tomograms of rat cardiac muscle in which subtomograms are extracted and compared and similar ones are averaged. We show that the Z-band comprises four to six layers of links, presumably α-actinin, linking antiparallel overlapping ends of the actin filaments from the adjoining sarcomeres. The reconstruction shows that the terminal 5-7nm of the actin filaments within the Z-band is devoid of any α-actinin links and is likely to be the location of capping protein CapZ.

Keywords: Z-disc; Z-line; electron microscopy; electron tomography; α-actinin.

Graphical abstract

Fig. 1

Electron micrographs of the Z-band in longitudinal sections of rat cardiac muscle. (a) A sarcomere showing two Z-bands bisecting the I-bands, A-band and M-band (M). Part of the Z-band on the left shows a clear lattice view of the Z-band tetragonal lattice. The Z-band on the right has a dense fuzzy appearance resulting from a projection off a lattice direction. (b–f) A panel of straight Z-bands showing patches of lattice view. (g and h) Plot profile of the Z-band obtained by averaging the plots of the Z-bands in (b) to (f). The width of the Z-band measured from the base of the plot is about 130 nm. In (g), the plot profile is reduced in size to exactly match the Z-band and I-band in (f), showing how the increase in density in the Z-band matches the projected image. Scale bar in (a) = 0.5 μm (applies to (b) – (f)).

Fig. 2

Electron tomography of rat cardiac muscle Z-band. (a) The Z-band tetragonal lattice and nomenclature of lattice views. The main figure illustrates a slightly oblique transverse section with actin filaments (grey) at the left and actin filaments at the right (red) interdigitate in the centre at the Z-band. Small patches of small-square lattice and basketweave Z-bands are shown, the former with sharply bent links and the latter with more gently curved links. Along the bottom, unit cells are outlined at the centre of the Z-band and at either side. Projecting about the major axes gives the lattice views we observe in longitudinal sections, like the 1,0 view and similar orthogonal 0,1 view. Projecting along the diagonal gives the 1,1 view. (b) Electron micrograph of the Z-band region used for the tomography in this study. The Z-band has a clear small-square lattice appearance. Dense glycogen granules (G) are present between the myofibrils. (c) 2D slice of the tomogram near the centre and (d) near one side. As expected from the figure in (a), there is a “small-square” lattice in the central slice (c) due to the overlapping ends of the actin filaments from the adjacent sarcomeres and a large-square lattice at the Z-band periphery (d) due to a single set of filaments from one sarcomere. Corresponding regions showing clear examples of the lattice are encircled in (b)–(d). A movie of the raw tomogram is shown in the supplementary data, Movie S1. Scale bar in (d) = 100 nm (applies to (b) and (c)).

Fig. 3

Surface-rendered stereo images of the small-square lattice Z-band in rat cardiac muscle following subtomogram averaging. Examples of the two sets of actin filaments of opposite polarity from sarcomeres below and above are labelled A and B, respectively. (a) Plan view. Links between the filaments are seen that run parallel with the lattice as found in small-square lattice Z-bands. (b) Oblique view showing more detail of the filaments and links. (c) Side view showing A and B polarity actin filaments with clear links (arrowheads) between them at a nominal periodicity of 38 nm. The centre of the Z-band is not at the centre of the tomogram; hence, we see the end of the A-filaments as they terminate inside the tomogram but the B-filaments are clipped at the bottom face of the tomogram. The terminal region of the A-filament marked with an asterisk (*) is devoid of links. We believe that this region may represent capping protein CapZ. (d) As (c) but 1 unit cell deeper into the average tomogram showing another clear example of an A-filament with a bare terminus region. In (a) and (b), prominent links (labelled P) occur between the same polarity B-filaments; these were called polar links in our previous study . Scale bar in (c) and (d) represents 10 nm in vertical direction.

Fig. 4

Interpretation of the rat cardiac Z-band tomogram by modelling. (a) Schematic model of the Z-band motif for small-square lattice Z-bands proposed by Morris etal. (1990) composed of two pairs of antiparallel actin filaments (magenta and cyan) linked by two α-actinin dimers shown in yellow and green. α-Actinin is a homodimer comprising a central axial rod domain (parallel with actin) and transverse actin binding domains (“struts”) with a relative twist of 90°. (b) Helical actin filament in which the monomers are related by an axial rise of 2.74 nm and a rotation of 167.1° so that every seventh actin subunit (red) is spaced 19.2 nm apart and rotated by 90° forming 4₃ screw symmetry-related binding sites for α-actinin. (c) Two sets of such α-actinin binding sites related to each other by 2.74 nm axial translation and 167.1° rotation about the filament axis form the basis of the repeating motif of the small-square Z-band lattice. (d) Overall symmetry of the repeating motif of the small-square Z-band illustrated with an end view of a schematic model comprising one up filament (magenta) and four down filaments (cyan) and associated α-actinin links (white). The 4₃ screw symmetry axes of the actin filaments combine with 2₁ screw symmetry axes midway between the two sets of filaments resulting an overall P4₃2₁2 symmetry. (e–j) Comparison of schematic model with the protein density of the subtomogram average in 1,0 (e and f), 0,1 (g and h) and 1,1 (i and j) views. Schematic models are shown separately (e, g and i) and in stereo view within the protein density (f, h and j). In (f), clear links are seen between the antiparallel filaments (double-headed arrows). In the 1,1 view (i and j), α-actinins show the 90° relative twist of the struts at each end of the rod. (f, h and j). This figure is shown as Movie S4 in the supplementary data.