Filamentous and Matrix Components of Skeletal Muscle Z-disks DOUGLAS E. KELLY AND MARY ANN CAHILL Department of Biological Structure, University of Miami School of Medicine, Miami, Florida and Department of Biological Structure, University of Washington, School of Medicine, Seattle, Washington ABSTRACT The fine structural appearance of Z-disk lattices in vertebrate skeletal “fast” muscle varies depending upon whether osmium or glutaraldehyde has been employed as the primary fixative. Prior investigators have attributed the differences to change in the extent of actin filament overlap within the Z-disk and/or to rearrangement of Z-disk filaments. Adult frog and young newt “fast” muscle has been studied under various degrees of stretch, with several different aldehyde and osmium fixation procedures, and after plastic section digestion techniques utilizing Pronase or pepsin. Serial cross sections of Z-disks were correlated with oriented cross and longitudinal sections. Fixation with collidine-buffered osmium and Verona1 acetate-buffered glutaraldehyde seems to provide the greatest and most distincitly contrasting differences. A consistently arranged phase, the filamentous lattice, can be discerned after either fixation. However, a second phase, termed “Z-disk matrix,” appears variable, perhaps due to extraction during primary osmium fixation procedures. Glutaraldehyde-fixed frog muscle Z-disks display a copious matrix, one which is seldom totally depleted by osmium fixation. In young newt muscle Z-disks, little matrix is present after glutaraldehyde fixation and none of it remains after primary osmium. In Z-disks fixed by either method, matrix that is retained appears to be deposited in lattice-like patterns. It is suggested that these matrix patterns, or their loss, are the basis for the varying images of Z-disks observed under different fixation conditions, and that the filamentous lattice js relatively stable. The Z-disk is more rapidly obliterated by Pronase or pepsin digestion than is any other muscle component, including actin (which appears notably unreactive). The rapid digestion effect is limited to the region postulated to include the matrix phase. Models for the structural interrelationship of filamentous and matrix phases are discussed and compared to prior Z-disk models. The fine structural architecture of skele- were based on cross-sectional images of tal muscle Z-disks in amphibians and mam- Z-disks disclosing a regular tetragonal mals has drawn increasing interest during or woven 200-240 A lattice, seemingly the past ten years. Most of this inquiry has formed of h e Glamentous material. Typirelated to Z-disks of “fast” skeletal muscle, cally, this lattice is oriented at an angle of and a number of models have been pro- 45” with respect to the rows of aligned dots posed to explain Zdisk images obtained by which represent actin filaments just as high resolution electron microscopical tech- they approach the faces of each Zdisk. It niques (Knappeis and Carlsen, ’62;Huxley, was assumed that within the Z-disk, this ’63; Franzini-Armstrong and Porter, ’64; relatively large, angled filament lattice Reedy, ’64; Kelly, ’67, ’69). Each of these serves to link, in one manner or another, early models was interpreted from similar Received July 16, ’71. Accepted Nov. 12, ’71. images of osmium-fixed material and in1 Present address: Department of Biological Struccorporated individual assumptions which ture, University of Miami School of Medicine, Miami, Florida 33152. could not be confirmed with the available 2 Present address: Fachbereich Biologie, Universitst techniques. Many of the interpretations Konstanz, D775 Konsl.anz, Germany. ANAT. REC., 172: 623-642. 623 624 DOUGLAS E. KELLY AND MARY A N N CAHILL the actin filaments of one sarcomere to hyde techniques, is more representative of the natural state. Moreover he has those of the adjacent sarcomere. More recently the problem has been com- suspected (personal communication) that pounded by the discovery of a second (and osmium-induced contracture (sarcomeric only occasionally coincident), smaller, lat- or within the Z-disk), coupled with loss of tice configuration in cross-sectioned Z-disks Z-disk materials, may promote the assump(Fardeau, '69; Landon, '70; MacDonald tion of the large, angled tetragonal configuand Engel, '71; Rowe, '71). This lattice is ration. By contrast, glutaraldehyde fixation also tetragonal, but measures only about does not produce tension at the Z-disk and 100-120 A, is oriented parallel to the it thereby promotes overlap of I-band actin nearby rows of aligned actin filament dots, filaments approaching the Z-disk from opand is characteristically obtained after al- posite directions. With this latter fixation, dehyde fixation procedures. Landon ('70) in Landon's view, the small lattice results has interpreted the large, 45" angled, tetra- from superimposition, within the thickness gonal lattice found after primary osmium of a specimen section, of two identical large fixation as being the result of a realignment (200-220 A), unangled (parallel) tetraof cross-linking filaments of the smaller gonal lattices, each filamentous and located Zdisk lattice. He has suggested that the near the two faces of the Z-disk. Their small lattice, as preserved by glutaralde- superimposition is parallel, but displaced Fig. 1 Electron micrograph of frog toe "fast" muscle. Myofibrils are seen i n cross section through the 2-disk region. Dense areas are the Z-disk proper, whereas lighter areas displaying actin dots are in the immediately adjacent I-band. This specimen has been fixed by collidine-buffered osmium tetroxide. The large rectangle displays faintly the large, angled (woven) lattice, whereas other dense areas display a small, parallel lattice (circle). Lines on the micrograph depict the alignment of actin dots outside the Z-disk as related to the orientation of lattices within the Z-disk. E n bloc uranyl acetate stain; lead section stain. x 135,000. SKELETAL MUSCLE Z-DISKS by half of the lattice period in both x and y axes so that in cross sections the small (+ 100 A), tetragonal lattice image results (see also Fardeau, ’69). MacDonald and Engel (’71), and more recently Rowe (’71 and personal communication), have also proposed models to explain the small lattice image. These authors’ models are constructed along the looping Z-disk filament concept proposed in our laboratory (Kelly, ’67), although the loops in their models 625 interlink differently :and there is a necessity of added looping filaments within the thickness of the Z-disk to account for the small lattice configuration. As an alternative to that type of proposa.1, MacDonald and Engel (’71) also offer, and tend to favor, a modified Knappeis-Carlsen (’62) configuration in which the Z-disk filaments are preserved by aldehyde fixation in an acutely bent (“swastika”) plosition to confer collectively the small lattice image. Fig. 2 Stereo electron micrograph pair showin: a cross section through the Z-disk of larval newt tailfin “fast” muscle after collidine-osmium fixation. The 2-disk proper is much less dense and more delicate than that seen in figure 1. It is composed entirely of the large, angled lattice which is disposed at -45” to the alignment of actin dots immediately adjacent to the Z-disk (see lines for orientation). In some areas, the large, angled lattice appears “woven” (top center). Close examination of the filament lattice within the circle reveals filaments that appear more ribbon-like than round. E n bloc uranyl acetate stain. x 135,000. This stereo illustration can be viewed either with a crosseyed viewing technique or by use of a cartographer’s stereo viewer. 626 DOUGLAS E. KELLY AND MARY ANN CAHILL Small and large lattice images were obtained in our laboratory during the study of both young newt and adult frog muscle of the “fast” variety. We have employed different approaches to study the problem, and as a result have obtained evidence suggesting an explanation that differs from the interpretations of the above named workers. Our interpretation is based upon recognition that the Z-disk in most skeletal muscles consists of two primary phases; a filamentous lattice and a surrounding, lessstructured matrix. It is suggested that the deposition or absence of the matrix after different fixation processes may be primarily responsible for the variable appearances of the Z-disk, but that the underlying filamentous lattice remains relatively unaltered. MATERIALS AND METHODS Skeletal muscle from both young newt and adult frog specimens was utilized and fixed with a wide variety of methods. The toe muscle (flexor digitorum) of Nembutal anesthetized adult frogs ( R a m pipiens) could easily be immobilized and fixed at rest length or in varying degrees of stretch. A similar opportunity was provided by the thin intermandibularis muscle in the chins of older larval or young postmetamorphic West Coast newts (Taricha torosa; collected as embryos in the Stanford, California, area). Newt intermandibularis muscle was exposed by incision and anchored or stretched by pinning the mandibles prior to in situ application of fixative. A similar procedure was employed for frog toe muscle. The approximate desired length Fig. 3 Cross section through Z-disks of frog toe muscle after glutaraldehyde fixation. The small (-110 A), parallel (circles) and large (-240 A), parallel (square) lattices are predominant in the field. The pleomorphic nature of the strands which make up these lattices is apparent (see square). A distinct angled lattice is more rarely observed in this material and is not displayed distinctly in this field. If present, it is apparently masked by other components, as for example in the very dense areas at the extreme left center of this field. En bEoc uranyl acetate stain; lead section stain. x 180,000. SKELETAL MUSCLE Z-DISKS 627 was maintained throughout fixation and lowing either fixation, tissue blocks were initial dehydration. In addition, muscle usually stained en bZoc 2 hours at room was obtained at random states of contrac- temperature in a 0.5% uranyl acetate solution from the tails and limbs of younger tion in Michaelis' buffer (pH 5.0). Tissue newt larvae. In all cases, the newts blocks were dehydrated in a graded series were anesthetized with MS-222 (tricaine- of ethanols and placed in propylene oxide methanesulfonate, Sandoz, Inc., New prior to embedding in Epon 812 (Luft, '6 1). York), after which whole larvae or excised Thin sections were cut with a diamond muscles were fixed. knife on a Porter-Blum MT-2 ultramicroWith both sources of muscle, the most tome, and usually stained additionally for informative and contrasting images were two to eight minutes with the alkaline-lead obtained from two fixation techniques : citrate procedure of Reynolds ('63). Where 3.75% osmium tetroxide in 0.05 M s-colli- prior en bloc staining had been omitted, dine buffer applied ice-cold for one-two lead staining of sections was preceded by hours (Bennett and Luft, '59); or 0.38% a two to eight minute treatment with halfghtaraldehyde in Michaelis' (veronal ace- saturated aqueous uranyl acetate. Microstate) buffer (at 275 milliosmoles) for a copy was done with Philips EM200 and similar period at room temperature. In the AEI-6B instruments. latter case, the tissues were rinsed in Newt intermandibularis muscle was parMichaelis' buffer and postfixed for one ticularly useful for precise longitudinal hour in iced collidine-osmium solution. Fol- sectioning to determine the extent of con- Fig. 4 Cross section through the Z-disk of a stretched frog toe myofibril after glutaraldehyde fixation. The image discloses an increased prevalance of large, angled 1.attice which in some areas (square) displays a woven pattern. The small -110 A, parallel lattice is also visible in other parts of the field (ovals) where the section plane has passed through the Z-disk near one of its faces (see discussion). Uranyl acetate stain and lead section stain. x 335,000. 628 DOUGLAS E. KELLY AND MARY ANN CAHILL traction or stretch in a given area of a fiber, after which the block could be rotated 90” for cross sectioning. This allowed close examination for any possible correlation between sarcomeric length and cross-sectional Z-disk morphology. Much of this study involved serial cross sectioning of Z-disks - a procedure which requires a series of at least three or four exceedingly thin (200-300 A) sections. Reconstruction from such series is most accurately accomplished by use of stacked lantern slide transparencies of Z-disk images. This type of analysis can be correlated and complemented by the simpler study of equally thin single cross sections, cut just slightly oblique to and running through the plane of a Z-disk. However, in the latter instances, individual filaments cannot be traced through the Z-disk, as is possible with certain of the serial section images. Some cross and longitudinal sections of frog and newt muscle were subjected to periodic acid oxidation and proteolytic hydrolysis extraction procedures (slightly modified from those developed by Anderson and Andr6 “681) prior to mounting on grids and section staining. The sections were floated on an oxidizing medium of 5.0% periodic acid for 10-20 minutes at 40°C and then transferred by a stainless steel wire loop to a 0.5% pepsin solution in 0.1 N HC1 or a 1.0% Pronase solution for 5, 10, 15, 25 or 45 minutes. Control sections were treated with 0.1 N HC1 and/or the oxidizing solution alone. OBSERVATIONS Three Z-disk lattice patterns are often distinguishable in cross sections of our material. They are: (1) the large (+ 220 A), square or “woven” lattice whose sides are angled 45” to the rows of parallel actin filaments seen as dots just outside the Z-disk; (2) a smalE (+ l l O A ) , square lattice whose sides lie parallel to the rows of actin filament dots; and ( 3 ) a large (+ 240 A ) , square lattice whose sides are also parallel to the dots. It will be seen that two and three above are closely related and that the small, parallel lattice depends upon presence of the large, parallel lattice for its expression. In collidine-osmium-fixed frog toe muscle, the large ( 4220 A), angled lattice is less commonly or distinctly observed in cross sections of Z-disks than it is in young newt material (figs. 1, 2 ) . As noted by Reedy (’64), if it is visible it is often twisted into a “basket-weave” pattern. Moreover, our observations correspond somewhat to those of Landon (’70) and Page (’65) in that, in this osmium-fixed material, the small (+ 110 A ) , parallel lattice is also at least as frequently observed (fig. 1 ) . The presence of the small lattice is rare in the similarly-fixed larval or young postmetamorphic newt muscle studied, the rule being a very delicate and distinct large ( 4220 A), angled lattice (fig. 2). Close examination of these images (particularly by stereo techniques) discloses that the filaments of the large lattice often have a thin ribbon-like appearance rather than being round. When glutaraldehyde fixation is used, frog toe muscle cross sections regularly display the now well-known small, parallel lattice. But in addition, interspersed patches of the large, parallel lattice are equally frequent (fig. 3 ) . Only occasionally is the large, angled (often woven) lattice also detectable. In stretched frog toe muscle (similarly fixed), the incidence of distinct side-by-side large, angled and small, parallel lattice in a given section is more frequent (figs. 4, 8 ) , although instances of seemingly pure small, parallel lattice have also been seen under these conditions when thicker sections were used. Newt muscle Z-disks do not display Fig. 5 Longitudinal section through a myofibril of stretched glutaraldehyde-fixed frog toe muscle. Parts of the Z-disk (circle, and another example in the inset) display a pattern suggesting the overlap of actin filaments from adjacent sarcomeres. However, evidence for an alternative explanation i s presented in this report. En bloc uranyl acetate stain; lead section stain. x 80,700. Inset x 148,000. Fig. 6 Longitudinal section through newt intermandibularis muscle, glutaraldehyde-fixed in a partially contracted state. A similar “overlapping” pattern (circle) is seen in the Z-disk i n this specimen and others fixed at various sarcomeric lengths. Uranyl acetate and lead section stain. x 80,000. Fig. 7 Longitudinal section through newt tailfin muscle fixed with collidine-osmium at approximately rest length. The Z-disk displays a typical zigzag appearance as well as other configurations, but it does not display the morhpology noted in figures 5 and 6. Uranyl acetate and lead section stain. x 80,000. I SKELETAL MUSCLE Z-DISKS 629 630 DOUGLAS E. KELLY AND MARY ANN CAHILL the same distinct or predominant small lattice pattern after any of several glutaraldehyde fixation procedures employed for either stretched or rest-length fibers. A large, angled lattice pattern prevails much in the same way that i t does after osmium fixation. Only sporadic and rather faint indications of small, or large, parallel lattices can be detected (fig. 9). The above observations suggest that the component common to both muscle SYStems is the large, angled lattice, and that it is structurally similar after both methods of fixation. It is also apparently little affected by stretch prior to and during fixation. Since this lattice is most distinct in young newt "fast" muscle, longitudinal sections of stretched and rest-length or shorter intermandibularis muscle sarcomeres were compared for differences in Z-disk images. The same range of possible images and dimensions (related to the angle of lateral view through the Z-disk) was found without significant variation. The longitudinal thickness of the Z-disk may decrease slightly during contraction, concomitant with its reported increase in girth (see Elliott et al., '67). However, the Z-disk has rather vague boundaries to measure precisely, and such a change was not detectable from the images studied. When the same tissue blocks were reoriented 90" for resectioning and the same fibers and sarcomeres observed in cross section, again no morphological difference in Z-disk lattice was apparent between known stretched and rest-length fibers. A slight expansion in the size of the lattice may occur with contraction (coincident to Z-disk girth increase), but it is still a -200-240 A large lattice, angled 45" to the rows of actin filament dots, and is not the small, parallel lattice. Since frog muscle fixed by glutaraldehyde (at rest length or stretched) most frequently and regularly displays the small lattice, this tissue was subjected to another line of scrutiny. Stretched fibers were examined in both longitudinal and near cross sections; in the latter instance, the plane of section was just slightly oblique to and included the Z-disks. In the longitudinal sections (stretched and glutaraldehyde-fixed), one Z-disk profile which often is visible displays a zig-zag Z-disk filament pattern, plus an apparent interdigitation of actin-filaments within the Z-disk (fig. 5). This image has been observed by ourselves and many other workers using muscle sarcomeres of shorter length. It has been interpreted by Landon ('70 and personal communication), MacDonald and Engel ('71), and Rowe ('71 and personal communication) as representing a fundamental change in Z-disk thickness and/or filament arrangement induced by glutaraldehyde fixation, and perhaps aided by lack of osmium-induced sarcomeric contracture. It is therefore surprising that in the current study, this image was obtained in both stretched and c m tracted fibers. In either instance one would expect that tension on the Z-disk would discourage actin interdigitation. Newt muscle also displays this image at various sarcomeric lengths (fig. 6 ) . In that tissue it is an image which does not frequently occur when primary osmium fixation is utilized (fig. 7). Cross sections (in this case, slightly oblique to the plane of the Z-disk) of stretched frog toe muscle fibers displayed the most frequent coincidental occurrence of large, angled and small, and large, parallel lattices. Several other features are also prominent, and it becomes obvious, especially in thinner sections, that all of these are arranged in a repeated sequence (fig. Fig. 8 A cross section running slightly obliquely through the 2-disk region of several frog toe muscle myofibrils. The muscle has been stretched and fixed i n glutaraldehyde. Where the section passes through the I-band, actin dots are seen i n random array (I, lower right). These become aligned near a face of a 2-disk (ZI). At the edge of the Z-disk face, interconnections among the aligned dots form the large (-240 A), parallel lattice (arrows). Within the same region (Z-I) other areas of small (- 110 A) lattice are visible. In the center of the Z-disk ( Z ) , the predominant large, angled lattice is visible ( i n this case displaying a woven pattern), as well as a dense, less easily interpretable meshwork. En bloc uranyl acetate stain; lead section 'stain. x 108,000. Fig. 9 A similar cross section taken from newt intermandibularis muscle fixed by glutaraldehyde. The same components of I-band, Zdisk face, and center Z-disk regions are visible, except that the matrix materials forming the large and small, parallel lattices a r e much more faint or scanty. The large, angled lattice in the center of the Z-disk, however, looks quite similar to that depicted in figure 8. Uranyl acetate and lead section stain. X 106,000. SKELETAL MUSCLE 2-DISKS 8). Where the section has passed through the I region, actin filaments are visible as randomly distributed dots. Where actin filaments approach the Z-disk, these become 63 1 arranged into parallel rows with dots equidistant and about 1240 A apart. Close to this point, they also acquire interconnecting dense strands which outline the mar- 632 DOUGLAS E. KELLY AND MARY ANN CAHILL - gins of 240 A squares linking the dots. This is the large, parallel lattice. Within the areas of the section presumed to occupy the Z-disk proper, these 240 A squares appear to be converted into the small ( 4110 A ) lattice, oriented parallel to the dot rows and margins of the -2240 A squares, OT into the large (+ 220 A), 45” angled (often woven) lattice, rn into a denser meshwork within which any one lattice cannot regularly be detected. Further, such sections suggest that the small and large, parallel lattices lie near the faces of the Z-disk, whereas the large, angled lattice may occupy a position near the center of the Z-disk. Similar, though less distinct, images and suggested sequences are observable in cross sections of glutaraldehyde-fixed newt muscle (fig. 9). The suggested sequences cannot be firmly established from single sections, but require corroboration by the use of serial cross sections to follow individual filaments or sets of filaments as they course through the Z-disk region. However, two prerequisites prevail: (1) that all the sections of a series be exceedingly thin (since the Z-disk proper is less than 800 A thick); and ( 2 ) that in a particular series, the sections are of appropriate thickness and in planes so as to contain individual, sequentially recognizable segments of the Z-disk system as it is traversed. These segments can then be accurately interpreted and reconstructed (fig. 16). Few series are obtained which simultaneously meet both criteria. Figures 10 and 11 depict portions of suitably thin and adequately positioned serial cross sections from stretched, glutaraldehyde-fixed frog toe muscle. Individual actin filaments, or small groups of them, can be traced into the Z-disk, whereupon their thinner filamentous appendages can be followed through to meet actin filaments of the adjacent sarcomere. Within this sequence, it is apparent that actin filaments are grouped into a squared (-2240 A) array of dots as they near the face of a Z-disk; and they become interconnected by fine strands of dense material to form the large, parallel 240 A tetragonal lattice in the immediate vicinity of the face. Such a lattice is not identical to the large, angled lattice deeper within the Z-disk, for not - - only is its orientation parallel to the rows of actin filaments, but in addition its strands are somewhat less regular in profile. This large, parallel lattice has not been incorporated into any of the earlier “osmium-based Z-disk models, but it seems to correspond to the “basic” lattice recognized by Landon ( ’ 7 0 ) as occupying either face of the Z-disk. He suggests that, when superimposed in a thicker section, the images of the two basic lattices of a Z-disk cast the collective small, parallel lattice profile. In the present study, the large, parallel lattice is usually the first one encountered as the sections progress into the Z-disk area, but it often (but not always) becomes permeated by a second pattern: the typical small, parallel lattice. The next sections can display the large, angled lattice (if suitaby positioned). And this lattice does appear to occupy the center of the Z-disk, for the small, and/or large, parallel lattices reappear in the subsequent section or sections. A similar analysis applied to collidineosmium-fixed larval newt muscle invariably shows a direct transition from parallel rows of actin dots into the large, angled lattice and back again to parallel actin dots. No small, or large, parallel lattices are seen near the face of a Z-disk (fig. 12). The small, parallel lattice has been termed filamentous by prior observers. In the present study, however, the pleomorphic nature of the density which forms the basis for both the small, and large, parallel lattices may be significant. These lattices are formed by strands of density which vary in diameter from about 20-60 A, and while their disposition is repeating, their Figs. 10 and 11 Serial cross sections traversing the thickness of the 2-disk reoion, and beyond, in stretched glutaraldehyde-fixed frog toe muscle. The circles and squares indicate areas of corresponding filaments and their appendages traced through the Z-disk region. Figure 11 contains serial sections that are thin enough and positioned with respect to sequentially recognizable segments of the Z-disk system. They show that the large, angled lattice lies in the center of the 2-disk ( C ) , while on either side of it the large and small, parallel lattices are visible (B and D ) . A similar, though less well defined, sequence is visible in the circles of figure 10A, B, and C. In the area of figure 10 depicted by the squares, the large and small parallel lattices are apparently minimally present. Uranyl acetate and lead section staining. Both figures x 124,000. ‘ SKELETAL MUSCLE 2-DISKS 633 634 DOUGLAS E. KELLY AND MARY ANN CAHILL presence varies greatly from place to place within a lattice and from specimen to specimen, even when glutaraldehyde has been used and other conditions have been kept as constant as possible (see fig. 3 ) . In short, the elements of the small, and large, parallel lattices do not maintain the same constancy of form as that encountered for the well-studied large, angled lattice. When pepsin or Pronase solutions are applied to preoxidized longitudinal Epon sections of frog muscle, an extraction of Zdisk density is observed within ten minutes (fig. 1 3 ) . It precedes removal of any other myofibrillar or membranous component (myosin filaments being the next most susceptible when treated with Pronase), and from its earliest manifestation any resultant electron lucency outside the A-band is sharply limited to the area enclosed within the faces of a Z-disk. In fact, actin filaments display a maximum tolerance to these treatments and never revealed any sign of dissolution. Since the enzymes attack the section from its surfaces, it is important to note that the same patterns of extraction are obtained with cross sections (fig. 14). Hence, the lack of response by actin filaments is not due to inaccessibility offered by their being buried within the Epon of the section. Within the Z-disk, in longitudinal or cross sections, digestion seems to clear all components equally, and with equal rapidity. It has not been possible to discern differential extraction of the various lattices described above, even with the shortest exposures to the enzymes. DISCUSSION The observations have led to the interpretation that in the Z-disk there are at least two phases of structure which have separate and identifiable characteristics : one which is filamentous and is similarly preserved by both osmium and glutaraldehyde fixation methods; and the other which is pleomorphic and seems susceptible to extraction under conditions of primary osmium fixation. Much of the discussion will revolve around the latter phase, for it is newest in concept. It is evident that the pleomorphic phase, which shall be termed “Z-disk matrix,” is more abundant in some muscles than others. Cardiac muscle Z-disks are very thick and dense, likely due to a generous matrix content, and mammalian “fast” skeletal muscle would appear to contain a heavier deposition of Z-disk matrix than comparable muscle from lower vertebrates. In cases studied here, the younger newt muscles seem to possess but a small fraction of matrix in their 2-disks, compared to the denser Z-disks of frog toe muscle. In either instance, depletion of the matrix phase is discernible after collidine-osmium fixation. However, the concentration of matrix material is great enough in frog toe muscle that appreciable quantities of the matrix phase remain visible after csmium fixation, whereas the presumably much smaller quantity in young newt muscle seems almost totally extracted (or rendered invisible) by such treatment. Figure 15 depicts, in three-dimensional schematic representation, one configuration of how these two phases might be positionally related in stretched, glutaraldehyde-fixed frog “fast” muscle, where matrix has been rendered most abundant and distinct. The filamentous component (“Z-filaments”) appears as it has been imaged in many prior studies, and for simplicity is here depicted according to the model of Knappeis and Carlsen (’62) as modified by Reedy (’64). The alternative looping filament Z-disk model (Kelly, ’67) could equally well be employed, in view of our present lack of determinative information. In either event, this filamentous phase is not significantly altered in morphology with the variations in fixation studied here. The matrix phase does appear to be altered according to fixation, and moreover, when present after glutaraldehyde fixation, this phase apparently can generate images which confuse interpretation of the filamentous phase. In figure 15 the matrix is depicted in an idealized fashion, and for clarity it has been totally excluded Fig. 12 Serial cross sections through the 2-disk region of myofibrils from newt tailfin muscle fixed in collidine-osmium. Sequential steps through the thickness of the 2-disk may be traced in many areas of these micrographs (for example, in the squared regions). In every case, a direct transition from aligned actin dots to the typical large, angled lattice is observed with no intervening matrix lattices of the small or large, parallel variety. E n bloc uranyl acetate stain. x 81,000. ‘ SKELETAL MUSCLE 2-DISKS Figure 12 635 636 DOUGLAS E. KELLY A N D MARY ANN CAHIU from the central zone occupied by Z-filaments. It should be realized that if in life it is a fundamentally non-structured matrix, its deposition in any symmetrical pattern may well be the result of its precipitation during fixation. In glutaraldehyde-fixed material, the matrix appears to be clumped more densely near faces of Z-disks, where it imparts the images of the large and small, parallel lattices, both of which can now be termed matrix lattices (figs. 15, 16). The small matrix lattice may well be an elaboration of, or added deposition upon, the large, parallel matrix lattice of a given face of the Z-disk. In addition, in a suitably thick section, the lattices of one Z-disk face may well reinforce the small lattice image of the other, as Landon suggests ('70). The intervening large, angled filamentous lattice might pose little impediment to such reinforcement if the angle of view is precisely perpendicular to the Z-disk plane. If the angle is slightly oblique, the total superimposed image can be confused and dense. Conceivably, stretching of the muscle fiber may elongate the Z-disk sufficiently to separate more adequately its matrix and filamentous segments. Because precise measurement of Z-disk components is difficult, i t cannot be determined in our images if much separation occurs, although the increased prevalence of side-byside large, angled, and small and large, parallel lattices in cross sections of stretched muscles would lend support to that interpretation. Furthermore, it does seem easier to achieve sequentially recognizable Z-disk segments in thin serial cross sections with stretched muscle (fig. 16). If, however, some collapse of the Z-disk does occur, as Landon ('70) suggests, one can appreciate how the two matrix-predominant segments might be drawn together so that even in a relatively thin section they would either reinforce or confuse (depending upon angle of view) each other's image and that of the Z-disk maments (fig. 17). In any section which includes all the Z-disk filamentous and matrix subcomponents, the resultant crosssectional image is more apt to be a very dense meshwork within which any repeat- ing lattice is obscured. This could account for the frequent dense Z-disk images encountered. Another explanation would be the assumption that fixation and other processing of the 2-disk can often precipitate matrix material into other, perhaps less regular, patterns than the ones which attract immediate attention. It is predictable that primary osmium fixation would aIlow extraction of some components which are retained when aldehyde procedures are employed. Other organelles, for example microtubules, display well-known and similar differential responses to such treatment. Levels of protein extraction after and during a variety of fixation and dehydration procedures have been quantitated (Wood and Luft, '65) ; the retentive limitations of osmium procedures and their immediate alteration of osmotic activities are well-recognized (reviewed by Bone and Denton, '71). If then the matrix phase is considered as relatively labile, the model in figure 15 depicts the interpretable results of but one fixation method - glutaraldehyde with veronal acetate buffer - after which, the maximum and most orderly array of filamentous and matrix components has been retained. At the other extreme, the young newt muscle (which has less visible matrix to start with), when fixed with collidine-buffered osmium, conforms to a similar model but notably lacks the matrix lattices. One must remember that the final Fig. 13 A longitudinal section of frog toe muscle, fixed in collidine-osmium. The Eponembedded section was treated with 0.5% pepsin for ten minutes according to the technique of Anderson and Andre (1968). The muscle was stretched prior to fixation to expose large extents of actin and myosin filaments separately. Note the lightened, extracted image in the region of the Z-disks ( Z ) , and also in areas of extracellular collagen fibrils ( C ) , resulting from this treatment. Similar images are obtained after Pronase treatment. Actin filaments are not affected by the treatment, and while myosin appears to be extracted after longer exposure to Pronase, such is not apparent in this preparation. The lightened Z-disk areas do not display discernible substructure. E n bloc uranyl acetate stain. x 54,000. Fig. 14 Frog toe muscle in cross section after fixation and extraction techniques identical to those described for figure 13. Note the lucent, extracted expanses of 2-disk ( 2 ) and unaffected I-band actin filaments (A). Only faint remnants of any Z-disk lattice are visible (arrows). E n bloc uranyl acetate stain. x 54,000. SKELETAL MUSCLE 2-DISKS 637 638 DOUGLAS E. KELLY AND MARY ANN CAHILL Fig. 15 Schematic representation illustrating one possible configuration of filamentous and matrix components within the Z-disk area which would conform to the findings in this investigation. This model is based on the filamentous fine structure proposed by Knappeis and Carlsen ('64), but with matrix materials added in the vicinity of the Z-disk faces. For clarity, matrix substance has been excluded from the center of the Z-disk in order to portray more adequately the filamentous structure there. Matrix materials may occupy the center of the Z-disk along with filaments, but the present study discloses a predominant deposition in the Zdisk face regions. There, matrix material seems to be deposited in a squared array between the actin filament tips, thus conferring the small and large matrix lattices (see text for a more complete discussion). image of a fixed component is not a guaranteed representative of the living state until confirmatory evidence with other fixatives or techniques (e.g., freeze-cleaving of unifixed tissue) is obtained. The frequently observed image (obtained from longitudinal sections of glutaraldehyde-fixed frog toe and young newt muscle) which displays an interdigitation of densities extending from actin filaments (figs. 5, 6 ) is of particular interest. Other workers have interpreted the image as suggesting a relaxation of tension upon the Z-disk (or added reinforcement within it) with consequent overlap of actin tips within the Z-disk (Landon, '70, and personal communication; MacDonald and Engel, '71; Rowe, '71). That such might occur in the absence of the familiar osmium-induced contracture seems plaus- ible, but the suggestion that glutaraldehyde does not provoke tension generation at fixation and/or does induce Z-disk actin filament overlap seems still debatable, since the same image can be observed in the present observations of glutaraldehydefixed, stretched frog toe muscle. Alternatively, it might be proposed that the apparent actin overlap is not that at all, but rather an image generated by the matrix material. The image appears regularly in glutaraldehyde or osmium-fixed frog muscle (where matrix is abundant and never totally extracted), as well as in glutaraldehyde-fixed young newt muscle. Its absence in osmium-fixed newt muscle (fig. 7) correlates with the loss of matrix components seen in cross section - presumably through extraction. It seems reasonable to suggest that the overlapping density in the SKELETAL MUSCLE Z-DISKS 639 '.. Fie. 16 An oriented lateral view of the Z-disk model Dortraved in figure 13. illustratine the appearance of selected thin sections at various levels through the thickness of the Z-disi area. Combining the cross-section images a t the bottom of the figure will give some indication of the appearance of cross-sectioned 2-disks of different thicknesses or different planes of cut. This diagram assumes no interdigitation of actin filaments within the thickness of the Z-disk. image may not be that of an actin filament tip, but rather a lateral view of the shelflike deposition of matrix material which lies in the same plane as the actin filament tip and extends beyond it (see figs. 15, 16). The chemical composition of any Z-disk component is far from clear (Huxley, '63; Pepe, '66; more recently reviewed by Pepe, '68; Landon, ' 7 0 ) , and although the suspicion that a matrix component may be involved seems implied by the illustrations and conclusions of prior observers (see e.g., MacDonald and Engel, '71), most discussion and speculation has concerned the nature of Z-disk :&laments or proposed crystal lattices. For example, Stromer et al. ('69) utilized dithiothreitol to extract Z-disk density from glycerinated skeletal muscle fibers. Sarcomeres were not thereby dissociated, and when a particular fraction of the extraction medium was returned, Z-disk density reappeared. While these workers discounted the presence of a tropomyosin crystal lattice as the basis of Z-disk structure, their evidence would appear interpretable as suggesting the presence of a removable and replaceable matrix. They concluded that the material in question was likely proteinaceous (pos- 640 DOUGLAS E. KELLY AND MARY ANN CAHILL Fig. 17 A similar diagram to figure 14, with the exception that the actin filaments have been compressed into the Z-disk, thus collapsing all Z-disk components into closer proximity. A relatively thin cross section would therefore portray superimposition of all matrix and filamentous components. If the Z-disk were not compressed, a much thicker cross section would be required to portray the image at the bottom of this figure. Compare this crosssectional image with the dense regions in the upper right of figure 8 and left center of figure 3. sibly actinin, but not tropomyosin or troponin). Other techniques for extracting Z-disks from unfixed muscle do so with a greater incidence of sarcomeric detachment (e.g., the pepsin extractions of Leduc and Holt, '65; or the urea methods of Rash et al., '68, '70), and it may be that these affect more structure than the matrix phase. Our results from Pronase and pepsin digestion of Epon-embedded muscle sections must be regarded with great caution and due attention to the limitations of the control procedures which accompany the technique. Nearly all filamentous or membranous components of muscle cells can be visibly digested if treatment is prolonged (especially with Pronase). Moreover, in our hands, even the periodic acid oxidizing pretreatment applied alone as a control will extract some connective tissue and muscle components (including Z-disks) if exposure is lengthy. Therefore, use of the added enzyme(s) as a means to assess the content of specific proteins seems questionable at the moment. However, several aspects of such enzymic digestion seem relevant where Z-disks are . SKELETAL MUSCLE Z-DISKS concerned. Firstly, Z-disks are digested more rapidly than any other myofibrillar component, and most notable is their distinctly different behavior compared to nearby actin filaments. While actin of the I-band remains intact, there is no discernable filamentous remnant within the borders of the Z-disks. This fact adds some doubt to the concepts of actin interdigitation within the Z-disk or of the filamentous lattice itself being composed of actin. Secondly, from its earliest appearance, digestion seems to occur homogeneously across the entire thickness of a Z-disk. It therefore embraces rather accurately the regions proposed as being occupied by matrix and filaments, but at the same time gives no differential information about these subcomponents. Additional efforts along these lines would appear warranted. In the meantime, recognition of the likelihood of a matrix phase (which after some methods of fixation displays discrete morphological patterns) is useful in considering past and future interpretations of Z-disk structure. Matrix additions to the filamentous lattice could, for example, account for the ribbonlike filament appearance described in figure 2. Matrix might also provide the basis for Franzini-Armstrong and Porter’s (’64) interpretation that amphibian skeletal muscle Z-disks have a membranous rather than filamentous basic fine structure. The relative proportions of matrix to filaments in the Z-disks may be quite variable among the broad spectrum of muscle fiber types in vertebrate and invertebrate organisms. Hence, Z-disks might well be expected to display diverse patterns of organization which are specialized evolutionary products serving diverse functional requirements. The invertebrate Z-disks which Ashhurst (’67, ’71) and others have studied, for example, may well have evolved along a line in which the matrix phase has been emphasized and in which a filamentous lattice had less adaptive value. Perhaps the matrix phase is the more primitive form of attachment among the members of a contractile system. Recently Rowe (’71) has provided a meticulous study of rat skeletal muscle and proposed a new looping Z-disk filament model which matches remarkably 64 1 his high-resolution electron micrographs. This model accounts for the presence of the small, parallel lattice without invoking either rearrangement of filamentous structure (as proposed by Landon, ’70) or the presence of matrix material (as proposed here). It also accounts well for the “interdigitating actin tip” Z-disk image seen in longitudinal sections and discussed above. However, Rowe’s model will portray the large, angled lattice only in a cross section through the exact central region of a Z-disk in which considerable overlap of actin filaments has occurred (see his fig. QA). In view of the prevalence of large, angled lattice in the stretched fibers (in which minimal interdigitation would be expected) of the current study, Rowe’s interpretation seems still open to question. Furthermore, his Z-disk model would not portray the large, parallel lattice in cross secticlns unless an added actin dot is present in the center of each square (see his fig 8A,B) - a property not seen in the observations of large, parallel lattice reported here. This type of discrepancy illustrates that additional insight is required before Z-disk architecture and evolution is sufficiently understood. It is hoped that the concept of filamentous and matrix phases in the system will stimulate further productive consideration and experimentation. ACKNOWLEDGMENTS The authors gratefully acknowledge the principal support provided by NSF research grants (GB-14098 and GB-20277) to Dr. Kelly, as well as USPHS grants GM-136 and HE-2698 (for general support of electron microscope facilities at the University of Washington, where portions of the work were clone). Drs. Richard L. Wood and Gary Hendrix kindly provided critical review of the manuscript. The technical help of Mrs. Annalena Charla, Mr. Edward Miller, and Mrs. Cynthia Bomar, as well as the secretarial skills of Miss Judith Anderson, are also greatly appreciated. LITERATURE CITED Anderson, A., and J. Andre 1968 The extraction of some cell components with pronase and pepsin from thin sections of tissue embedded 642 DOUGLAS E. KELLY AND MARY ANN CAHILL in a n epon-araldite mixture. J. Microscopie, 7: 343-354. Ashhurst. D. E. 1967 2-line of the flight muscle of 'Belostomatid water bugs. J. Moiec. Biol. 27: 385-389. 1971 The Z-line in insect flight muscle. J. Molec. Biol., 55: 283-285. Bennett, H. S., and J. H. Luft 1959 s-Collidine as a basis for buffering fixatives. J. Biophys. Biochem. Cytol., 6: 113-114. Bone, Q., and E. J. Denton 1971 The osmotic effects of electron microscope fixatives. J. Cell Biol., 49: 571-581. Elliott, G. F., J. Lowy and B. M. Willman 1967 Low angle X-ray diffraction studies of living striated muscle during contraction. J. Molec. 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