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Immunological Techniques in Fluorescence and Electron Microscopy Applied to Skeletal Muscle Fibers

Frank A. Pepe

10.1002/cphy.cp100104

Source: Supplement 27: Handbook of Physiology, Skeletal Muscle

Originally published: 1983

Published online: January 2011

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Myofibrillar Proteins
1.1 A Band
1.2 M Band
1.3 I Band
1.4 Z Band
2 Nonmyofibrillar Proteins
Figure 1. Figure 1.

Sarcomere (repeat unit of striated myofibril) from muscle of the freshwater killifish (Fundulus diaphanus). a: Longitudinal section. Z, Z band; I, I band; A, A band; H, H band; bz, bare zone; M, M band, b: Cross section through A band in region of overlap of thin and thick filaments. Thick filaments have solid circular profiles. Some appear to have triangular profiles, c: Cross section through A band in region of H band (nonoverlap region). Only thick filaments are present. Thick filaments have solid circular profiles, d: Cross section through A band in region of bare zone immediately adjacent to M band. Thick filaments have solid triangular profiles, e: Cross section through A band in region of M band. Each thick filament is connected to each of its 6 neighboring thick filaments by M bridges. Some filaments are clearly hollow and have circular profiles, f. Cross section through I band. Only thin filaments are present. [From Pepe 125.]
Figure 2. Figure 2.

Negative‐stained natural myosin filaments. Note rough surface of filament where myosin cross bridges are present and smooth bare‐zone region (bz) in middle of filament where there are no myosin cross bridges.
Figure 3. Figure 3.

Computer processing of images of transverse sections of individual myosin filaments by rational averaging. Contour density maps of rotational averaging of 6 combined images are shown. Left, bold straight lines join centers of subfilaments. Right, same map without these lines. There are 12 subfilaments hexagonally packed with 3 located centrally and 9 located peripherally, forming a triangular profile with a subfilament missing at each apex. Diagram at bottom is model proposed by Pepe 120,122, in which the predicted arrangement and number of subfilaments are similar to those observed in the rotationally averaged images. [From Pepe 131.]
Figure 4. Figure 4.

Antibody specific for fast‐twitch and slow‐twitch myosins. A and B: specific antibody to fast‐twitch myosin (Ab‐F) gives a single precipitin line when immunodiffused against a mixture of fast‐twitch and slow‐twitch myosins (central well). This line fuses with 1 of the 2 lines obtained when a mixture of antibody to fast‐twitch myosin (Ab‐F) and slow‐twitch myosin (Ab‐S) is diffused against the mixture of the 2 myosins. The other line fuses with the single line obtained with specific antibody to slow‐twitch myosin (Ab‐S). Immunodiffusion patterns are shown in A and diagramed in B. C: frozen sections of chicken sartorius muscle stained with fluorescein‐labeled Ab‐S show that some cells contain slow‐twitch myosin (bright fluorescent cells), whereas other cells do not (unstained cells). D: serial section of same muscle fibers now stained with Ab‐F; the cells in C that contained slow myosin are unstained, whereas the cells in C that were unstained now stain brightly, indicating presence of fast myosin. Therefore in the sartorius muscle 2 types of muscle fibers are present: those containing fast‐twitch myosin and those containing slow‐twitch myosin.

A and B from Arndt and Pepe 2. Micrographs C and D courtesy of N. Rubinstein
Figure 5. Figure 5.

Fluorescent antimyosin staining of chicken fibrils. All staining was done in 25% glycerol containing 7.5 × 10−2 M KCl, 7.5 × 10−4 M MgCl2, 7.5 × 10−3 M phosphate buffer at pH 7.0. A: antimyosin contaminated with anti‐C protein. Staining of fibrils at 2.5 μm and 1.8 μm sarcomere lengths. B: antilight meromyosin staining of fibrils obtained by absorption of antimyosin with heavy meromyosin contaminated with C protein. C: anti‐heavy meromyosin staining of fibrils obtained by absorption of antimyosin with light meromyosin contaminated with C protein. D: anti‐heavy meromyosin and anti‐C‐protein staining of fibrils obtained by absorption of antimyosin with light meromyosin that was not contaminated with C protein. E: specific anti‐C protein isolated from antimyosin contaminated with anti‐C protein. Same pattern is observed with specific anti‐C protein isolated from IgG fraction of antiserum to C protein.
Figure 6. Figure 6.

A: antimyosin and anti‐C‐protein staining observed in electron microscopy. Antimyosin contaminated with anti‐C protein. The 7 dense bands in middle third of each half of the A band are due to C protein 19,108,127,135. C protein is present at intervals determined by myosin molecules to which it binds 13,147. B: anti‐C protein isolated by affinity purification from mixture of antimyosin and anti‐C protein used in A. Note the 7 dense bands due to C protein. Weak 8th band and strong 9th band are due to 2 other proteins (see refs. 19,135). C: antimyosin staining of A band. With heavy staining, normally less‐dense nonoverlap region becomes indistinguishable from the rest of the A band because of the large amount of antibody protein introduced. D: unstained sarcomere. Note clearly less‐dense nonoverlap region of the A band without heavy antimyosin staining.
Figure 7. Figure 7.

Immunodiffusion of antimyosin and anti‐C‐protein mixture against purified myosin, purified C protein, and myosin contaminated with C protein. Mixture of antimyosin and anti‐C protein is in center well (15 mg/ml IgG). The 2 wells at top right (not visible) contain C protein at 1 mg/ml. The 2 wells at bottom right contain column‐purified myosin at 5 mg/ml. The 2 wells at left contain myosin contaminated with C protein at 6 mg/ml. Note the 2 lines with myosin contaminated with C protein and the fusion of 1 of these with the line formed with pure myosin and fusion of the other with the line formed with pure C protein.
Figure 8. Figure 8.

Fluorescent antibody staining of myofibrils with polyclonal antibodies isolated by sequential affinity binding. A: specific antimyosin isolated by binding to column‐purified myosin coupled to agarose. A‐band‐staining patterns are shown for sarcomere lengths of 2.8 μm (upper) and 1.8 μm (lower). Far right, Coomassie blue‐stained SDS gel containing protein controls in unlabeled lane. Controls were bovine serum albumin (68,000 daltons) and soybean trypsin inhibitor (22,000 daltons). Second lane contains protein contaminants (I) removed from myosin preparations on column purification on DEAE‐Sephadex A‐50. Other lanes contain heavy meromyosin S2 fragment of myosin (S2), light meromyosin fragment (L), heavy meromyosin S1 fragment of myosin (S1), and myosin (M), respectively. Similar gels were transferred to nitrocellulose sheets 172, and bands were stained by immunoperoxidase techniques. This was done similarly for B through D where only immunoperoxidase‐stained sheets are shown. Specific antimyosin clearly does not react with protein controls or contaminants (I) removed by column purification. However, it does react with S2, L, S1, and myosin. Fluorescent antimyosin‐staining pattern is similar to that previously described for antimyosin contaiminated with anti‐C protein (Fig. 5A). B: fluorescent antibody staining of myofibrils with antibody for an antigenic region specific to S2. Upper sarcomere length, 2.0 μm. Staining is restricted primarily to a band near each A‐I junction. Lower sarcomere length, 2.6 μm. Staining in middle of A band corresponding to nonoverlap region of A band occurs in addition to staining near each A‐I junction. Antibody binds only to the S2 fragment on nitrocellulose sheet. Occasionally the myosin, because of its high chain weight, does not transfer to the nitrocellulose sheet properly, as apparently happened here. This transfer difficulty was not encountered with other proteins. C: fluorescent antibody staining of myofibrils with antibody for antigenic determinants on S1 and S2. Upper sarcomere length, 1.8 μm. Total A‐band staining with unstained gap in middle of A band corresponding to bare‐zone region of myosin filaments. Lower sarcomere length, 2.7 μm. Essentially uniform staining throughout A band with unstained gap in middle of A band corresponding to bare‐zone region of myosin filaments. There is a small increase in brightness corresponding to nonoverlap region of A band. This antibody gave a precipitin line only with S1 and myosin on immunodiffusion but clearly also shows a reaction with the S2 band on the nitrocellulose sheet. D: fluorescent antibody staining of myofibrils with antibody for an antigenic region specific to LMM. Upper sarcomere length, 1.7 μm; lower sarcomere length, 2.9 μm. In both, staining is restricted primarily in a single band near each A‐I junction. This antibody stained only the LMM fragment of myosin and myosin on the nitrocellulose sheet. A‐D: × 4,500.
Figure 9. Figure 9.

Models for structure of M band. A: model proposed by Knappeis and Carlsen 71. There are 6 M bridges attached to each myosin filament at each level in M band where an M line is observed. Only middle 3 levels contributing to middle 3 M lines are shown here. An M filament parallel to myosin filaments attaches perpendicular to M bridges between any 2 myosin filaments. B: model proposed by Luther and Squire 84 is similar to that proposed by Knappeis and Carlsen 71 except that there is a Y‐shaped element that attaches to each of 3 M filaments by 1 of 3 arms of Y as shown in diagram. This Y‐shaped element is positioned relative to the 1st and 3rd M‐line positions but a little closer to the center of the M band than the M lines themselves. C: model proposed by Pepe 128 is considerably different from the other 2 models in that there are no M filaments and M bridges at each level are all oriented similarly, but those at one level are oriented 60° relative to those at neighboring level. In transverse section 6 M bridges are observed on each filament, 1 bridging to each of neighboring filaments. In longitudinal section, depending on plane of section, M band consists of different patterns of M lines.
Figure 10. Figure 10.

M‐band patterns observed in longitudinal sections. A: longitudinal sections through M band. Long axis of fibrils is horizontal. The most clearly observed patterns of lines in the M band are shown. From left to right these consist of 3 centrally placed lines, 5 lines generally with central 3 more distinct, 4 lines, 2 lines 1 of which is centrally placed, and 2 distinct lines placed 1 on each side of less distinct (or absent) central line. Lateral muscle of freshwater killifish (Fundulus diaphanus). B: different M‐band patterns can be observed in sarcomeres of same myofibril. Note pattern of 3 lines in M band of sarcomere on left and pattern of 2 lines in M band of sarcomere on right. C: different patterns can also be observed in M bands of neighboring sarcomeres of different myofibrils in same cell. Note 2 neighboring lines are observed in sarcomere of myofibril at top, 1 of 2 lines being to left of central line. In neighboring sarcomere of myofibril on bottom, again 2 lines are observed in M band; however, 1 line is to right of central line of M band. These differences in pattern of lines in M band are what would be expected for M‐band model proposed by Pepe 128.
Figure 11. Figure 11.

Models for fine structure of thin filament. A: model proposed by Ebashi et al. 25. B: model amended by Ohtsuki 115 indicating position of joint between 2 adjacent tropomyosin molecules and bestowing helical symmetry on troponin molecules, so that 2 troponin molecules opposite each other are apart by a distance equal to diameter of actin molecule. C: model for fine location of troponin subunits proposed by Ohtsuki (I and C, troponin I and troponin C; T1 and T2, portions of troponin T corresponding to T1 and T2 fragments, respectively) in relation to tropomyosin (TM). (There remains a possibility that positions of troponin C and troponin I would be interchanged.) Z and H bands exist on far right and left sides, respectively. [From Ebashi 24.]
Figure 12. Figure 12.

Troponin binding in tropomyosin paracrystals. A: edge of tropomyosin paracrystal; paracrystal terminates at extreme edge of narrow dark band, × 121,000. B: broken portion of tropomyosin paracrystal. × 153,000. C: tropomyosin paracrystal; arrangement of tropomyosin molecules, represented as bars with arrowheads, shown schematically, × 160,000. D: troponin‐tropomyosin paracrystal; troponin is localized in middle of wide bright band (indicated by arrows). × 160,000. [From Ebashi et al. 29.]
Figure 13. Figure 13.

Schematic illustration of antibody distribution of troponin components along thin filament. [From Ohtsuki 117.]


Figure 1.

Sarcomere (repeat unit of striated myofibril) from muscle of the freshwater killifish (Fundulus diaphanus). a: Longitudinal section. Z, Z band; I, I band; A, A band; H, H band; bz, bare zone; M, M band, b: Cross section through A band in region of overlap of thin and thick filaments. Thick filaments have solid circular profiles. Some appear to have triangular profiles, c: Cross section through A band in region of H band (nonoverlap region). Only thick filaments are present. Thick filaments have solid circular profiles, d: Cross section through A band in region of bare zone immediately adjacent to M band. Thick filaments have solid triangular profiles, e: Cross section through A band in region of M band. Each thick filament is connected to each of its 6 neighboring thick filaments by M bridges. Some filaments are clearly hollow and have circular profiles, f. Cross section through I band. Only thin filaments are present. [From Pepe 125.]



Figure 2.

Negative‐stained natural myosin filaments. Note rough surface of filament where myosin cross bridges are present and smooth bare‐zone region (bz) in middle of filament where there are no myosin cross bridges.



Figure 3.

Computer processing of images of transverse sections of individual myosin filaments by rational averaging. Contour density maps of rotational averaging of 6 combined images are shown. Left, bold straight lines join centers of subfilaments. Right, same map without these lines. There are 12 subfilaments hexagonally packed with 3 located centrally and 9 located peripherally, forming a triangular profile with a subfilament missing at each apex. Diagram at bottom is model proposed by Pepe 120,122, in which the predicted arrangement and number of subfilaments are similar to those observed in the rotationally averaged images. [From Pepe 131.]



Figure 4.

Antibody specific for fast‐twitch and slow‐twitch myosins. A and B: specific antibody to fast‐twitch myosin (Ab‐F) gives a single precipitin line when immunodiffused against a mixture of fast‐twitch and slow‐twitch myosins (central well). This line fuses with 1 of the 2 lines obtained when a mixture of antibody to fast‐twitch myosin (Ab‐F) and slow‐twitch myosin (Ab‐S) is diffused against the mixture of the 2 myosins. The other line fuses with the single line obtained with specific antibody to slow‐twitch myosin (Ab‐S). Immunodiffusion patterns are shown in A and diagramed in B. C: frozen sections of chicken sartorius muscle stained with fluorescein‐labeled Ab‐S show that some cells contain slow‐twitch myosin (bright fluorescent cells), whereas other cells do not (unstained cells). D: serial section of same muscle fibers now stained with Ab‐F; the cells in C that contained slow myosin are unstained, whereas the cells in C that were unstained now stain brightly, indicating presence of fast myosin. Therefore in the sartorius muscle 2 types of muscle fibers are present: those containing fast‐twitch myosin and those containing slow‐twitch myosin.

A and B from Arndt and Pepe 2. Micrographs C and D courtesy of N. Rubinstein


Figure 5.

Fluorescent antimyosin staining of chicken fibrils. All staining was done in 25% glycerol containing 7.5 × 10−2 M KCl, 7.5 × 10−4 M MgCl2, 7.5 × 10−3 M phosphate buffer at pH 7.0. A: antimyosin contaminated with anti‐C protein. Staining of fibrils at 2.5 μm and 1.8 μm sarcomere lengths. B: antilight meromyosin staining of fibrils obtained by absorption of antimyosin with heavy meromyosin contaminated with C protein. C: anti‐heavy meromyosin staining of fibrils obtained by absorption of antimyosin with light meromyosin contaminated with C protein. D: anti‐heavy meromyosin and anti‐C‐protein staining of fibrils obtained by absorption of antimyosin with light meromyosin that was not contaminated with C protein. E: specific anti‐C protein isolated from antimyosin contaminated with anti‐C protein. Same pattern is observed with specific anti‐C protein isolated from IgG fraction of antiserum to C protein.



Figure 6.

A: antimyosin and anti‐C‐protein staining observed in electron microscopy. Antimyosin contaminated with anti‐C protein. The 7 dense bands in middle third of each half of the A band are due to C protein 19,108,127,135. C protein is present at intervals determined by myosin molecules to which it binds 13,147. B: anti‐C protein isolated by affinity purification from mixture of antimyosin and anti‐C protein used in A. Note the 7 dense bands due to C protein. Weak 8th band and strong 9th band are due to 2 other proteins (see refs. 19,135). C: antimyosin staining of A band. With heavy staining, normally less‐dense nonoverlap region becomes indistinguishable from the rest of the A band because of the large amount of antibody protein introduced. D: unstained sarcomere. Note clearly less‐dense nonoverlap region of the A band without heavy antimyosin staining.



Figure 7.

Immunodiffusion of antimyosin and anti‐C‐protein mixture against purified myosin, purified C protein, and myosin contaminated with C protein. Mixture of antimyosin and anti‐C protein is in center well (15 mg/ml IgG). The 2 wells at top right (not visible) contain C protein at 1 mg/ml. The 2 wells at bottom right contain column‐purified myosin at 5 mg/ml. The 2 wells at left contain myosin contaminated with C protein at 6 mg/ml. Note the 2 lines with myosin contaminated with C protein and the fusion of 1 of these with the line formed with pure myosin and fusion of the other with the line formed with pure C protein.



Figure 8.

Fluorescent antibody staining of myofibrils with polyclonal antibodies isolated by sequential affinity binding. A: specific antimyosin isolated by binding to column‐purified myosin coupled to agarose. A‐band‐staining patterns are shown for sarcomere lengths of 2.8 μm (upper) and 1.8 μm (lower). Far right, Coomassie blue‐stained SDS gel containing protein controls in unlabeled lane. Controls were bovine serum albumin (68,000 daltons) and soybean trypsin inhibitor (22,000 daltons). Second lane contains protein contaminants (I) removed from myosin preparations on column purification on DEAE‐Sephadex A‐50. Other lanes contain heavy meromyosin S2 fragment of myosin (S2), light meromyosin fragment (L), heavy meromyosin S1 fragment of myosin (S1), and myosin (M), respectively. Similar gels were transferred to nitrocellulose sheets 172, and bands were stained by immunoperoxidase techniques. This was done similarly for B through D where only immunoperoxidase‐stained sheets are shown. Specific antimyosin clearly does not react with protein controls or contaminants (I) removed by column purification. However, it does react with S2, L, S1, and myosin. Fluorescent antimyosin‐staining pattern is similar to that previously described for antimyosin contaiminated with anti‐C protein (Fig. 5A). B: fluorescent antibody staining of myofibrils with antibody for an antigenic region specific to S2. Upper sarcomere length, 2.0 μm. Staining is restricted primarily to a band near each A‐I junction. Lower sarcomere length, 2.6 μm. Staining in middle of A band corresponding to nonoverlap region of A band occurs in addition to staining near each A‐I junction. Antibody binds only to the S2 fragment on nitrocellulose sheet. Occasionally the myosin, because of its high chain weight, does not transfer to the nitrocellulose sheet properly, as apparently happened here. This transfer difficulty was not encountered with other proteins. C: fluorescent antibody staining of myofibrils with antibody for antigenic determinants on S1 and S2. Upper sarcomere length, 1.8 μm. Total A‐band staining with unstained gap in middle of A band corresponding to bare‐zone region of myosin filaments. Lower sarcomere length, 2.7 μm. Essentially uniform staining throughout A band with unstained gap in middle of A band corresponding to bare‐zone region of myosin filaments. There is a small increase in brightness corresponding to nonoverlap region of A band. This antibody gave a precipitin line only with S1 and myosin on immunodiffusion but clearly also shows a reaction with the S2 band on the nitrocellulose sheet. D: fluorescent antibody staining of myofibrils with antibody for an antigenic region specific to LMM. Upper sarcomere length, 1.7 μm; lower sarcomere length, 2.9 μm. In both, staining is restricted primarily in a single band near each A‐I junction. This antibody stained only the LMM fragment of myosin and myosin on the nitrocellulose sheet. A‐D: × 4,500.



Figure 9.

Models for structure of M band. A: model proposed by Knappeis and Carlsen 71. There are 6 M bridges attached to each myosin filament at each level in M band where an M line is observed. Only middle 3 levels contributing to middle 3 M lines are shown here. An M filament parallel to myosin filaments attaches perpendicular to M bridges between any 2 myosin filaments. B: model proposed by Luther and Squire 84 is similar to that proposed by Knappeis and Carlsen 71 except that there is a Y‐shaped element that attaches to each of 3 M filaments by 1 of 3 arms of Y as shown in diagram. This Y‐shaped element is positioned relative to the 1st and 3rd M‐line positions but a little closer to the center of the M band than the M lines themselves. C: model proposed by Pepe 128 is considerably different from the other 2 models in that there are no M filaments and M bridges at each level are all oriented similarly, but those at one level are oriented 60° relative to those at neighboring level. In transverse section 6 M bridges are observed on each filament, 1 bridging to each of neighboring filaments. In longitudinal section, depending on plane of section, M band consists of different patterns of M lines.



Figure 10.

M‐band patterns observed in longitudinal sections. A: longitudinal sections through M band. Long axis of fibrils is horizontal. The most clearly observed patterns of lines in the M band are shown. From left to right these consist of 3 centrally placed lines, 5 lines generally with central 3 more distinct, 4 lines, 2 lines 1 of which is centrally placed, and 2 distinct lines placed 1 on each side of less distinct (or absent) central line. Lateral muscle of freshwater killifish (Fundulus diaphanus). B: different M‐band patterns can be observed in sarcomeres of same myofibril. Note pattern of 3 lines in M band of sarcomere on left and pattern of 2 lines in M band of sarcomere on right. C: different patterns can also be observed in M bands of neighboring sarcomeres of different myofibrils in same cell. Note 2 neighboring lines are observed in sarcomere of myofibril at top, 1 of 2 lines being to left of central line. In neighboring sarcomere of myofibril on bottom, again 2 lines are observed in M band; however, 1 line is to right of central line of M band. These differences in pattern of lines in M band are what would be expected for M‐band model proposed by Pepe 128.



Figure 11.

Models for fine structure of thin filament. A: model proposed by Ebashi et al. 25. B: model amended by Ohtsuki 115 indicating position of joint between 2 adjacent tropomyosin molecules and bestowing helical symmetry on troponin molecules, so that 2 troponin molecules opposite each other are apart by a distance equal to diameter of actin molecule. C: model for fine location of troponin subunits proposed by Ohtsuki (I and C, troponin I and troponin C; T1 and T2, portions of troponin T corresponding to T1 and T2 fragments, respectively) in relation to tropomyosin (TM). (There remains a possibility that positions of troponin C and troponin I would be interchanged.) Z and H bands exist on far right and left sides, respectively. [From Ebashi 24.]



Figure 12.

Troponin binding in tropomyosin paracrystals. A: edge of tropomyosin paracrystal; paracrystal terminates at extreme edge of narrow dark band, × 121,000. B: broken portion of tropomyosin paracrystal. × 153,000. C: tropomyosin paracrystal; arrangement of tropomyosin molecules, represented as bars with arrowheads, shown schematically, × 160,000. D: troponin‐tropomyosin paracrystal; troponin is localized in middle of wide bright band (indicated by arrows). × 160,000. [From Ebashi et al. 29.]



Figure 13.

Schematic illustration of antibody distribution of troponin components along thin filament. [From Ohtsuki 117.]

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Article Title: Immunological Techniques in Fluorescence and Electron Microscopy Applied to Skeletal Muscle Fibers
 

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Frank A. Pepe. Immunological Techniques in Fluorescence and Electron Microscopy Applied to Skeletal Muscle Fibers. Compr Physiol 2011, Supplement 27: Handbook of Physiology, Skeletal Muscle: 113-141. First published in print 1983. doi: 10.1002/cphy.cp100104