The Insect Flight Muscle Sarcomere as a Model System for Immunolocalization

METHODS: A Companion to Methods in Enzymology 10, 219–233 (1996) Article No. 0097

The Insect Flight Muscle Sarcomere as a Model System for Immunolocalization Mary C. Reedy* and Belinda Bullard† *Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710; and †EMBL, Heidelberg, Germany

Highly ordered insect flight muscle provides an excellent system for coordinated immunolocalization of sarcomeric proteins at increasing levels of resolution, from fluorescent and goldtagged secondary antibodies to 3- and 5-nm gold directly coupled to Fab fragments. The penetration of antibody probes of various sizes into native and preserved muscle or tissue sections is compared. Factors affecting resolution and labeling efficiency are examined, such as probe size, and removal of uncoupled Fc and gold particles. The quality of preservation and the level of structural detail achieved with ice and plastic embedding media and different labeling methods are explored. We discuss problems encountered at the highest level of immunoelectron microscopy using gold/Fab to visualize the probe in relation to wellcontrasted, in situ myosin and troponin molecules in 25-nm-thin epoxy sections by transmission EM. q 1996 Academic Press, Inc.

The sarcomere of striated muscle represents both an opportunity and a test for immunolocalization because of the highly regular and compact arrangement of the numerous component proteins. Actin in striated muscle forms long filaments made up of helically arranged monomers. The filaments are complexed with regulatory strands of tropomyosin associated with troponin; these filaments containing actin, troponin, and tropomyosin are termed ‘‘thin filaments.’’ Arrays of oppositely polarized thin filaments are anchored in the Zbands, the structures that cross-link the ends of the thin filaments. Each thin filament–Z-band array (I-ZI segment) is interdigitated with a hexagonal array of bipolar myosin-containing thick filaments, whose midpoints or bare zones are aligned in the M-band. Numerous other proteins are complexed with thick or thin filaments that probably modify and regulate the structure and function of the myosin cross-bridges and actin in ways that have yet to be defined. The interdigitated thick and thin filament arrays bounded by Z-bands form the sarcomere (Fig. 1). Variations in this ordered

arrangement and in component proteins allow regulation of the force and velocities produced by the actomyosin motors, tailoring the form and function of muscles to suit such different tasks as lifting a 200-lb. weight or powering the wings of an insect at 200 strokes/s. In this chapter, we will focus on the methods we have used to immunolocalize some of the proteins of highly ordered indirect flight muscles (IFM) of the waterbug, Lethocerus, and the fruitfly, Drosophila. There are several differences in organization and physiology between IFM and vertebrate striated muscles. In all striated muscle thick filaments, the tails of the dimeric myosin molecules are packed into the thick filament shaft such that the two heads project from the thick filament along helical tracks. The helically arranged myosin heads form the cross-bridges that link thick and thin filaments. In contrast to the 43-nm helical repeat of vertebrate thick filaments, the helical repeat of myosin molecules in IFM thick filaments is 38.7 nm, which matches the helical repeat of actin subunits in the thin filament. The regulatory proteins, troponin and tropomyosin, have the same 38.7-nm periodicity in the thin filaments. This may be one reason that IFM gives the best view of actin, myosin cross-bridges, and troponin in vivo of any muscle (16, 17, 21, 22). Another reason for this clarity is the filament arrangement of IFM, compared to vertebrate striated muscle. In IFM, thin filaments occupy dyad positions between two thick filaments. Therefore, in sufficiently thin sections (25 nm), cross-bridges originating from those two thick filaments and the thin filaments to which they bind lie entirely within the section and can be seen in one view, with no other cross-bridges or filaments intruding into the section from layers above or below. This view occurs in 25-nm longitudinal sections of alternating myosin and actin filaments (myac layers in Fig. 1). In vertebrate striated muscle, thin filaments occupy trigonal positions where they receive myosin cross-bridges from three surrounding thick filaments that lie outside of the section plane. 219

1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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The most ordered state in IFM occurs in the absence of ATP (rigor), when the maximum number of myosin heads attach to actin. Rigor is induced by removing all muscle membranes with Triton X-100 and glycerol in a buffered salt solution that contains no ATP. Such glycerination allows direct access to the contractile machinery and control of the cross-bridges, which can be held in rigor, relaxed by MgATP, or exposed to numerous other experimental variations. For immunolocalization, glycerinated fibers are ideal because the intact contractile machinery in the muscle fibers is completely accessible to antibodies without further permeabilization. Rigor is studied because it is a stable, highly ordered state that produces and holds tension and presumably reflects important properties of the actomyosin motor. Rigor crossbridges in IFM form the characteristic double chevron motif composed of ‘‘lead’’ and ‘‘rear’’ cross-bridge pairs (seen in Fig. 1). The arrowhead appearance of the rigor double chevron motif is conferred by the angled lead chevron, the chevron closer to the M-line. Each lead bridge contains two myosin heads from one molecule binding to successive actin monomers along one actin strand. The less angled ‘‘rear’’ cross-bridge contains a single myosin head and appears most often as a bead over the thin filament, with only weak lateral bridging bars to the thick filament. The rear bridge may be single headed because the azimuth of its actin target is out of reach of the second head or attachment of the second head may be blocked by proximity to the large troponin complex of IFM, a subject we will address below. In IFM, thin longitudinal sections can also isolate layers of thin filaments only from the hexagonal lattice, which include the portions of the cross-bridges binding to actin, termed actin layers (Fig. 1). In contrast to the helical register of actin filaments in vertebrate muscle, IFM thin filaments are arranged in staggered rotational alignment; the repeat of adjacent filaments is staggered by 12.9 nm, thus dividing the 38.7-nm axial repeat into three 12.9-nm axial repeats. The cross-bridges are seen in actin layers as 39-nm periodic beads and ‘‘ladder rungs’’ connecting adjacent filaments. In 15-nm cross sections, the double chevrons form a ‘‘flared X’’ motif, in which the arms of the X are formed by the lead and rear cross-bridges binding to four of the six actin filaments surrounding the thick filament (Fig. 1). The unoccupied filaments flanking the flared

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X are the interdoublet gap between double chevrons. These thin cross sections display the entire 38.7-nm axial repeat as three separate 12.9-nm levels: lead and rear bridges and interdoublet gap. IFM contraction requires calcium binding to the thin filament regulatory proteins and to myosin in the thick filaments. In addition, IFM must be slightly stretched to exert full tension; that is, it is ‘‘stretch activated.’’ IFM thin filament regulation, as in vertebrate muscle, involves calcium binding to the troponin complex. The troponin complex in striated muscle is composed of TnC, the calcium-binding subunit; TnI, the inhibitory subunit, and TnT, the subunit that binds the complex to tropomyosin. Tropomyosin forms strands that lie in the helical grooves of the actin filament. IFM has a component in the troponin complex that is not found in vertebrate troponin. This extra component is termed heavy troponin (TnH) (or heavy tropomyosin) (3). Drosophila TnH is a natural fusion protein between tropomyosin and a proline-rich carboxy-terminal extension of Ç230 residues (11). TnH colocalizes with the troponin complex every 38.7 nm on the thin filament and antibody labeling shows that there is an epitope very close to the rear chevron in rigor IFM. TnH has been proposed to act as a ‘‘stretch sensor’’ linking the troponin/tropomyosin complex to the cross-bridges, thereby playing a role in stretch activation (15). IFM oscillates rapidly at nearly full overlap of thick and thin filaments and does not show the I-bands characteristic of vertebrate muscle (Fig. 1A). This creates a general problem, in that there is virtually no I-band in which to view thin filaments free of cross-bridges. This represented a significant problem for detailed immunolocalization of thin filament proteins because crowding in the A-band, which contains both thick and thin filaments, limits accessibility of antibody and the staggered alignment of adjacent thin filaments requires an immuno-probe that gives a resolution better than 12.9 nm to resolve closely packed epitopes. This problem has been partly solved by artifically stretching glycerinated, ATP-relaxed IFM fibers to create I-bands, pulling portions of the thin filaments clear of thick filaments and cross-bridges, as seen in Figs. 1B and 1C, in which rigor has been induced by washing away the ATP following the stretch. Studies of IFM with partially overlapped filaments allowed the contribution of cross-bridges and troponin to the 38.7-nm periodic

FIG. 1. Gallery of EM images showing ultrathin section orientations of Lethocerus IFM. (A) Low magnification of an unstretched myofibril flanked by mitochondria (m) showing a whole sarcomere of a myac layer, bounded by Z-lines (Z), displaying A-bands (A) and the M-line (M). Note that these intact, resting length sarcomeres do not have I-bands. Near the top, a half-sarcomere of an actin layer is seen, with the M-line at the top. At the bottom a half-sarcomere of another myac layer is seen. Parts of several large mitochondria can be seen flanking the myofibril. (B) A higher magnification of a half-sarcomere of an actin layer from a partially stretched fiber. (C) A high-magnification view of an actin layer. (D) A myac layer from a partially stretched fiber. (E) High-magnification view of a myac layer showing double chevrons. (F) A 15-nm cross section showing flared Xes.

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beads on the thin filaments to be resolved separately. These studies revealed that, in contrast to the lateral register of troponin in vertebrate muscle, troponin also has a staggered arrangement in IFM, following the staggered rotational alignment of the actin filaments. Cross-bridges follow this same lattice in binding to actin. This means that cross-bridges and troponin in IFM are arranged on congruent lattices, making it likely that some components of the troponin complex could interact with cross-bridges in IFM. It then became an important question as to where cross-bridges are located in relation to troponin, and specifically, the relative locations of cross-bridges, TnH, and TnT (the larger components of the troponin complex). The crystalline regularity of IFM offers the opportunity to map with high precision the relative locations of the molecules that interact and/or regulate contraction and stretch activation, if sufficiently high-resolution immunoEM localization can be obtained.

to label the primary antibody; other classes of rat antibody react weakly with protein A.

IMMUNOFLUORESCENCE Antibodies are then tested by immunofluorescence on isolated myofibrils. This step can give the overall

IMMUNOLOCALIZATION OF IFM PROTEINS Immunolocalization techniques confront conflicting requirements for speed and simplicity versus resolution and structural preservation. To balance these requirements, we pursue a multistep approach that attempts to maximize the advantages presented by IFM and retain the simplicity and speed of previously developed approaches, such as immunofluorescence. The issues we will discuss in this section are: how much should structures be fixed for stabilization, when to fix structures for stabilization, the race between good structure and reduced antigenicity, resolution vs signal intensity, and antibody access (the penetration problem). The first step in immunolocalization is to obtain specific, high-affinity antibodies. Our search for antibodies was motivated by the need to identify the components of features in the IFM sarcomere, such as the location and composition of the large troponin complex relative to cross-bridges; i.e., is troponin present in the beads in actin layers and at the rear crossbridge in the double chevron? To help answer these questions, Geoff Butcher and Belinda Bullard have raised a series of rat monoclonal antibodies against components of Lethocerus IFM proteins. Many of these antibodies, when tested in Western blots, recognized familiar proteins in the troponin complex or other identified bands in gels of IFM proteins. The next step is to determine if the class of the antibodies of interest is IgG, IgM, or one of the subclasses of IgG. The large size of IgM antibodies, which are composed of five Ig subunits, can present penetration problems and they are not appropriate when high resolution is needed. If the antibodies are IgG2c , protein A–gold can be used

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FIG. 2. Fluorescent micrographs showing the exposure of antigen in isolated myofibrils. (A) Myofibril labeled by soaking in anti-TnT (IgG), followed by fluorescent second antibody. (B) Myofibril labeled with anti-p200 (IgM) and second antibody. Confocal micrographs were taken of sections 0.4 mm thick through the near side (first panel), the middle (second panel), and the far side (third panel) of a myofibril; the bottom panel in B is a composite cross-sectional image of 10 longitudinal sections through a single bright region (A-band) and shows that the antibody is confined to the periphery of the myofibril. (C) A frozen section of myofibrils labeled with anti-p200 and second antibody, showing labeling in the A-band. The anti-TnT IgG penetrated the intact myofibril better than the larger anti-p200 IgM. The true distibution of p200 is shown in frozen sections, such as these, where the IgM has access to the A-band. Arrows indicate the Z-line. Scale bar, 3 mm.

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distribution of the epitope recognized by the antibody and some indication of the affinity of the antibody and the sensitivity of the antigen to the level of fixation. For example, in testing 14 monoclonal antibodies that reacted against components of the troponin complex in Western blots, 10 gave intense fluorescent staining of myofibrils. However, of the 14, only 4 gave strong enough labeling to use for EM localization. The myofibrils used in the immunofluorescent assay are usually not fixed before exposure to primary and secondary antibodies. However, after mild fixation of the myofibrils, most antibodies show good fluorescent staining. All of the anti-troponin antibodies gave fluorescence throughout the A-band of the sarcomeres, except in the central M- and H-band regions, consistent with a distribution along the length of the thin filaments. Further experience with other antibodies raised an important and interesting issue, as to whether antibodies penetrate evenly into the myofibril. Figure 2A shows an immunofluorescence image obtained with a conventional fluorescence microscope of a myofibril soaked in anti-TnT antibody, showing uniform brightness in the A-bands (except in the central M-band). Figure 2B shows a myofibril reacted with an IgM antibody to a 200-kDa cytoskeletal protein (p200), viewed in a confocal fluorescent microscope. At the surface of the myofibril, the overall fluorescence in the A-bands appears even and dim, suggesting either that the antibody does not bind well or that very few epitopes and

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protein molecules are present. However, in a synthetic cross section generated by combining multiple optical sections though the myofibril, the fluorescence appears bright around the edge and absent in the middle. This suggests a third possibility, that the epitope recognized by the antibody is distributed around the outside of the myofibril and sarcomeres. However, soaking with antibody for a long period (8–10 h), instead of the usual 15–30 min, produces uniform bright fluorescence throughout the myofibril. This means that the antibody does not penetrate quickly throughout the myofibril, even though the sarcolemma and mitochondria have been completely removed by glycerination and it is no longer packed into a fiber with hundreds of other myofibrils. Such penetration problems can be avoided by staining sections of myofibrils with antibodies. Interior sites can be exposed to antibodies by freezing fibers or myofibrils, thereby providing a support firm enough to obtain thin sections. The exposed interior sites can then be labeled with antibodies. Comparison of the labeling patterns of the fluorescent tagged IgM antibody in soaked myofibrils with the labeling on frozen sections confirms that the ring-like staining is due to penetration of only the outermost regions of the myofibril (Fig. 2C). The epitope is actually distributed throughout the myofibril along most of the A-bands in the frozen section. This is also true of other antibodies; epitope staining apparently limited to the periphery may be due to lack of antibody penetration. On the other hand, strong labeling of

FIG. 3. Localization of antigen in frozen sections. Sections were labeled with (A) anti-TnH and (B) anti-zeelin 2, and then with a bridging antibody and protein A–gold (9 nm). The lack of background labeling enables the distribution of the two antigens across the sarcomere to be distinguished (anti-TnH labels a wider band than anti-zeelin 2). However, no periodicity is seen in the gold label on these sections. Z, Z-band; H, H-band. Scale bar, 500 mm.

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peripheral structures can give the appearance of uniform epitope may be localized, thereby reducing the resolution. fluorescent staining when viewed in a conventional fluo- In general, as probe size increases, spatial resolution is rescence microscope. reduced. Frozen sections are very useful in retaining antigenicity and exposing interior sites. Moreover, using ice as the embedding medium does allow very thin frozen CRYOIMMUNOEM sections to be obtained, thereby permitting high-resolution immunoEM. Sections of only a single layer of filaFrozen sections are also used to localize antigens at ments can be obtained, which are similar to those dethe EM level, in which case the antibodies are tagged scribed below for Lowicryl-embedded fibers in Figs. 5 directly or indirectly with gold particles, which are visi- and 6. However, frozen sections have the disadvantage ble in the EM. Figure 3 shows frozen sections of lightly that structural detail is limited and great skill is fixed IFM fibers labeled with anti-TnH or with anti- needed to obtain thin sections. zeelin, tagged with protein A–gold, showing the distribution of the epitopes along the A-band. One advantage of freezing the tissue to obtain sections is that this IMMUNOEM ON LOWICRYL-EMBEDDED method requires little or no fixation. This means that SECTIONS one can obtain access to the interior sites without having to cross-link the tissue, thereby retaining antigenicLowicryl is an embedding medium that polymerizes ity. Some antigens, such as anti-a actinin and spectrin, are very sensitive to fixation and will not bind antibod- at low temperature so that some antigenicity of the ies if the tissue is fixed. (The opposite is also true, tissue is preserved. Structural preservation is better however; some antigens do not bind antibodies unless than in frozen sections. Lowicryl allows very thin secthe tissue has been treated with acetone or methanol, tions to be obtained more easily than with frozen tissue but the antigenicity of most epitopes is reduced in Lowapparently because this exposes the epitope.) A disadvantage of frozen sections is that they must be icryl sections, due to the relatively greater cross-linking thawed before antibody staining. With little or no fixation, and dehydration required with the use of this embedthe fine structure of frozen tissue does not survive the ding medium compared to freezing. Figure 5 shows an ideal Lowicryl section of Lethocerus thawing and even with fixation and contrasting after antibody staining, preservation of fine structural details, such IFM labeled with anti-TnH. This image was obtained by as cross-bridges, is poor relative to conventional methods. lightly fixing stretched fibers and labeling thin sections (This can be appreciated by comparing the fine structure of sarcomeres in Fig. 3 with those in Fig. 5 in Lowicryl sections and those in Fig. 11 in Araldite sections.) When an antigen must be localized as precisely as possible, frozen sections are labeled with primary antibody or Fab fragment directly complexed with a gold particle. However, the most commonly used method of labeling the antibody with a gold probe involves the use of primary antibody, followed by a secondary antibody complexed with gold or by protein A–gold. In the case of relatively rare epitopes or those concentrated in a particular region, enhancement of the signal from an epitope can be performed on frozen sections, using the sandwich technique. The number of gold particles that label one epitope can be increased by labeling with primary antibody, followed by a bridging antibody and then protein A–gold. The probe is relatively large but there is no penetration problem on FIG. 4. Comparison of probe sizes. Scale diagram of probes used frozen sections. Figure 4A shows that the maximum ra- in immunolabeling. (A) Primary antibody (IgG), bridging antibody, dius of the probe with 9-nm gold is about 30 nm (the and protein A–gold (9 nm). (B) Primary antibody (IgG) and 9-nm distance between the antibody-combining site and the cen- gold. (C) Fab and 3-nm gold. (D) Nanoprobe with Fab covalently ter of the gold particle). The sandwich technique is particu- bound to 1.4-nm gold; the gold particle is surrounded by an organic shell, giving the particle a total diameter of 2.7 nm. Maximum radii lary useful in labeling epitopes in dense structures such (distance from the center of the gold particle to the antibody-combinas Z-discs. However, the amplification of signal obtained ing site) of the probes are given; in practice the radii may be smaller by using multiple probes expands the area in which the (e.g., the radius of Fab and 5-nm gold is estimated to be 4.5 nm).

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with primary antibody followed by a bridging antibody and protein A–gold. The TnH epitope is distributed all along the thin filaments, throughout the I-band and in the short region of thin and thick filament overlap. In some places there is a periodicity in the gold label of Ç40 nm, which corresponds to the spacing of troponin on the thin filaments; this could not be seen in frozen sections. Figure 6A shows a Lowicryl section so thin that it includes only a single layer of filaments. This is an oblique section in which the section plane shifts from a myac layer containing thick and thin filaments, through a split myosin layer that cuts through the thick filaments, to an actin layer, containing thin filaments only. The shifting

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of the section plane gives a moire ´ appearance to the image. The anti-paramyosin antibody probe illustrated here recognizes a protein in the center of the thick filament. Where the sectioning has scraped away the thick filament surface, the epitope is exposed. The gold probe is confined exclusively to the transitional split myosin layers, leaving the actin and myac layers unlabeled. A similar effect is seen with antibody to zeelin 2, a protein associated with the thick filament (Fig. 6B). Lowicryl embedding has the disadvantages that optimal fixation cannot be used if antigenicity is to be preserved for section labeling, thereby lowering structural preservation. Compared with Araldite embedding, the

FIG. 5. Labeling on sections of a Lowicryl-embedded fiber. The fiber was stretched to pull thin filaments free of thick filaments. The section was labeled with anti-TnH, bridging antibody, and protein A gold (9 nm). The label is on the thin filaments and extends into the overlap region. Filaments are better preserved than in frozen sections and in some places there are short stretches of periodic labeling (arrows). I, I-band; A, A-band; Z, Z-band. Scale bar, 500 mm.

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embedding procedure is time consuming and extra skill is necessary to obtain thin Lowicryl sections.

PREEMBEDDING LABELING FOR IMMUNOEM IFM offers an advantage that releases us from the restrictions involved in labeling sections with antibod-

ies. Since the sarcomere in a fiber demembranated by glycerination is a stable structure accessible to antibodies, we can soak unfixed, glycerinated fibers directly in antibody and fix and contrast only after the immunoprobe has bound. This allows unaltered antigenicity of the epitopes during labeling, followed by cross-linking and contrasting during fixation. Araldite embedding

FIG. 6. Labeling on thin oblique sections. Sections of fibers embedded in Lowicryl were labeled with (A) anti-paramyosin and (B) antizeelin 2, then with bridging antibody and protein A–gold (9 nm). The sections cut through myosin layers and actin layers and at the edge of the myosin layers (split myosin regions), the center of the thick filaments is exposed. Paramyosin in the core of the thick filament is labeled in the split myosin region. Zeelin 2 is also in the split myosin region but extends further into the actin layer than paramyosin. (C) Diagram explaining the labeling patterns in oblique sections. The top diagram shows an oblique section through thick and thin filaments labeled with anti-paramyosin and anti-zeelin 2; the bottom diagram shows the section seen from the top. Split myosins are labeled on one side of the section only. Z, Z-band; Sm, split myosin. Scale bar, 500 mm.

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after presoaking in antibody gives the best resolution of antibody label and best preservation of structure (15) and has the advantage that obtaining 25-nm sections is easy. The extensive cross-linking and dehydration required for Araldite embedding drastically reduce the antigenicity of most epitopes in Araldite sections. Therefore, antibody labeling of fibers is completed by presoaking in antibody before they are cross-linked and contrasted with a fixation protocol, using glutaraldehyde, tannic acid, osmium, and uranyl acetate. This procedure has been X-ray monitored to document the fidelity of muscle preservation. Well-preserved, labeled fibers are then embedded in Araldite. From 25-nm sections, thick and thin filaments and cross-bridges can be clearly seen in relation to the antibody probe. High-resolution immunoEM of IFM in Araldite requires a probe small enough both to penetrate the close quarters of the overlap zone of the sarcomere and to localize with high definition within a small radius, yet large and dense enough to detect at a magnification of Ç18,0001 by EM. Many epitopes in the sarcomere are numerous and arranged on a regular lattice, making it desirable to use the smallest probe with the finest resolution. We compared the penetration and labeling efficiency of IgG, Fab, and gold/Fab into unfixed fibers stretched to create an I-band (Fig. 7). Although IgG will penetrate through the myofibrils and sarcomeres in an overnight soak, IgG cross-links adjacent thin filaments and obscures periodicity finer than 38.7 nm. Because the 12.9-nm stagger in rotational alignment between thin filaments effectively divides into thirds the 38.7-nm repeat, relevant features within the 38-nm repeat are only separated by 12.9 nm from the nearest neighbor. This means that the immunoprobe must have a radius

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of less than 12.9 nm (see Fig. 4), so that image features in the sarcomere lattice separated by less than 12.9 nm may be distinguished. The small Fab fragment (Mr 50 kDa) has a maximum diameter of Ç5 nm and penetrates well, but adds only the density equivalent to about one actin monomer and is difficult to detect even when it labels a periodic feature with high efficiency (Fig. 7B). Fab directly coupled to 3- or 5-nm gold (see methods) is easily recognized as a small, well-defined dense area that labels with high efficiency. This is dramatically illustrated in the I-band shown in Fig. 7C, where the dense gold beads label most of the thin filaments, revealing the Ç39-nm period. Figure 8 shows 15-nm cross sections of presoaked, Araldite embedded fibers, comparing rigor flared Xes to those labeled by IgG or Fab. From cross sections, it is clear that IgG cross-links adjacent filaments and obscures the rigor flared X pattern (Fig. 8A). However, anti-TnT or anti-TnH Fab only subtly enhance the density of the 12.9 levels corresponding to lead crossbridge, rear cross-bridge, or interdoublet gap in the flared X, even when optical filtering is used to reduce noise in the images. The tentative conclusion reached from the analysis of many such images was that both anti-TnT and anti-TnH Fab labelled epitopes close to the rear chevron. However, plain Fab labeling is too subtle to be used routinely and a gold-labeled probe is required. The relative positions of the components of the troponin complex were determined by using two different size gold particles directly coupled to anti-TnH and anti-TnT Fab to label thin filaments in fibers that were stretched to create I-bands. Two epitopes can be resolved if they are further apart than twice the probe

FIG. 7. Araldite embedding of IFM showing I-bands of stretched fibers comparing penetration and labeling by presoaking in anti-TnH (A) IgG, (B) Fab, or (C) gold/Fab. Z-band is at the top.

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radius. First, the possible resolution and efficiency of binding of the chosen 3- and 5-nm gold/Fab probes had to be determined. Thin filaments isolated from IFM provide a useful model for estimating the labeling efficiency of Fab coupled to different sizes of gold particles (14). Quantita-

tive comparison of labeling efficiency of different sizes of gold give values for 3-nm gold/Fab of 90%, for 5-nm gold/Fab of 70%, and for 10-nm gold/Fab of 30%. The efficiency of labeling by 5-nm gold is 20% less than that of 3-nm gold. One should be mindful of this point when estimating the relative amounts of one epitope versus

FIG. 8. High-magnification views of 15-nm flared X cross sections of presoaked, Araldite-embedded fibers, comparing the labeling of IgG (A), to a rigor control (B), and to Fab-labeling (C and D). The Fab labeling by anti-TnH (C) and anti-TnT (D) are compared in relation to 12.9-nm levels corresponding to lead (L) and rear (R) cross-bridges and interdoublet gap (i). Individual flared Xes are highlighted by pentagons in (B) and (D) and a square in (C). Optically filtered images of rigor (bi), TnH (ci), and TnT (di) labeled flared Xes are shown in the insets and the level in the flared X to which density is subtly increased by Fab is depicted in inset diagrams. In (A) the flared Xes are viewed looking toward the M-band, while in (B–D), they are viewed looking toward the Z-line. The labeling by the large IgG tends to obscure the structure of the flared Xes and appears random. On the other hand, the small amount of mass added by Fab (equal to approximately one actin monomer) is almost undetectable when added to the substantial mass of a cross-bridge arm of the flared X (which includes three actin monomers and one or two myosin heads in the depth of the 15-nm section). Analysis of many similar images indicated that anti-TnH labels at the rear chevron level and anti-TnT labels very close by but slightly Z-ward of the rear cross-bridge, often enhancing density in the interdoublet gap in cross sections. In longitudinal sections (Fig. 11), Fab or gold/Fab appears to label close to the rear chevron, but the division of the 39-nm repeat into three 12.9-nm levels in the flared X suggests that TnH is closer to the rear cross-bridge than TnT.

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another based on the relative number of different size gold particles. The minimum separation distance between epitopes can be determined by statistical analysis of the spacing between labels on thin filaments even if all sites are not completely labeled by gold/Fab or where the labeling appears random (14). Analysis of the interparticle distances on isolated thin filaments labeled with anti-TnH gold/Fab (Fig. 9) gives a value for the minimum separation of successive TnH epitopes along one thin filament of Ç40 nm, in close agreement with the expected 38.7nm spacing of the epitopes. The measured 40-nm separation is the same for 3-, 5-, and 10-nm gold probe sizes, although labeling efficiency varies, as noted above. The actual size of the gold/Fab probe is important for detecting smaller spacings, such as the separation between TnT and TnH epitopes in the same troponin complex. The smallest gold particle that can be easily seen by transmission EM in stained sections of muscle is Ç3 nm. Figure 4 shows a 3-nm gold/Fab probe as it is commonly depicted, with a radius of about 6.5 nm. Baschong and Wrigley (1) estimate that the maximum radius is 8 nm). However, negatively stained images of colloidal gold complexed with Fab show a protein shell of 2 nm thick that is independent of the size of gold particle (Fig. 9) (14). Our measurements gave a radius of the 3-nm gold/Fab probe as 3.5 nm, and the radius of a 5-nm gold/Fab as 4.5 nm. These measurements are about half that expected if the gold binds to the end of the Fab furthest from the antibody-combining site (1) (Fig. 4). The gold particle may bind to the side of the Fab, bringing the antibody-combining site closer to the gold particle than predicted. When thin filaments are labeled with anti-TnH 5-nm gold/Fab, the gold particle falls anywhere within a sphere of radius 4.5 nm around the epitope. The error in measurements of TnH periodicity on isolated thin filaments (standard deviation 4.2 nm) is consistent with the measured probe radius of 4.5 nm. It therefore appeared that 3- and 5-nm gold/ Fab probes could give sufficient resolution to resolve epitopes in TnT and TnH within the same 39-nm repeat. Using the technique of double labeling thin filaments in the I-bands of stretched IFM with anti-TnT 3-nm

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gold/Fab and anti-TnH 5-nm gold/Fab (Fig. 10), we showed that the 5- and 3-nm probes excluded one another from the same 39-nm repeat, very seldom binding in close succession. The 3- and 5-nm probes bound alternately in successive 39-nm repeats, in ‘‘runs’’ of either 3- or 5-nm particles along a filament, or at the same level on several adjacent filaments. The mutual exclusion of the 3- and 5-nm gold probes suggests that the epitopes are close together; the radii of the probes are 3.5- and 4.5-nm, respectively. Therefore, the epitopes are likely to be less than 8 nm apart. The penetration of gold probes into the A-band remains problematic, as illustrated in Fig. 11. The size of the probe is not the main factor preventing penetration since large molecules, such as IgG and myosin subfragment-1, will penetrate the A-band. The I-band in stretched fibers is completely penetrated by the 5- and 3-nm gold/Fab probes, but neither gold/Fab will go far into the thick and thin filament overlap region. Gold/ Fab with one 3-nm gold particle per Fab measures Ç7 nm in diameter, while the space between thick and thin filaments is 16 nm, so there seems to be ample room for the probe. However, the gold/Fab probe behaves as if it had a much larger diameter in the close confines of the overlap zone. Colloidal gold is charged and it appears likely that residual charge on the surface of colloidal gold is not effectively neutralized by a coat of protein and water, so charge interactions hamper the penetration of the gold probe into the overlap zone. Recently, small gold clusters have been covalently attached to antibodies for use in immunolabeling (7– 10). The largest gold cluster commercially available has a gold core of 1.4 nm, surrounded by an organic shell, giving an overall diameter of 2.7 nm. This Nanogold can be covalently linked to a single Fab molecule, giving a probe with a maximum radius of 6.3 nm, which is comparable to 5-nm colloidal gold/Fab probe (Fig. 4). Therefore the two probes should give the same resolution of the antigen. Moreover, a nanogold probe is not charged like a colloidal gold probe and penetrates tissues better (20). It might be expected that nanogold probes would penetrate the overlap region of the myofibril. The serious disadvantage of the 1.4-nm Nanogold

FIG. 9. Periodic labeling on isolated thin filaments. (A) Negatively stained thin filament labeled with anti-TnH 10-nm gold/Fab. The minimum spacing between gold particles is 40 nm and analysis of many filaments shows that the most frequent spacing is the minimum spacing. About 30% of the troponin sites are labeled by 10-nm gold/Fab. Scale bar, 100 nm. (B) Negatively stained image of a 10-nm colloidal gold particle complexed with Fab. The protein shell is about 2 nm thick. Scale bar, 20 nm.

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probe is that it is too small to be visible by transmission EM (TEM). Silver enhancement has been used to enlarge the gold particles after binding and, indeed, shows that the Nanogold probe penetrates the overlap region. However, in our hands, the silver reacts with contrasting agents, such as osmium, to produce diffuse density that obscures details. This may happen because of the heavy labeling of the epitopes and resulting high concentration of gold particles in the sarcomere. If the contrasting and fixative components are eliminated, the structural preservation is poor. Nanogold probes are visible by scanning transmission EM (STEM) in specimens lacking other contrasting agents, but very little of the fine structure of the sarcomeres is visible under these conditions with STEM. A covalent gold

probe with a gold cluster of about 3 nm that can be seen by TEM is needed.

IMMUNOLABELING TECHNIQUES Fluorescence In the immunofluorescent assay, intact isolated myofibrils are examined using epifluorescent optics with a Zeiss microscope. Isolated myofibrils are prepared by homogenizing and washing glycerinated muscle and adhering the isolated myofibrils to a glass slide. Myofibrils may be reacted with antibody without fixation or fixed with 2.5–6% formaldehyde or up to 1% glutaral-

FIG. 10. High-magnification view of I-bands from highly stretched IFM fibers, presoaked in antibody and embedded in Araldite, showing (A) single labeled anti-TnT 3-nm gold/Fab (double arrows) and (B) double-labeled 3-nm anti-TnT gold/Fab (smaller arrow) and 5-nm antiTnH gold/Fab (larger arrow).

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FIG. 11. Illustration of the problem of gold/Fab penetration into A-band of sarcomeres even after 10 h of presoaking in antibody probes. (A) Araldite embedded rigor myac layer with double chevron highlighted by a box. (B) Anti-TnT Fab labeled fiber with double chevron highlighted by a box. Fab adds only some diffuse density to the double chevron. (C) In some areas where gold/Fab piles up in the I-band and M-line (open arrows), gold/Fab penetrates the outermost thin filaments in the overlap zone, but gold labeling appears less periodic than in areas where gold particles in the I-band clearly show a 39-nm repeat, as in (D). Small solid arrow points to gold particle. (D) Anti-TnH gold/Fab labels the

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dehyde before reacting with antibody. The degree of fixation depends on the antigen. Fixed or unfixed myofibrils are rinsed well, blocked, soaked in primary antibody for 30 min, and then rinsed and soaked in secondary fluorescent-tagged antibody for 30 min. For initial tests of the antibody, a hybridoma cell supernatant is used if the antibody is monoclonal. Unfixed myofibrils are preferable because of their native antigenicity. However, the binding of some antibodies can lead to solubilization and loss of the target protein in unfixed myofibrils, so if tissue is soaked for longer periods in antibody, fixation may be necessary to hold the target protein in place. Note also that penetration is slower if the myofibrils are fixed. The position of fluorescent label in the sarcomere is determined by phase-contrast microscopy, which is used to identify Z-discs and A-bands. The relative intensity of fluorescence in different parts of the sarcomere is usually due to differences in exposure of the antigen, not to relative amounts of the antigen. This can be checked by extracting some of the proteins from the myofibril to see if there is any change in the labeling pattern. The contraction state of the myofibrils can change the apparent distribution of the antigen due to change in availability of antigen. Antigen has been observed to migrate in unfixed glycerinated muscle after 4 weeks storage at 0807C. The resolution achieved with fluorescence microscopy is about 80 nm, which is the width of the Z-disc in Lethocerus IFM. The resolution is not dependent on the size of the probe, because the probe is well below 80 nm in diameter. The extent of antibody penetration can be measured using a confocal microscope to look at fluorescence at different levels throughout the myofibril. Typically fluorescence from a series of sections 0.4 mm thick is recorded and converted into a synthetic cross section, as in Fig. 2. Synthetic cross sections can be used to test time and conditions needed for complete penetration of antibody. Cryo-sections of myofibrils give the best exposure of antigens. A pellet of myofibrils is obtained by centrifuging washed, glycerinated myofibrils. The pellet is fixed and embedded as for cryo-EM (see below) and 1-mm thick sections are cut. Sections are labeled with primary antibody and secondary fluorescent-tagged antibody and examined in a fluorescence microscope. Fixation, which reduces antigenicity, is necessary because

thin filaments in the I-band (lower solid arrow) but only penetrates a short way into the A-band (broken line level with rear chevrons). (E) Anti-TnT gold/Fab also shows gold probe in the I-band (lower solid arrow), but only the first two chevrons into the A-band are labeled by gold (the four rear chevrons to the left of upper arrow).

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sections of pellets otherwise fall apart. The method involves more effort than labeling intact myofibrils and resolution is no better, but antigens are uniformly exposed on the surface of the sections and the same blocks can be used for high-resolution EM. EM on Cryo-sections Fiber bundles containing four or five fibers are dissected from glycerinated or fresh muscle and processed on ice in a multiwell plate. Fresh fibers are skinned in rigor solution with 1% Triton X-100 for 15 min. Glycerinated fibers may be mounted on U-pins and stretched a measured amount in relaxing solution with glycerol (15). After washing, fibers are lightly fixed with 2 to 6% paraformaldehyde or 0.1% glutaraldehyde, or both, in rigor or relaxing solution for 30 min. The strength of fixation depends on the sensitivity of the antigen. Fibers are infused with 2.1% sucrose in rigor solution as cryoprotectant for 15 min at 257C and then mounted on copper stubs and immersed in liquid nitrogen. Cryosections (50–100 nm thick) are cut on a Reichert FC4E or RMC CR2000 ultramicrotome at 01107C, picked up in a loop with sucrose in rigor solution, and adhered to 200 mesh copper grids coated with Formvar and carbon. Grids are inverted on a drop of rigor buffer in a petri dish, and sections are stored for less than an hour before immunolabeling at 257C by the method of Tokuyasu (23) modified by Griffiths et al. (6). The sections are blocked with 5% FCS in rigor solution and labelled with primary antibody, bridging antibody, and protein A–gold (9 nm), each for 30 min. Triton X-100 (0.1%) may be included in blocking and antibody solutions. Bridging antibody (e.g., goat anti-rat) is necessary because protein A–gold does not react well with rat antibody (except subclass IgG2c) and all our monoclonals are raised in rats. The advantage of protein A is that it combines with the Fc region of IgG and does not interfere with the antigen-binding site. Grids are stained by floating on 2% methyl cellulose with 0.3% uranyl acetate. This gives a low-contrast image that shows gold particles clearly. EM on Lowicryl Sections Glycerinated or fresh fibers are fixed and frozen as for cryosectioning and then freeze substituted and embedded in Lowicryl HM20 by a modification of the method of van Genderen et al. (24). Fibers are freezesubstituted with methanol and 0.5% uranyl acetate at 0907C for 48 h, followed by a rise in temperature to 0457C over 24 h. After washing in methanol at 0457C, fibers are infiltrated with Lowicryl/methanol mixtures with resin:methanol ratios (vol/vol) of 1:1 and 2:1, and with pure resin for 2 h each with one change. Fibers are infiltrated with Lowicryl for a further 12 h and then polymerized with UV light for 48 h, all at 0457C.

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Sections 40–80 nm thick are cut at 257C and mounted on 200 mesh copper grids coated with Formavar and carbon. After rehydrating for 10 min with a drop of rigor solution, sections are labeled with antibody and protein A–gold by the method used for cryosections and stained with 4% uranyl acetate for 10 min. The methods used for immunoEM with cryo- and Lowicryl-embedded sections of IFM and the modifications needed for particular antibodies are described by Lakey et al. (12, 13) and Ferguson et al. (4). EM with Preembedding Labeling Bundles of approximately five glycerinated fibers are mounted on U-pins and left at resting length or stretched by a desired amount and placed in a multiwell plate. After four rinses in appropriate physiological buffered solution (rigor or relaxing solution), fibers on U-pins are blocked in 0.1% Triton X-100 in the same solution and soaked in antibody overnight at 107C. Fibers are rinsed three times for one-half hour in rigor or relaxing solution. Fibers can be double-labeled by incubating sequentially with antibodies complexed with 3- and 5-nm gold particles. Fibers may be fixed in cold 0.2% tannic acid in buffer for 30 min, rinsed in water, and postfixed in 2% uranyl acetate in water for 30 min at 47C, or fixed in 3% glutaraldehyde, 0.2% tannic acid in a buffered solution (usually lowionic-strength rigor solution at pH 6.8) for 30 min at room temperature. Fibers are rinsed in buffer and then in 100 mM KPO4 , 10 mM MgCl2 , pH 6.0, postfixed in 1% OsO4 in the same KPO4 buffer (ice cold) for 20 min rinsed in deionized water three times, block-stained in 2% uranyl acetate in water for 30 min, and rinsed in water. All fibers are dehydrated in a graded series of ethyl alcohol. Fibers are infiltrated with accelerated Araldite 506 for 30 min at 657C and drained, and fresh resin is added and incubated for 20 min at 657C. The fibers are cut into 1-mm lengths, arranged in rafts on polyethylene sheets in tiny puddles of fresh Araldite, and prepolymerized until tacky, and BEEM capsules filled with fresh resin are inverted over the rafts of approximately three fiber segments. Blocks are polymerized at 657C or 807C for 2 days. Preparation of Fab and Gold/Fab Probes Fab is prepared from IgG isolated from ascitic fluid. IgG is purified on a DEAE column and digested with papain. The conditions used to digest IgG into Fab and Fc fragments differ for different subclasses of rat IgG (18). Fab is separated from Fc by gel filtration on an FPLC Superose 12 column or by ion exchange on a DE-52 column. The yield of Fab from the Superose 12 column is lower than that from the DE-52 column but gel filtration is quicker because additional concentrating and desalting steps are not needed (14).

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The smallest probe we have used to identify troponin epitopes is 3-nm gold/Fab, which is visible in EMs of stained muscle sections. Particles of colloidal gold 2–3 nm in diameter are prepared by reducing HAuCl4 with NaSCN (2); 5-nm particles are prepared by reducing HAuCl4 with tannic acid and sodium citrate (19). Larger 10-nm particles are prepared by reducing HAuCl4 with sodium citrate alone (5). The method of Baschong et al. (2) and Baschong and Wrigley (1) for titrating gold with Fab was modified for use with our Fab (14). The gold colloid is titrated with Fab in a microtiter plate to determine the minimum amount of Fab needed to prevent the gold from flocculating when 10% NaCl is added. The end point is estimated by examining samples in the EM to see the extent of aggregation. The conjugate is prepared by adding Fab in 2 mM Mops, pH 7.0, to the gold colloid (pH 7.2) with addition of 0.05% polyethylene glycol to prevent flocculation. The conjugate is centrifuged at 150,000g for 30 min in a Beckman Airfuge. This procedure produces a loose light-brown pellet, which is the gold/Fab, and a compact dark brown pellet, which is excess gold colloid. The loose pellet is dissolved in rigor solution with 0.05% polyethylene glycol and examined in the EM to ensure that the probe is not aggregated and that there are no clumps of uncomplexed gold. The probe is used within a week of preparation. Baschong and Wrigley (1) conjugated 3-nm gold particles with between one and five Fab molecules. The 2-nm shell of protein around 3nm particles would correspond to about three Fabs. However, not all the Fabs may remain active.

SOURCES Protein A gold: Dr. Posthuma, University of Utrecht, Holland. Secondary anti-rat antibodies: Rabbit anti-rat IgG, Cappel, Durham, North Carolina. FITC-labeled goat anti-rat antibody, Sigma Chemical Co., St. Louis, Missouri. Fetal calf serum, Sigma. DEAE, and FPLC Superose 12, Pharmacia, Piscataway, New Jersey. DE52, Whatman, Maidstone, England. Glutaraldehyde, EM grade, Tousimis Research Corp., Rockville, Maryland. Osmium tetroxide, Electron Microscopy Sciences (EMS), Fort Washington, Pennsylvania. Tannic acid and Araldite 506, EMS. Lowicryl K4M, Polaron, BioRad, Hatfield, Pennsylvania. Glycerol, Serva, Crescent Chem. Co, Hauppauge, New York. Colliodal gold, Nanoprobes, Loop Rd., Stony Brook, New York. Diamond knives, Diatome U.S., Fort Washington, Pennsylvania.

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ACKNOWLEDGMENT We thank Charles Ferguson for expert technical assistance and for providing the images in Figs. 3, 5, and 6.

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