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Identification of a connecting filament protein in insect fibrillar flight muscle
J. MoZ. Biol.
(1981)
153, 661-679
Identification of a connecting Filament Protein in Insect Fibrillar Flight Muscle JUDITH
Department
(Received
of Physiology, 80 East Concord 16 March
I).
SAIDE
Boston University School of Medicine Street, Boston, Mass., U.S.A.
1981, and in revised form
18 ilugust
1981)
A major component on sodium dodecyl sulfate-containing gels of solubilized isolated Z-discs, purified from honeybee flight muscle, migrates with an apparent molecular weight of 360,000. Antibodies to this high molecular weight polypeptide have been prepared by injecting rabbits with homogenized gel slices containing the protein band. With indirect, immunofluorescence microscopy these antibodies are localized to a region extending from the edge of the Z-band to the A-band in shortened or stretched sarcomeres. Similarly, glycerinated flight muscle treated with antiserum and prepared for electron microscopy shows enhanced density from the ends of the thick filaments to the I-Z junction regardless of sarcomere length. Evidence indicates that antiserum is directed toward a structural protein of connecting filaments, which link thick filaments to the Z-band in insect fibrillar muscle, rather than to a thin filament component. In Ouchterlony double-diffusion experiments a single precipitin band is formed when antiserum is diffused against solubilized Z-discs ; no reaction occurs between antiserum and proteins from native thin filaments prepared from honeybee flight muscle. Further, antibody stains the I-band in flight muscle fibrils from which thin filaments are removed. Finally, honeybee leg muscle myofibrils, in which connecting filaments have not been observed, are not, labelled with antibody. Since antibody binds to the short projections which extend from the flat surfaces of isolated Z-discs, these projections are assumed to be remnants of connecting filaments and the source of the 360,000 M, protein. The amino acid composition of this high molecular weight material, purified by Sepharose chromatography, is presented. The protein has been named “projectin”.
1. Introduction The indirect flight muscles of insects from the orders Diptera, Hemiptera, Coleoptera and Hymenoptera have evolved with unusual physical properties. The muscles can contract at rates several hundred times per second, much faster than the frequency of nerve stimulation. If these fibers are glycerinated and attached to a resonant lever system, they will oscillate for hours in the presence of ATP and calcium (Jewel1 & Ruegg, 1966). This mechanical behavior has been ascribed to “stretch activation”, a delayed change of active tension following a change of length. Although the molecular basis of stretch activation is not understood, it has 661 0022-2836/81/350661-19
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1981
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Press
Inc.
(London)
Ltd.
J. D. SAIDE
662
been suggested that structural elements called “connecting filaments”, which link thick filaments to the Z-band in insect oscillatory flight muscle, may play a role in the process. Connecting filaments are thought to influence the activity of crossbridges by transmitting stress to the thick filaments during stretch (but see Wray, 1979). They are also assumed to contribute to the high stiffness of resting muscle (Pringle, 1978; Garamviilgyi & Belagyi, 1968). The chemical nature of connecting filaments has not been described. In this paper we present evidence that these structures contain an extremely high molecular weight protein, estimated to be 360,000. This component has been identified in sodium dodecyl sulfate-containing gels of solubilized isolated Z-disc preparations, and it is shown to originate from isolated Z-disc surface projections. A summary of this work has appeared in abstract form (Saide, 1980).
2. Materials
and Methods
(a) Preparations (Apis meZZ$e’era) myofibrils were purified
Honeybee from flight muscle homogenates by differential and sucrosedensity gradient centrifugation and stored at - 20°Cin 2.0 M-sucrose (Saide & Ullrick, 1974). Myofibrils from giant waterbug (Lethocerus masimus) flight muscle, honeybee leg muscle and rabbit psoas muscle were prepared from glycerinated samples (Abbott, 1973) by homogenization in storage buffer (50% (v/v) gly cerol, 61 M-KCl, 002 M-potassium phosphate, pH 6%). Fibrils were washed repeatedly by centrifugation (17OOg, 30 min) and resuspension in modified Hodge’s solution (91 M-KCl, 0601 M-MgCl,, 25 mM-EGTAt, pH 7.0). Lethmerus muscle homogenates were sometimes agitated in modified Hodge’s solution containing 1% (v/v) Triton X-100 (Fisher Scientific, Fairlawn, N.J.) to remove mitochondria associated with myofibrils. Isolated Z-discs were prepared from honeybee myofibrils with 0.5% (v/v) lactic acid (Ernst et al., 1958 ; Guba et al., 1960 ; Garamviilgyi at al., 1962), and purified by procedures described in a previous publication (Saide & Ullrick, 1974). Native thin filaments were isolated from honeybee myofibrils using the method of SzentGyorgyi et al. (1971) as modified by Goldberg & Lehman (1978). (b) Sodium dodecyl sulfate/poZyacryZamide gel electrophoresis Samples were dissolved in 905 M-Tris-sulfate (pH 6.1) (gel sample buffer) containing 8 Murea, 91% (v/v) p-mercaptoethanol and 1% (w/v) SDS. They were electrophoresed on 5% (w/v) polyacrylamide gels using the discontinuous buffer system of Neville ( 197 1) Details of gel and sample preparations, staining and destaining procedures, and molecular weight determinations are published elsewhere (Saide & Ullrick, 1974). For densitometry studies, gels were stained with Fast green FCF (Fisher Scientific, Fairlawn, N.J.) and scanned with a 0.2 mm slit at 640 nm. (c) Antibody The 360,000 M, protein used for antibody gels in which 50 pg samples of solubilized electrophoresis the gels were pierced with a the tracking dye. They were then frozen on t Abbreviations sodium
dodecyl
production production was sliced from SDS/polyacrylamide isolated Z-discs were electrophoresed. Following needle dipped in India ink to mark the position of solid CO, and stored at - 2O”C, while sample gels
used: EGTA, ethyleneglycol-bis-(fi-amiuoethyl
sulfate;
Tris-sulfate,
di tris(hydroxymethy1)
ether)-N.N’-tetraecetic
aminomethane
sulfate.
acid; SDS,
CONNECTING
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PROTEIN
663
were stained to determine the migration distance of the 360,000 M, protein relative to the tracking dye. After the appropriate band had been sectioned from each unstained, frozen gel, the remaining gel segments were stained to ensure that no unwanted protein had been included in the slice removed. Selected gel slices were combined and homogenized in 1 ml of PBS (015 hf-NaCl, 601 Msodium phosphate, pH 7.4) and 0.25 mg heat-killed Mycobacterium tuberculosis (Difco Laboratories, Detroit, Mich.). The suspension, containing approx. 15Opg of antigen, was injected intradermally at multiple sit#es along the back of a rabbit. The animal was given booster injections with the same quantity of protein 3 months later and was bled from the central artery of the ear 3 times at weekly intervals thereafter. (d) Gamma globulin purification Serum was diluted 1 : 1 with PBS and brought to 50% ammonium sulfate saturation. The precipitated fraction was dialyzed against PBS and stored at - 20°C. or purified further by chromatography on DEAE-cellulose (Sober & Peterson, 1958). (e) Adsorption
of antiserum
Approximately 3 mg of isolated Z-discs were sedimented from a suspension in water by centrifugation at 100,OOOg for 1 h at 4°C. The Z-discs were mixed with 2.5 mM-EDTA (pH 7.0), a solution which caused them to aggregate. They were then compacted (12,000g for 2 min) and mixed with 05 ml of antiserum. After 15 h of gentle agitation at 4”C, the mixture was briefly centrifuged, and the supernatant, containing “adsorbed” serum, was withdrawn and frozen. The pellet was washed repeatedly in PBS containing 2.5 mM-EDTA until the absorbance of the supernatant at 280 nm was zero. The Z-discs were then stirred with 1 ml of 0.5% lactic acid which released bound antibody without dispersing Z-disc structural proteins. Following centrifugation, the supernatant, containing purified immunoglobulins, was dialyzed against PBS containing 2.5 mM-EDTA and stored at -20°C. (f) lmmunodiffusion
tests
Antibody specificity was tested by the immunodiffusion method of Ouchterlony (1953). Diffusion dishes contained 1% agarose in 1 mw-Tris-acetate (pH 7.5) 2.5 mM-EDTA. Test substances (25 ~1) were added to wells which were arranged in the agarose at 1 cm spacings. Dishes were incubated at 4°C in a moist environment for 1 week, washed in several changes of PBS for 48 h at 4°C and stained with Coomassie Brilliant Blue R-250. (g) Radioimmunostaining experiments An IgG fraction (100 pg) of sheep anti-rabbit immunoglobulins (Cappel Laboratories, Cochranville, PA) was iodinated using an “‘1 radioiodination kit (NEZ-151) from New England Nuclear (Boston, MA). Labeled protein was separated from reactants on a 2 ml AGl-X8 column (Bio-Rad Laboratories, Richmond, CA.) equilibrated with PBS containing 1 mg bovine serum albumin/ml. Solubilized Z-discs or purified myofibrils (Saide & Ullrick. 1974) were electrophoresed in SDS on a 1 mm thick 5% polyacrylamide slab gel (12 cm x 10 cm) using the discontinuous buffer system of Neville (1971). After the position of the tracking dye was marked with India ink, the gel was frozen on solid COz and divided into 5-mm wide vertical strips. The strips were soaked in 40% methanol/lO”/e acetic acid to precipitate proteins and to remove SDS, and then equilibrated with PBS containing 2.5 mM-EDTA. The gel strips were placed into Plexiglass wells (96 cm x 0.6 cm x 10 cm) which were filled with a solution of gamma globulin (1 mg/ml in PBS, 2.5 mM-EDTA) prepared from either antiserum or non-immune rabbit serum by ammonium sulfate fractionation. The wells were enclosed in a moist environment and agitated gently for 24 h at room temperature. Gel strips were washed again for 48 h at 4°C
664
J. D. SAIDE
with several changes of buffer and then incubated as before with a solution containing lz51labeled sheep anti-rabbit immunoglobulin (5 x lo6 cts/min per ml) or [‘251]protein A (New England Nuclear, Boston, MA) (7.5 x lo5 cts/min per ml) in 1 mg bovine serum albumin/ml, PBS and 2.5 mM-EDTA. After additional exhaustive washing in buffer at 4”C, the gel strips were frozen on solid CO,, and l-mm slices were counted in a gamma spectrometer. To locate band positions and to ensure that protein had not been eluted from the gels, some gel strips were stained after being taken through the procedure in buffer only (Burridge, 1976; Wang et al., 1979). (h) Indirect immunojlwrescence Myofibrils, which had bound to the surface of glass slides after settling out of suspension, were washed in PBS and incubated for 30 min at room temperature with pm-immune serum, antiserum or gamma globulin fractions. Fibrils were washed for 30 min with 3 changes of PBS and incubated for 30 min with a 1 : 32 dilution of a fluorescein-conjugated IgG fraction of goat anti-rabbit gamma globulin (Cappel Laboratories Inc., Downington, PA). They were washed as before and mounted under coverslips with 10% (v/v) glycerol in PBS. Fibrils were examined with a Zeiss phase contrast microscope equipped with epifluorescence optics. Photographs were taken with Kodak Tri-X pan film at ASA 1600 and developed in Diafine. Exposure times for photographing and printing fluorescent images of control and antibody-labeled fibrils were identical. (i) Immunostaining and electron microscopy (i) Flight muscle Jibers Honeybee longitudinal flight muscles with attached exoskeleton were glycerinated (Abbott, 1973) and kept in storage buffer at -20°C for several weeks. Before the experiment small fiber bundles were soaked for 90 min in ice-cold relaxing solution consisting of @l MKCl, 0001 iv-MgCl,, 2 mM-EGTA, 10 mM-ATP (pH 6%). Each bundle was transferred onto a small block of paraffin wax and anchored between 2 stainless steel spring clamps. One clamp was pinned to the paraffin block: the other was connected to a manually operated micropositioner which was used to stretch the muscle 0045 mm/min to lengths up to 200% rest length. Fibers were held at a given length by pinning the second clamp in position. The muscle was continually moistened with relaxing solution during this procedure. An incubation well was formed by encircling the preparation with a ridge of hot, melted paraffin wax. When the wax cooled, 125 ~1 of test solution was added to the well to cover the fibers. Preparations were incubated with undiluted antiserum or gamma globulin purified from antiserum (4.5 mg/ml in PBS, 5 mM-EDTA). Control fibers were similarly antiserum, or purified non-immune treated with either pre-immune serum, “adsorbed” gamma globulin (4.5 mg/ml in PBS, 5 mM-EDTA). Samples were incubated at 2°C in a moist environment for 48 h. They were subsequently washed for 48 h by floating the inverted paraffin blocks over ice-cold PBS, 25 mM-EDTA. For electron microscopy, fibers were fixed in their incubation wells for 2 h at room temperature with 3% glutaraldehyde in stock buffer (0.067 M-sodium phosphate, pH 7.4). They were then cut from their attachments and postfixed for 1 h in ice-cold 3% (w/v) osmium tetroxide. Samples were dehydrated in graded concentrations of alcohol, taken through propylene oxide and embedded in Araldite: DDSA (Ted Pella, Inc., Tustin, CA) (1.3 : 1). Thin sections were stained with uranyl acetate and lead citrate and examined with a Siemens (1A) electron microscope (60 kV) calibrated with a diffraction grating replica. (ii) Isolated honeybee Z-discs Purified isolated Z-discs suspended in water were mixed with an equal volume of normal or immune rabbit gamma globulin (4 mg/ml in 2 x concentrated PBS with 5 mM-EGTA). The mixture was shaken gently at room temperature for 1 h. Z-discs were subsequently sedimented at 1200g for 10 min and washed repeatedly in stock buffer until no soluble
CONNECTING
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protein was detected in the supernatant by measuring absorbance at 280 nm. Z-disc pellets were subsequently treated as solid tissue blocks and prepared for electron microscopy by procedures described above. Z-disc proteins Isolated Z-discs were dissolved in a solution of 7 M-guanidine . hydrochloride, 0.2% pmercaptoethanol, 25 mm-EDTA (pH 7.0) and concentrated in dialysis tubing against Aquacide IIA (Calbiochem-Behring Corp., La Jolla, CA). The solution was dialyzed against gel sample buffer containing 8 M-urea and 61% /3-mercaptoethanol. After addition of SDS to a final concentration of lo/o, the solution was clarified by centrifugation at 9000 g for 30 min. Then 400 ~1 of supernatant (2 mg/ml) was loaded onto a 409 mm x 12 mm Sepharose 4B column equilibrated with 61% SDS in gel sample buffer. 65 ml volumes were collected at a rate of 10 ml/h, and portions from alternate fractions were analyzed by SDS/polyacrylamide gel electrophoresis. The column void volume was determined in a separate run from the elution position of Blue Dextran 2000 (Pharmacia, Uppsala, Sweden). (j) Gel @ration
of
(k) Amino acid analysis Samples were dialyzed against water, lyophilized, and hydrolyzed in 2 ml 6 M-HCl for 20 h at 105°C in ampules sealed under vacuum. The hydrolyzates were dried under nitrogen, then dissolved in 601 M-HCl, and analyzed with a Beckman 119 CL analyzer equipped with a model 126 integrator. Serine, threonine and tyrosine values were not corrected for losses during hydrolysis. Estimates for methionine and half-cystine include their oxidation products, methionine sulfoxide and cysteic acid. (1) Protein determinations Protein concentrations were estimated according to the procedures of Lowry et aZ. (1951). Bovine serum albumin was used as a standard. Concentrations of gamma globulin solutions were determined by absorbance measurements at 280 nm assuming an extinction of 1.4 for a 1 mg/ml solution. Based on Lowry determinations, the extinction coefficient at 280 nm for solubilized Z-discs (1 mg/ml) was estimated at 1.46.
3. Results (a)
Characterization
of the 360,000
M, protein
SDS/polyacrylamide gel electrophoresis of solubilized isolated Z-disc preparations resolves several polypeptides. The molecular weights of three of these components are estimated to be 87,000, 158,000, and 175,000 (Saide & Ullrick, 1974; and Fig. 3(a)), and immunofluorescence studies on honeybee flight muscle fibrils confirm that these proteins originate from the Z-band (Saide & Ericson, 1979). A fourth band, which penetrates only a few millimeters into 5% gels, has an extrapolated molecular weight of 360,000. Gel densitometry studies indicate that it accounts for as much as 30% of electrophoresed material. Although it was suspected that this high molecular weight component represented aggregated protein, subsequent studies have shown it to be a unique polypeptide species. (i) Antibody speci$city The specificity of antibodies Ouchterlony double-diffusion
raised against the 360,000 M, protein was tested in experiments. Z-disc proteins dispersed in 8 M-Urea
666
J. D. SAIDE 30
25 I.7 I II x
20
.-: \ 2 2
15
a c .-> tg .-0
10
B 5 ,
10
20
30
40
50
60
70 t
80
Slice number
360~000
1751000
871000
1. Tests for specificity of immunoglobulins raised against the 360,000 M, protein. Radioactivity vwsw gel slice plot. (0) Gel treated with a 50% ammonium sulfate fraction from antiserum (1.3 mg/ml in PBS, 25 IIIM-EDTA); (0) control gel treated with gamma glbbulin fractionated from non-immune rabbit serum (1-3 mg/ml). Gel strips were subsequently incubated with lz51-labeled sheep anti-rabbit immunoglobulin (5 x lo6 cts/min per ml) in PBS, 2.5 mmEDTA, 1 mg bovine serum albumin/ml. The stained gel below the radioactivity profiles indicates the position of isolated Z-disc protein bands. The arrow marks the position of the tracking dye. Inset: Ouchterlony diffusion plate. (z) Solubilized isolated Z-discs (1.4 mg/ml) in 8 M-urea, gel sample buffer, 0.1% fl-mercaptoethanol; (a) gamma globulins fractionated from antiserum and dissolved in PBS to twice their concentration in serum (9 mg/ml). Indicated dilutions were made with PBS. (b) PBS: (n) gamma globulin fractions from normal rabbit serum twice concentrated as in (a). RG.
formed a single precipitin line when diffused in agarose against immunoglobulins ; no reaction was observed in control experiments in which immunoglobulins were replaced with non-immune gamma globulins (Fig. 1, inset). While these results suggested that antibodies recognized a single antigen in Z-disc preparations, we noted that the precipitin line formed in these studies was not hyper-sharp. We, therefore, tested immunologic specificity with the more sensitive technique of radioimmunostaining. The results of such studies in which antigen was localized on slab gels of Z-disc proteins are shown in Figure 1. A single sharp peak of radioactivity is found at the
CONNECTING
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PROTEIN
16
3211 10
20
30
40
50
Slice number
60
70
A
80
I
FIG. 2. Radioactivity VWSU~ gel slice plots. Gel strips were incubated with a 50% fraction (0.8 mg/ml in PBS) from antiserum against the 360,000 ilf, protein. They treated with [ 1551]protein A (7.5 x l-0’ cts/min per ml) in PBS, 2.5 mM-EDTA, albumin/ml. Radioactivity profiles (not shown) of control gels, treated with a 50% fraction of pre-immune serum (68 mg/ml in PBS), show no evidence of peaks at 360,000 M, protein. Gel electrophoresis patterns of solubilized Z-discs isolated in solutions (below, -O-O--) and solubilized myofibrils (above, - - 0 - - 0 -
ammonium sulfate were subsequently 1 mg bovine serum ammonium sulfate the position of the high ionic strength - ).
position of the 360,000 M, protein. The curve rises steeply, but its descending limb is skewed, reaching baseline at a position on the gel corresponding to a molecular weight of about 175,000. This finding suggests that the 360,000 M, protein may be unstable, giving rise to fragments of lower molecular weight. The absence of a radioactivity peak at any other position on the gel, however, demonstrates that the high molecular weight polypeptide is a unique component, immunologically distinct from other major Z-disc proteins in our preparation. To rule out the possibility that the 360,000 Mr protein was an artifact resulting from exposure of myofibrils to dilute acid, we probed two additional slab gel strips
668
J. D. SAIDE
with antibody, one containing electrophoresed proteins of purified myofibrils and the other proteins of honeybee Z-discs isolated by exposing fibrils to solutions of high ionic strength (Bullard & Sainsbury, 1977). In both cases antibody recognized polypeptides migrating with an apparent molecular weight of 360,000 (Fig. 2). Both the position and shape of the radioactivity peaks in Figures 1 and 2 are comparable. Together, these Figures demonstrate not only that the 360,000 H, protein is a component of myofibrils, but that it remains with isolated Z-discs when fibrils are extracted with either dilute lactic acid or concentrated salt solutions. and amino acid composition of the 360,000 M, protein (ii) Pur@cation The 360,000 M, protein was partially purified by chromatography of solubilized Z-discs on Sepharose4B in the presence of 91% SDS. Analysis of column fractions by SDS/polyacrylamide gel electrophoresis indicates that the high molecular weight material elutes near the void volume. Early fractions have two major components; later fractions contain, in addition, a third minor polypeptide (Fig. 3, lanes c to g). Those fractions pooled for amino acid analysis did not include the minor protein. (Similarly, this component was avoided when SDS-containing gels
a
b
c
d
e
f
9
360 vOO0
175,000 158,000
-
8 7,000
FIG. 3. SDS/5% polyacrylamide gel electrophoresis patterns of solubilized isolated Z-discs (lane a); sequential Sepharose 4B column fractions eluted near the void volume (lanes b to g). Column load: 400 ~1 of isolated Z-discs (2 mg/ml) in 8 M-We&, gel sample buffer, @l% SDS. Elution buffer: gel sample buffer with 0.1% SDS.
CONNECTING
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Amino
acid composition
669
PROTEIN
1
of the 360,000
M, protein
Residues/1000 (meanfs.D.) 82k 109.3f12.2 66.3+ 74.2* 1248k 75.5* 93.3* 67.55 53.1* 11.5+ 36.3k 685+ 26.9f 32.0+ 87.2k 23.3+ 505f
Cyel2 ASP Thr Ser Glu Pro Gly Ala Val Met Ile Leu TY~ Phe LYS His A%
32) 1.8 1.5 59 1.9 8.1 2.0 1,o 1.7 1.7 7% 1.2 3.4 64 1.1 45
The amino acid composition of the 360,000 bf, protein averaged from 3 different preparations. Values for methionine and half-cystine include the oxidation products methionine sulfoxide and cysteic acid. The tryptophan content w&e not measured.
of isolated Z-discs were sectioned to isolate the 360,000 M, protein for antibody production.) Fractions enriched in the high molecular weight protein are contaminated with a component which does not penetrate 5% gels. The contaminant is assumedto be a glycoprotein, since it contains oxidizable carbohydrate residues, which were detected on polyacrylamide gels by the method of Eckhardt et al. (1976) (S. G. Ericson & J. D. Saide, unpublished results). Although densitometry studies indicate that the impurity may range from 10 to 30% of total protein in pooled fractions containing the 360,000 M, material, such fractions (Fig. 3, lanes c to e) were analyzed for amino acid composition. Results tabulated from three different preparations are presented in Table 1. Since standard deviations are lessthan 10% of the meansfor most amino acids, it appears that the small but variable amount of contaminant does not significantly influence the amino acid profile. The analyses reveal no unusual features in amino acid composition except for a high proline content, which has also been observed in the amino acid composition of intact isolated Z-discs (Saide & Ullrick, 1974). (b) Localization
of the 360,000
M, component
in myo$brils and isolated
Z-discs
(i) Indirect immunojluorescence microscopy The staining pattern of honeybee myofibrils treated with antiserum to the 360,000 M, protein is unlike that observed with antiserum to other isolated Z-disc
670 (a)
J. D. SAIDE
(b)
(d)
FIG. 4. Phase (top) and fluorescence (bottom) micrographs of (a) rest length, and (b) stretched honeybee myofibrils stained with anti-serum to the 360,000 M, protein. Fibrils were stretched by gently moving the coverslip over the slide. (0) Control fibril treated with pre-immune serum. (d) Glycerin&& Lethoceruaflight muscle fibril stained with a gamma globulin fraction from antiserum (1 mg/ml in PBS). (e) Control Ledhocerua fibril treated with gamma globulin (1 mgjml) from non-immune rabbit serum. (fj Phase (left) and fluorescence (right) micrographs of glycerinated honeybee leg muscle myofibrils treated with antiserum. Magnifications: (a) to (e) 3040 x ; (f) 1440 x
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proteins (Saide & Ericson, 1979). Antibody labeling appears on either side of the Zband, not at its center. In rest length fibrils where the A-band occupies nearly the entire sarcomere, fluorescence appears as a doublet, with a narrow band of staining along each edge of the Z-band (Fig. 4(a)). In stretched sarcomeres the band of fluorescence is broader, extending from the edge of the Z-band to the border of the A-band at all carcomere lengths (Fig. 4(b)). N o o th er region of the fibril is labeled. Qualitatively similar results are found with immune globulins purified from antiserum either by ammonium sulfate precipitation or by adsorption onto isolated Z-discs (see Materials and Methods, section (e)). Fibrils treated with pre-immune serum (Fig. 4(c)) or a pre-immune gamma globulin fraction show no significant labeling. Rest length Lethocerus myofibrils also reveal the characteristic doublet following exposure to antibody (Fig. 4(d)). However, no antibody binding has been demonstrated in myofibrils from either honeybee leg muscle (Fig. 4(f)) or rabbit psoas muscle (not shown). (ii) Immunostaining and electron microscopy The pattern of antibody labeling in honeybee flight muscle fibers examined by electron microscopy is consistent with results of indirect immunofluorescence studies. Antibody accumulates in the region between the Z-band and the A-band, extending from the edge of the Z-band to the tips of the thick filaments where it penetrates slightly beyond the tapered ends (Fig. 5(b)). In unstretched fibers this staining is usually very intense, and the narrow bands of bound antibody which flank the Z-band have a density comparable to it. There is no evidence of antibody binding within the Z-band, however, or around its circumference, and in some myofibrils there appear to be very narrow, unstained regions at the lateral (I-Z) margins of the Z-band (Fig. 5(b)). In stretched fibers, less intensely staining patches of antibody are scattered throughout the I-band, creating a grainy appearance (Fig. 6(b)). As in rest length sarcbmeres, staining appears to overlap the tips of the thick filaments. No protein binding has been detected in stretched or unstretched control fibers treated with either pre-immune serum (Fig. 5(a)), “adsorbed” serum from which immunoglobulins have been removed (Fig. 6(a)), or gamma globulin fractions prepared from non-immune serum. Two possibilities are suggested by these results. One is that the 360,966 Jf, protein is associated with thin filaments, but that antibody binding sites are accessible only outside the A-band. The other is that the high molecular weight protein is a structural component of connecting filaments. Several findings indicate that the latter is true. (1) In Ouchterlony doublediffusion experiments native honeybee thin filament proteins, dispersed in 8 Murea, do not form precipitin lines with antiserum to the 366,666 M, protein under conditions in which Z-disc preparations react with antibody. (2) Immunoglobulins can be shown to accumulate in the region between the Z-band and the ends of thick filaments in myofibrils from which thin filaments are missing (Fig. 7)t. 7 Unexpectedly, thin filaments disappeared in fibers incubated with unfractionated antiserum. They were unaffected, however, by pm-immune serum, antiserum adsorbed with isolated Z-discs, or gamma globulins purified from antiserum. We suspect that the loss of thin filaments may have resulted from an activation of proteases of the complement system.
J. D. SAIDE
(bl
FIG. 5. (a) Longitudinal section of a rest length control myofibril treated with pre-immune serum. (Control fibrils treated with pre-immune serum, gamma globulin purified from non-immune serum (46 mgiml) or antiserum adsorbed with isolated Z-discs are indistinguishable.) (b) Honeybee myofibril labeled with gamma globulins (46 mg/ml) purified from antiserum to the 36O,OOOM, protein. Magnification : 9000 x
(3) Honeybee leg muscle myofibrils, which have broad I-bands but no apparent connections between thick filaments and the Z-line (Candia Carnevali et al., 1980), are not labeled by antibody in indirect immunofluorescence studies (Fig. 4(f)). Taken together, these observations provide compelling evidence that the 360,000 M, component is a connecting filament protein. In light of these results it seemed likely that the protein would be found in the projections which have been observed to extend from the surfaces of isolated Zdiscs (Saide & Ullrick, 1974). In fact, when isolated Z-discs are incubated with gamma globulin fractions prepared from antiserum, antibody coats this surface
CONNECTING
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PROTEIN
(a)
1 IJm FIG. 6. (a) Longitudinal section of a stretched control honeybee myofibril treated with antiserum adsorbed with isolated Z-discs. (b) Slightly oblique section through a lightly stained stretched honeybee myofibril treated with gamma globulin (46 mg/ml) purified from antiserum to the 360,000 M, protein. Magnification : 10,000 x
material, effectively sandwiching the Z-disc backbone between layers of densely staining protein (Fig. 8(b)). As in labeled myofibrils, there is no evidence of antibody accumulation within the Z-disc backbone, and in many sections pale lines of reduced binding appear at the junction between the backbone and the projections (Fig. 8(b)). No labeling has been detected in control Z-discs, treated with non-immune gamma globulins (Fig. 8(a)). Such results indicate that isolated Z-disc surface projections are remnants of connecting filaments, rather than stubs of thin filaments as was suggested earlier (Saide & Ullrick, 1974). This conclusion is supported by gel densitometry studies in which protein with the molecular weight
674
J. D. SAIDE
FIG. 7. Longitudinal adsorbed with isolated filaments in the labeled
of actin isolated
sections of slightly stretched honeybee Z-discs (left), or unfractionated antiserum myofibril. Magnification : 19,600 x
has been found to account in dilute lactic acid.
for less than
myofibrils (right).
treated with antiserum Note the absence of thin
2% of the total
protein
in Z-discs
4. Discussion The presence of connections between thick filaments and the Z-band in insect fibrillar flight muscle is now well established. Connecting filaments were noted first in Diptera, by Auber & Couteaux (1962,1963) and later in Apis (Garamviilgyi, 1965,1966u,b,1969; Trombitas & Tigyi-Sebes, 1972,1974,1979; Saide & Ullrick, 1973) and Lethocerus muscle (Zebe et al., 1968; Reedy, 1971; White & Thorsen, 1973 ; Ullrick et al., 1977). In Lethocerus, the most convincing evidence is based on electron micrographs of fibers stretched under rigor conditions so that thin filaments are torn from the Z-band (Reedy, 1971). Traversing the regions between the Z-band and the broken ends of thin filaments are fine thread-like structures, which, in cross section, are found to occupy the same lattice positions as thick filaments in the A-band (White & Thorson, 1973). In oblique sections of stretched honeybee (Apis) flight muscle, filament counts in equal areas of the I-band and overlap zone of the A-band yield similar numbers (Saide & Ullrick, 1973). Moreover, longitudinal sections of stretched fibrils, treated to remove thin filaments, also reveal thick filament continuity with the Z-band (Trombitas & Tigyi-Sebes, 1972,1974).
CONNECTING
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PROTEIN
(a
O-5 urn FIG. 8. Isolated Z-discs, viewed on edge, treated with (a) gamma globulin fractionated from nonimmune rabbit serum (2 mg/ml) or (b) gamma globulin prepared from antiserum to the 360,000 ,Wr protein (2 mg/ml).
676
J.
D. SAIDE
Although it is generally agreed that the connecting material in stretched muscle is filamentous, there has been some dispute about its form in unstretched fibers (see Ashhurst, 1977). Pringle has proposed that the connecting substance is an unstructured gel, which becomes oriented into filaments only under axial stress (Pringle, 1974,1977). This view is based on Ashhurst’s report that in oblique sections through unstretched Hemiptera fibers, connecting filaments are absent from their expected lattice position in the I-band (Ashhurst, 1967). Although irregularly shaped structures, interpreted as connecting filaments, have been described in oblique sections of rest length Diptera (Auber & Couteaux, 1962,1963) and Hymenoptera muscle (Saide & Ullrick, 1973), Ashhurst suggests that these apparent filaments are more likely to be protrusions of Z-band material into the narrow I-band (Ashhurst, 1977). In the present study evidence indicates that the material linking thick filaments to the Z-line is filamentous even in the absence of stress effects. This conclusion is based on the appearance of isolated Z-disc surface projections which our results suggest are derived from this connecting substance. In the micrograph of Z-discs, treated with gamma globulin solutions, the projections are aggregated and are not shown to advantage. However, in untreated Z-discs in which the projections are well-preserved and favorably oriented, they are found to be filamentous (Plate VII, Saide & Ullrick, 1974). These structures are not as distinct or rod-like as thin or thick filaments. They have a fluffy quality, and they appear flexible. It may be that they do not remain rigidly oriented in the unstretched myofibril and consequently are sometimes lost from their appropriate lattice position in oblique sections. Our conclusion that unstretched connecting material is in the form of filaments is also supported by the observations of Trombitas & Tigyi-Sebes (1979). These authors have published micrographs of honeybee flight muscle thick filaments which arise from adjacent sarcomeres and are joined at their tapered ends by slender, threadlike connections. In this investigation immunostaining techniques have provided evidence that connecting filaments in flight muscles from representatives of two insect orders, Hymenoptera (Apis) and Hemiptera (Lethocerus), contain a 360,000 M, protein. The protein has been localized to a region between the lateral margins of the Z-band and the ends of the myosin-containing filaments. Although the component extends a short distance beyond the tapered portions of these filaments toward the M-line, it is not clear how it is integrated within the thick filament structure. We are also uncertain how the protein is associated with Z-band components. It is likely that the 360,000 M, protein does not actually penetrate the Z-band, since no antibody binding has been observed here. Although enhanced density at the Z-line might go undetected because of its intrinsic opacity, the absence of labeling is further suggested by the failure of antibody to accumulate at the outer surface (perimeter) of the Z-line. The frequent appearance of a narrow, unstained region at the I-Z junction of labeled myofibrils and isolated Z-discs also supports the view that the 360,000 M, protein is restricted to filaments lying outside the Z-band. The possibility cannot be ruled out, however, that this protein is in fact assembled within the Z-band lattice but is masked from antibody by associated proteins.
CONNECTING
FILAMENT
PROTEIN
677
We do not know whether the high molecular weight material is the only component in connecting filaments or whether it is complexed with one or more additional proteins. Bullard et al. (1977a,b) (see also Trombitas et al., 1977) reported that antibodies raised against insect paramyosin preparations stained the region between thick filaments and the Z-band. Since the antigenic species located in this area was not found to be paramyosin, it was assumed to be a contaminant in the paramyosin preparation. This material has not been identified, however. While it may be that the contaminating antigen is a protein distinct from the 360,000 M, material, the possibility exists that it is the same component or a breakdown product. We noted earlier that the high molecular weight protein is apparently degraded to lower molecular weight, immunologically similar polypeptides. The properties of the 360,000 M, protein and other proteins that may be associated with it in connecting filaments should be of interest since these structures have been shown to have unusual physical characteristics. In honeybee myofibrils, connecting filaments can be extended to well over ten times their rest length and can support stresses which cause thick filaments to pull apart (Garamviilgyi, 1965,1966a,b,1969). Their restoring force has been dramatically demonstrated in experiments in which relaxed muscle is stretched and then allowed to go into rigor. When such preparations are released, the recoil forces of the connecting filaments cause the sarcomeres to shorten, and thin filaments, prevented from sliding by rigor links, are found crumpled against the Z-band (Trombitas & Tigyi-Sebes, 1977). The highly compliant nature of connecting filaments suggested to us that the amino acid composition of the 360,000 M, protein might be similar to that of rubber-like proteins such as resilin or elastin (Seifter & Gallop, 1966). However, our results indicate that proline is no more than 7.5% of the total residues, that hydroxyproline is absent, and that hydrophobic residues are not in unusually high proportion. The amino acid composition, then, offers no clues to explain the exceptional extensibility of these structures. Wang el al. (1979) identified three extremely large polypeptides (titin) with molecular weights exceeding 400,000 in chicken skeletal myofibrils. Immunostaining studies demonstrated that they were widespread in vertebrate and invertebrate striated muscles and, under various conditions, distributed in myofibrils at the Z-bands, M-lines, A-I junctions, or throughout the A-band. Although the molecular weights of titin and the 360,000 M, protein are similar, we find that antibodies to the latter component bind exclusively in the I-band, and label only those fibers that have been demonstrated to have connecting filaments. These observations suggest that titin and the high molecular weight material are unrelated. To summarize, a 360,006 M, protein has been identified in gels of isolated Z-discs. With immunostaining techniques we have provided evidence that it is located in the connecting filaments, which join thick filaments to the Z-band in insect oscillatory flight’ muscle, and that it arises from isolated Z-disc surface projections. For purposes of reference, and because of its origin we suggest that this protein be called “projectin”.
6’78
J. D. SAIDE
I thank MS Margaret Phillips, MS Jean Maloney and MS I. Weiss for their excellent technical assistance, Dr David Sack for sectioning tissue blocks, Dr Carl Franzblau for making available his amino acid analyzer, Mr George Crombie for performing the analyses, and Dr Benjamin Kaminer for helpful suggestions during the preparation of this manuscript. This research was supported by grants from the Muscular Dystrophy Association and the National Institutes of Health (HL22985). The work was done during the tenure of an Established Investigatorship Award from the American Heart Association.
REFERENCES Abbott, R. H. (1973). J. PhysioZ. 231, 195-208. Ashhurst, D. E. (1967). J. Mol. Biol. 27, 385-389. Ashhurst, D. E. (1977). In Znsect FZight Mu.scZe (Tregear, R. T., ed.), pp. 117-196, Elsevier, North Holland, Amsterdam. Auber, J. & Couteeux, R. (1962). C.R.H. Acad. Sci. 254, 34253426. Auber, J. 8: Couteaux, R. (1963). J. Microscopic, 2, 309-324. Bullard, B. & Sainsbury, G. M. (1977). B&hem. J. 161, 399-403. Bullard, B., Hammond, K. & Luke, B. (1977a). J. Mol. BioZ. 115, 417-440. Bullard, B., Bell, J. L. & Luke, B. M. (19773). In Znsect Flight Muscle (Tregear, R. T., ed.), pp. 41-52, Elsevier, North Holland, Amsterdam. Burridge, K. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 4457-4461, Candia Carnevali, M. D., deEquileor, M. & Valvassori, R. (1980). J. Submicros. Cytol. 12, 427-446. Eckhardt, A. E., Hayes, C. E. & Goldstein, I. J. (1976). Anal. Biochem. 73, 192-197. Ernst, E., Garamvijlgyi, N. & Guba, F. (1958). Acta PhysioZ. Hung. 14, (suppl. 40). Garamvolgyi, N. (1965). J. Ultrastruct. Res. 13, 409-424. Geramvijlgyi, N. (1966~). Actu B&him. Biophys. Acod. Sci., Hung. 1, 89-100. Garamvijlgyi, N. (19665). Actu Biochim. Biophys. Acad. Sci., Hung. 1, 293-297. GaramvSlgyi, N. (1969). J. Ultrastruct. Res. 27, 462-471. Garamviilgyi, N. & Belagyi, J. (1968). Acta Biochim. Biophys. Acod. Sci., Hung. 3, 195-204. Garamviilgyi, N., Metzger-TiirGk, G. & Tigyi-Sebes, A. (1962). Acta Physiol. Acud. Sci., Hung.
22, 223-233.
Goldberg, A. & Lehman, W. (1978). Biochem. J. 171, 413-418. Guba, F., Garemviilgyi, N. & Ernst, E. (1960). Proc. 4th Int. Congr. Electron Microscopy, vol. 2, pp. 324-325, Springer-Verlag, Berlin. Jewell, B. R. & Ruegg, J. C. (1966). Proc. Roy. Sot. ser. B, 164, 428-459. Lowry, D. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 193, 265-275. Neville, D. M. (1971). J. BioZ. Chem. 246, 6328-6334. Ouchterlony, 0. (1953). Actu Puthol. Microbial. Scund. 32, 231-240. Pringle, J. W. S. (1974). Symp. BioZ. Hung. 17, 67-78. Pringle, J. W. S. (1977). In Insect FZight Muscle (Tregear, R. T., ed.), pp. 177-196, Elsevier, North Holland, Amsterdam. Pringle, J. W. S. (1978). Proc. Roy. Sot. ser. B, 201, 1077130. Reedy, M. K. (1971). In Contractility of Muscle Cells and Related Processes (Podolsky, R. J., ed.), pp. 229-246, Prentice-Hall, Englewood Cliffs, New Jersey. Saide, J. D. (1980). J. Cell BioZ. 87, 269a. Saide, J. D. L Ericson, S. G. (1979). Biophys. J. 25, 242a. Saide, J. D. & Ullrick, W. C. (1973). J. Mol. BioZ. 79, 329-337. Saide, J. D. & Ullrick, W. C. (1974). J. Mol. BioZ. 87, 671-683. Seifter, S. & Gallop, P. M. (1966). In The Proteins (Neurath, H., ed.) vol. 4, 2nd edit., pp. 153458, Academic Press, N.Y. and London. Sober, H. A. & Peterson, E. A. (1958). Fed, Proc. Fed. Amer. Sot. Exp. BioZ. 17, 1116-1126. Szent-Gyorgyi, A. G., Cohen, C. & Kendrick-Jones, .J. (1971). .I. Mol. BioZ. 56, 239-258.
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Trombitas, C. &, Tigyi-Sebes, A. (1977). In Insect Ftight Muscle (Tregear, R. T., ed.), pp. 7980, Elsevier, North Holland, Amsterdam. Trombitas, C., Tigyi-Sebes, A. & Pallai, G. (1977). In Insect FZigfr,t Muscle (Tregear, R. T., ed.), pp. 53356, Elsevier, North Holland, Amsterdam. Trombitas, K. & Tigyi-Sebes, A. (1972). Acta B&him. Biophys. Acud. Sci., Hung. 7, 193194. Trombitas. K. & Tigyi-Sebes, A. (1974). Acta B&him. Biophys. Acad. Sci., Hung. 9, 243253. Trombitas, K. & Tigyi-Sebes, A. (1979). Nature (London), 281, 319-320. Ullrick, W. C.. Toselli, P. A., Chase, D. & Dasse, K. (1977). J. Ultrastruct. Res. 69, 263-271. Wang, K., McClure. J. & Tu, A. (1979). Proc. Nut. ACT. Sci., U.S.A. 76, 3698-3702. White, D. C. S. & Thorson, J. (1973). Progr. Biophys. Mol. Biol. 27, 173-255. Wray, J. S. (1979). Nature (London), 289, 325-326. Zebe, E.. Meinrenkev. W. & Ruegg, I. C. (1968). 2. Zellforsch. 87. 603-621. Edited
by H. E. Huxley
(1981)
153, 661-679
Identification of a connecting Filament Protein in Insect Fibrillar Flight Muscle JUDITH
Department
(Received
of Physiology, 80 East Concord 16 March
I).
SAIDE
Boston University School of Medicine Street, Boston, Mass., U.S.A.
1981, and in revised form
18 ilugust
1981)
A major component on sodium dodecyl sulfate-containing gels of solubilized isolated Z-discs, purified from honeybee flight muscle, migrates with an apparent molecular weight of 360,000. Antibodies to this high molecular weight polypeptide have been prepared by injecting rabbits with homogenized gel slices containing the protein band. With indirect, immunofluorescence microscopy these antibodies are localized to a region extending from the edge of the Z-band to the A-band in shortened or stretched sarcomeres. Similarly, glycerinated flight muscle treated with antiserum and prepared for electron microscopy shows enhanced density from the ends of the thick filaments to the I-Z junction regardless of sarcomere length. Evidence indicates that antiserum is directed toward a structural protein of connecting filaments, which link thick filaments to the Z-band in insect fibrillar muscle, rather than to a thin filament component. In Ouchterlony double-diffusion experiments a single precipitin band is formed when antiserum is diffused against solubilized Z-discs ; no reaction occurs between antiserum and proteins from native thin filaments prepared from honeybee flight muscle. Further, antibody stains the I-band in flight muscle fibrils from which thin filaments are removed. Finally, honeybee leg muscle myofibrils, in which connecting filaments have not been observed, are not, labelled with antibody. Since antibody binds to the short projections which extend from the flat surfaces of isolated Z-discs, these projections are assumed to be remnants of connecting filaments and the source of the 360,000 M, protein. The amino acid composition of this high molecular weight material, purified by Sepharose chromatography, is presented. The protein has been named “projectin”.
1. Introduction The indirect flight muscles of insects from the orders Diptera, Hemiptera, Coleoptera and Hymenoptera have evolved with unusual physical properties. The muscles can contract at rates several hundred times per second, much faster than the frequency of nerve stimulation. If these fibers are glycerinated and attached to a resonant lever system, they will oscillate for hours in the presence of ATP and calcium (Jewel1 & Ruegg, 1966). This mechanical behavior has been ascribed to “stretch activation”, a delayed change of active tension following a change of length. Although the molecular basis of stretch activation is not understood, it has 661 0022-2836/81/350661-19
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1981
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Press
Inc.
(London)
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been suggested that structural elements called “connecting filaments”, which link thick filaments to the Z-band in insect oscillatory flight muscle, may play a role in the process. Connecting filaments are thought to influence the activity of crossbridges by transmitting stress to the thick filaments during stretch (but see Wray, 1979). They are also assumed to contribute to the high stiffness of resting muscle (Pringle, 1978; Garamviilgyi & Belagyi, 1968). The chemical nature of connecting filaments has not been described. In this paper we present evidence that these structures contain an extremely high molecular weight protein, estimated to be 360,000. This component has been identified in sodium dodecyl sulfate-containing gels of solubilized isolated Z-disc preparations, and it is shown to originate from isolated Z-disc surface projections. A summary of this work has appeared in abstract form (Saide, 1980).
2. Materials
and Methods
(a) Preparations (Apis meZZ$e’era) myofibrils were purified
Honeybee from flight muscle homogenates by differential and sucrosedensity gradient centrifugation and stored at - 20°Cin 2.0 M-sucrose (Saide & Ullrick, 1974). Myofibrils from giant waterbug (Lethocerus masimus) flight muscle, honeybee leg muscle and rabbit psoas muscle were prepared from glycerinated samples (Abbott, 1973) by homogenization in storage buffer (50% (v/v) gly cerol, 61 M-KCl, 002 M-potassium phosphate, pH 6%). Fibrils were washed repeatedly by centrifugation (17OOg, 30 min) and resuspension in modified Hodge’s solution (91 M-KCl, 0601 M-MgCl,, 25 mM-EGTAt, pH 7.0). Lethmerus muscle homogenates were sometimes agitated in modified Hodge’s solution containing 1% (v/v) Triton X-100 (Fisher Scientific, Fairlawn, N.J.) to remove mitochondria associated with myofibrils. Isolated Z-discs were prepared from honeybee myofibrils with 0.5% (v/v) lactic acid (Ernst et al., 1958 ; Guba et al., 1960 ; Garamviilgyi at al., 1962), and purified by procedures described in a previous publication (Saide & Ullrick, 1974). Native thin filaments were isolated from honeybee myofibrils using the method of SzentGyorgyi et al. (1971) as modified by Goldberg & Lehman (1978). (b) Sodium dodecyl sulfate/poZyacryZamide gel electrophoresis Samples were dissolved in 905 M-Tris-sulfate (pH 6.1) (gel sample buffer) containing 8 Murea, 91% (v/v) p-mercaptoethanol and 1% (w/v) SDS. They were electrophoresed on 5% (w/v) polyacrylamide gels using the discontinuous buffer system of Neville ( 197 1) Details of gel and sample preparations, staining and destaining procedures, and molecular weight determinations are published elsewhere (Saide & Ullrick, 1974). For densitometry studies, gels were stained with Fast green FCF (Fisher Scientific, Fairlawn, N.J.) and scanned with a 0.2 mm slit at 640 nm. (c) Antibody The 360,000 M, protein used for antibody gels in which 50 pg samples of solubilized electrophoresis the gels were pierced with a the tracking dye. They were then frozen on t Abbreviations sodium
dodecyl
production production was sliced from SDS/polyacrylamide isolated Z-discs were electrophoresed. Following needle dipped in India ink to mark the position of solid CO, and stored at - 2O”C, while sample gels
used: EGTA, ethyleneglycol-bis-(fi-amiuoethyl
sulfate;
Tris-sulfate,
di tris(hydroxymethy1)
ether)-N.N’-tetraecetic
aminomethane
sulfate.
acid; SDS,
CONNECTING
FILAMENT
PROTEIN
663
were stained to determine the migration distance of the 360,000 M, protein relative to the tracking dye. After the appropriate band had been sectioned from each unstained, frozen gel, the remaining gel segments were stained to ensure that no unwanted protein had been included in the slice removed. Selected gel slices were combined and homogenized in 1 ml of PBS (015 hf-NaCl, 601 Msodium phosphate, pH 7.4) and 0.25 mg heat-killed Mycobacterium tuberculosis (Difco Laboratories, Detroit, Mich.). The suspension, containing approx. 15Opg of antigen, was injected intradermally at multiple sit#es along the back of a rabbit. The animal was given booster injections with the same quantity of protein 3 months later and was bled from the central artery of the ear 3 times at weekly intervals thereafter. (d) Gamma globulin purification Serum was diluted 1 : 1 with PBS and brought to 50% ammonium sulfate saturation. The precipitated fraction was dialyzed against PBS and stored at - 20°C. or purified further by chromatography on DEAE-cellulose (Sober & Peterson, 1958). (e) Adsorption
of antiserum
Approximately 3 mg of isolated Z-discs were sedimented from a suspension in water by centrifugation at 100,OOOg for 1 h at 4°C. The Z-discs were mixed with 2.5 mM-EDTA (pH 7.0), a solution which caused them to aggregate. They were then compacted (12,000g for 2 min) and mixed with 05 ml of antiserum. After 15 h of gentle agitation at 4”C, the mixture was briefly centrifuged, and the supernatant, containing “adsorbed” serum, was withdrawn and frozen. The pellet was washed repeatedly in PBS containing 2.5 mM-EDTA until the absorbance of the supernatant at 280 nm was zero. The Z-discs were then stirred with 1 ml of 0.5% lactic acid which released bound antibody without dispersing Z-disc structural proteins. Following centrifugation, the supernatant, containing purified immunoglobulins, was dialyzed against PBS containing 2.5 mM-EDTA and stored at -20°C. (f) lmmunodiffusion
tests
Antibody specificity was tested by the immunodiffusion method of Ouchterlony (1953). Diffusion dishes contained 1% agarose in 1 mw-Tris-acetate (pH 7.5) 2.5 mM-EDTA. Test substances (25 ~1) were added to wells which were arranged in the agarose at 1 cm spacings. Dishes were incubated at 4°C in a moist environment for 1 week, washed in several changes of PBS for 48 h at 4°C and stained with Coomassie Brilliant Blue R-250. (g) Radioimmunostaining experiments An IgG fraction (100 pg) of sheep anti-rabbit immunoglobulins (Cappel Laboratories, Cochranville, PA) was iodinated using an “‘1 radioiodination kit (NEZ-151) from New England Nuclear (Boston, MA). Labeled protein was separated from reactants on a 2 ml AGl-X8 column (Bio-Rad Laboratories, Richmond, CA.) equilibrated with PBS containing 1 mg bovine serum albumin/ml. Solubilized Z-discs or purified myofibrils (Saide & Ullrick. 1974) were electrophoresed in SDS on a 1 mm thick 5% polyacrylamide slab gel (12 cm x 10 cm) using the discontinuous buffer system of Neville (1971). After the position of the tracking dye was marked with India ink, the gel was frozen on solid COz and divided into 5-mm wide vertical strips. The strips were soaked in 40% methanol/lO”/e acetic acid to precipitate proteins and to remove SDS, and then equilibrated with PBS containing 2.5 mM-EDTA. The gel strips were placed into Plexiglass wells (96 cm x 0.6 cm x 10 cm) which were filled with a solution of gamma globulin (1 mg/ml in PBS, 2.5 mM-EDTA) prepared from either antiserum or non-immune rabbit serum by ammonium sulfate fractionation. The wells were enclosed in a moist environment and agitated gently for 24 h at room temperature. Gel strips were washed again for 48 h at 4°C
664
J. D. SAIDE
with several changes of buffer and then incubated as before with a solution containing lz51labeled sheep anti-rabbit immunoglobulin (5 x lo6 cts/min per ml) or [‘251]protein A (New England Nuclear, Boston, MA) (7.5 x lo5 cts/min per ml) in 1 mg bovine serum albumin/ml, PBS and 2.5 mM-EDTA. After additional exhaustive washing in buffer at 4”C, the gel strips were frozen on solid CO,, and l-mm slices were counted in a gamma spectrometer. To locate band positions and to ensure that protein had not been eluted from the gels, some gel strips were stained after being taken through the procedure in buffer only (Burridge, 1976; Wang et al., 1979). (h) Indirect immunojlwrescence Myofibrils, which had bound to the surface of glass slides after settling out of suspension, were washed in PBS and incubated for 30 min at room temperature with pm-immune serum, antiserum or gamma globulin fractions. Fibrils were washed for 30 min with 3 changes of PBS and incubated for 30 min with a 1 : 32 dilution of a fluorescein-conjugated IgG fraction of goat anti-rabbit gamma globulin (Cappel Laboratories Inc., Downington, PA). They were washed as before and mounted under coverslips with 10% (v/v) glycerol in PBS. Fibrils were examined with a Zeiss phase contrast microscope equipped with epifluorescence optics. Photographs were taken with Kodak Tri-X pan film at ASA 1600 and developed in Diafine. Exposure times for photographing and printing fluorescent images of control and antibody-labeled fibrils were identical. (i) Immunostaining and electron microscopy (i) Flight muscle Jibers Honeybee longitudinal flight muscles with attached exoskeleton were glycerinated (Abbott, 1973) and kept in storage buffer at -20°C for several weeks. Before the experiment small fiber bundles were soaked for 90 min in ice-cold relaxing solution consisting of @l MKCl, 0001 iv-MgCl,, 2 mM-EGTA, 10 mM-ATP (pH 6%). Each bundle was transferred onto a small block of paraffin wax and anchored between 2 stainless steel spring clamps. One clamp was pinned to the paraffin block: the other was connected to a manually operated micropositioner which was used to stretch the muscle 0045 mm/min to lengths up to 200% rest length. Fibers were held at a given length by pinning the second clamp in position. The muscle was continually moistened with relaxing solution during this procedure. An incubation well was formed by encircling the preparation with a ridge of hot, melted paraffin wax. When the wax cooled, 125 ~1 of test solution was added to the well to cover the fibers. Preparations were incubated with undiluted antiserum or gamma globulin purified from antiserum (4.5 mg/ml in PBS, 5 mM-EDTA). Control fibers were similarly antiserum, or purified non-immune treated with either pre-immune serum, “adsorbed” gamma globulin (4.5 mg/ml in PBS, 5 mM-EDTA). Samples were incubated at 2°C in a moist environment for 48 h. They were subsequently washed for 48 h by floating the inverted paraffin blocks over ice-cold PBS, 25 mM-EDTA. For electron microscopy, fibers were fixed in their incubation wells for 2 h at room temperature with 3% glutaraldehyde in stock buffer (0.067 M-sodium phosphate, pH 7.4). They were then cut from their attachments and postfixed for 1 h in ice-cold 3% (w/v) osmium tetroxide. Samples were dehydrated in graded concentrations of alcohol, taken through propylene oxide and embedded in Araldite: DDSA (Ted Pella, Inc., Tustin, CA) (1.3 : 1). Thin sections were stained with uranyl acetate and lead citrate and examined with a Siemens (1A) electron microscope (60 kV) calibrated with a diffraction grating replica. (ii) Isolated honeybee Z-discs Purified isolated Z-discs suspended in water were mixed with an equal volume of normal or immune rabbit gamma globulin (4 mg/ml in 2 x concentrated PBS with 5 mM-EGTA). The mixture was shaken gently at room temperature for 1 h. Z-discs were subsequently sedimented at 1200g for 10 min and washed repeatedly in stock buffer until no soluble
CONNECTING
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665
protein was detected in the supernatant by measuring absorbance at 280 nm. Z-disc pellets were subsequently treated as solid tissue blocks and prepared for electron microscopy by procedures described above. Z-disc proteins Isolated Z-discs were dissolved in a solution of 7 M-guanidine . hydrochloride, 0.2% pmercaptoethanol, 25 mm-EDTA (pH 7.0) and concentrated in dialysis tubing against Aquacide IIA (Calbiochem-Behring Corp., La Jolla, CA). The solution was dialyzed against gel sample buffer containing 8 M-urea and 61% /3-mercaptoethanol. After addition of SDS to a final concentration of lo/o, the solution was clarified by centrifugation at 9000 g for 30 min. Then 400 ~1 of supernatant (2 mg/ml) was loaded onto a 409 mm x 12 mm Sepharose 4B column equilibrated with 61% SDS in gel sample buffer. 65 ml volumes were collected at a rate of 10 ml/h, and portions from alternate fractions were analyzed by SDS/polyacrylamide gel electrophoresis. The column void volume was determined in a separate run from the elution position of Blue Dextran 2000 (Pharmacia, Uppsala, Sweden). (j) Gel @ration
of
(k) Amino acid analysis Samples were dialyzed against water, lyophilized, and hydrolyzed in 2 ml 6 M-HCl for 20 h at 105°C in ampules sealed under vacuum. The hydrolyzates were dried under nitrogen, then dissolved in 601 M-HCl, and analyzed with a Beckman 119 CL analyzer equipped with a model 126 integrator. Serine, threonine and tyrosine values were not corrected for losses during hydrolysis. Estimates for methionine and half-cystine include their oxidation products, methionine sulfoxide and cysteic acid. (1) Protein determinations Protein concentrations were estimated according to the procedures of Lowry et aZ. (1951). Bovine serum albumin was used as a standard. Concentrations of gamma globulin solutions were determined by absorbance measurements at 280 nm assuming an extinction of 1.4 for a 1 mg/ml solution. Based on Lowry determinations, the extinction coefficient at 280 nm for solubilized Z-discs (1 mg/ml) was estimated at 1.46.
3. Results (a)
Characterization
of the 360,000
M, protein
SDS/polyacrylamide gel electrophoresis of solubilized isolated Z-disc preparations resolves several polypeptides. The molecular weights of three of these components are estimated to be 87,000, 158,000, and 175,000 (Saide & Ullrick, 1974; and Fig. 3(a)), and immunofluorescence studies on honeybee flight muscle fibrils confirm that these proteins originate from the Z-band (Saide & Ericson, 1979). A fourth band, which penetrates only a few millimeters into 5% gels, has an extrapolated molecular weight of 360,000. Gel densitometry studies indicate that it accounts for as much as 30% of electrophoresed material. Although it was suspected that this high molecular weight component represented aggregated protein, subsequent studies have shown it to be a unique polypeptide species. (i) Antibody speci$city The specificity of antibodies Ouchterlony double-diffusion
raised against the 360,000 M, protein was tested in experiments. Z-disc proteins dispersed in 8 M-Urea
666
J. D. SAIDE 30
25 I.7 I II x
20
.-: \ 2 2
15
a c .-> tg .-0
10
B 5 ,
10
20
30
40
50
60
70 t
80
Slice number
360~000
1751000
871000
1. Tests for specificity of immunoglobulins raised against the 360,000 M, protein. Radioactivity vwsw gel slice plot. (0) Gel treated with a 50% ammonium sulfate fraction from antiserum (1.3 mg/ml in PBS, 25 IIIM-EDTA); (0) control gel treated with gamma glbbulin fractionated from non-immune rabbit serum (1-3 mg/ml). Gel strips were subsequently incubated with lz51-labeled sheep anti-rabbit immunoglobulin (5 x lo6 cts/min per ml) in PBS, 2.5 mmEDTA, 1 mg bovine serum albumin/ml. The stained gel below the radioactivity profiles indicates the position of isolated Z-disc protein bands. The arrow marks the position of the tracking dye. Inset: Ouchterlony diffusion plate. (z) Solubilized isolated Z-discs (1.4 mg/ml) in 8 M-urea, gel sample buffer, 0.1% fl-mercaptoethanol; (a) gamma globulins fractionated from antiserum and dissolved in PBS to twice their concentration in serum (9 mg/ml). Indicated dilutions were made with PBS. (b) PBS: (n) gamma globulin fractions from normal rabbit serum twice concentrated as in (a). RG.
formed a single precipitin line when diffused in agarose against immunoglobulins ; no reaction was observed in control experiments in which immunoglobulins were replaced with non-immune gamma globulins (Fig. 1, inset). While these results suggested that antibodies recognized a single antigen in Z-disc preparations, we noted that the precipitin line formed in these studies was not hyper-sharp. We, therefore, tested immunologic specificity with the more sensitive technique of radioimmunostaining. The results of such studies in which antigen was localized on slab gels of Z-disc proteins are shown in Figure 1. A single sharp peak of radioactivity is found at the
CONNECTING
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667
PROTEIN
16
3211 10
20
30
40
50
Slice number
60
70
A
80
I
FIG. 2. Radioactivity VWSU~ gel slice plots. Gel strips were incubated with a 50% fraction (0.8 mg/ml in PBS) from antiserum against the 360,000 ilf, protein. They treated with [ 1551]protein A (7.5 x l-0’ cts/min per ml) in PBS, 2.5 mM-EDTA, albumin/ml. Radioactivity profiles (not shown) of control gels, treated with a 50% fraction of pre-immune serum (68 mg/ml in PBS), show no evidence of peaks at 360,000 M, protein. Gel electrophoresis patterns of solubilized Z-discs isolated in solutions (below, -O-O--) and solubilized myofibrils (above, - - 0 - - 0 -
ammonium sulfate were subsequently 1 mg bovine serum ammonium sulfate the position of the high ionic strength - ).
position of the 360,000 M, protein. The curve rises steeply, but its descending limb is skewed, reaching baseline at a position on the gel corresponding to a molecular weight of about 175,000. This finding suggests that the 360,000 M, protein may be unstable, giving rise to fragments of lower molecular weight. The absence of a radioactivity peak at any other position on the gel, however, demonstrates that the high molecular weight polypeptide is a unique component, immunologically distinct from other major Z-disc proteins in our preparation. To rule out the possibility that the 360,000 Mr protein was an artifact resulting from exposure of myofibrils to dilute acid, we probed two additional slab gel strips
668
J. D. SAIDE
with antibody, one containing electrophoresed proteins of purified myofibrils and the other proteins of honeybee Z-discs isolated by exposing fibrils to solutions of high ionic strength (Bullard & Sainsbury, 1977). In both cases antibody recognized polypeptides migrating with an apparent molecular weight of 360,000 (Fig. 2). Both the position and shape of the radioactivity peaks in Figures 1 and 2 are comparable. Together, these Figures demonstrate not only that the 360,000 H, protein is a component of myofibrils, but that it remains with isolated Z-discs when fibrils are extracted with either dilute lactic acid or concentrated salt solutions. and amino acid composition of the 360,000 M, protein (ii) Pur@cation The 360,000 M, protein was partially purified by chromatography of solubilized Z-discs on Sepharose4B in the presence of 91% SDS. Analysis of column fractions by SDS/polyacrylamide gel electrophoresis indicates that the high molecular weight material elutes near the void volume. Early fractions have two major components; later fractions contain, in addition, a third minor polypeptide (Fig. 3, lanes c to g). Those fractions pooled for amino acid analysis did not include the minor protein. (Similarly, this component was avoided when SDS-containing gels
a
b
c
d
e
f
9
360 vOO0
175,000 158,000
-
8 7,000
FIG. 3. SDS/5% polyacrylamide gel electrophoresis patterns of solubilized isolated Z-discs (lane a); sequential Sepharose 4B column fractions eluted near the void volume (lanes b to g). Column load: 400 ~1 of isolated Z-discs (2 mg/ml) in 8 M-We&, gel sample buffer, @l% SDS. Elution buffer: gel sample buffer with 0.1% SDS.
CONNECTING
FILAMENT TABLE
Amino
acid composition
669
PROTEIN
1
of the 360,000
M, protein
Residues/1000 (meanfs.D.) 82k 109.3f12.2 66.3+ 74.2* 1248k 75.5* 93.3* 67.55 53.1* 11.5+ 36.3k 685+ 26.9f 32.0+ 87.2k 23.3+ 505f
Cyel2 ASP Thr Ser Glu Pro Gly Ala Val Met Ile Leu TY~ Phe LYS His A%
32) 1.8 1.5 59 1.9 8.1 2.0 1,o 1.7 1.7 7% 1.2 3.4 64 1.1 45
The amino acid composition of the 360,000 bf, protein averaged from 3 different preparations. Values for methionine and half-cystine include the oxidation products methionine sulfoxide and cysteic acid. The tryptophan content w&e not measured.
of isolated Z-discs were sectioned to isolate the 360,000 M, protein for antibody production.) Fractions enriched in the high molecular weight protein are contaminated with a component which does not penetrate 5% gels. The contaminant is assumedto be a glycoprotein, since it contains oxidizable carbohydrate residues, which were detected on polyacrylamide gels by the method of Eckhardt et al. (1976) (S. G. Ericson & J. D. Saide, unpublished results). Although densitometry studies indicate that the impurity may range from 10 to 30% of total protein in pooled fractions containing the 360,000 M, material, such fractions (Fig. 3, lanes c to e) were analyzed for amino acid composition. Results tabulated from three different preparations are presented in Table 1. Since standard deviations are lessthan 10% of the meansfor most amino acids, it appears that the small but variable amount of contaminant does not significantly influence the amino acid profile. The analyses reveal no unusual features in amino acid composition except for a high proline content, which has also been observed in the amino acid composition of intact isolated Z-discs (Saide & Ullrick, 1974). (b) Localization
of the 360,000
M, component
in myo$brils and isolated
Z-discs
(i) Indirect immunojluorescence microscopy The staining pattern of honeybee myofibrils treated with antiserum to the 360,000 M, protein is unlike that observed with antiserum to other isolated Z-disc
670 (a)
J. D. SAIDE
(b)
(d)
FIG. 4. Phase (top) and fluorescence (bottom) micrographs of (a) rest length, and (b) stretched honeybee myofibrils stained with anti-serum to the 360,000 M, protein. Fibrils were stretched by gently moving the coverslip over the slide. (0) Control fibril treated with pre-immune serum. (d) Glycerin&& Lethoceruaflight muscle fibril stained with a gamma globulin fraction from antiserum (1 mg/ml in PBS). (e) Control Ledhocerua fibril treated with gamma globulin (1 mgjml) from non-immune rabbit serum. (fj Phase (left) and fluorescence (right) micrographs of glycerinated honeybee leg muscle myofibrils treated with antiserum. Magnifications: (a) to (e) 3040 x ; (f) 1440 x
CONNECTING
FILAMENT
PROTEIN
671
proteins (Saide & Ericson, 1979). Antibody labeling appears on either side of the Zband, not at its center. In rest length fibrils where the A-band occupies nearly the entire sarcomere, fluorescence appears as a doublet, with a narrow band of staining along each edge of the Z-band (Fig. 4(a)). In stretched sarcomeres the band of fluorescence is broader, extending from the edge of the Z-band to the border of the A-band at all carcomere lengths (Fig. 4(b)). N o o th er region of the fibril is labeled. Qualitatively similar results are found with immune globulins purified from antiserum either by ammonium sulfate precipitation or by adsorption onto isolated Z-discs (see Materials and Methods, section (e)). Fibrils treated with pre-immune serum (Fig. 4(c)) or a pre-immune gamma globulin fraction show no significant labeling. Rest length Lethocerus myofibrils also reveal the characteristic doublet following exposure to antibody (Fig. 4(d)). However, no antibody binding has been demonstrated in myofibrils from either honeybee leg muscle (Fig. 4(f)) or rabbit psoas muscle (not shown). (ii) Immunostaining and electron microscopy The pattern of antibody labeling in honeybee flight muscle fibers examined by electron microscopy is consistent with results of indirect immunofluorescence studies. Antibody accumulates in the region between the Z-band and the A-band, extending from the edge of the Z-band to the tips of the thick filaments where it penetrates slightly beyond the tapered ends (Fig. 5(b)). In unstretched fibers this staining is usually very intense, and the narrow bands of bound antibody which flank the Z-band have a density comparable to it. There is no evidence of antibody binding within the Z-band, however, or around its circumference, and in some myofibrils there appear to be very narrow, unstained regions at the lateral (I-Z) margins of the Z-band (Fig. 5(b)). In stretched fibers, less intensely staining patches of antibody are scattered throughout the I-band, creating a grainy appearance (Fig. 6(b)). As in rest length sarcbmeres, staining appears to overlap the tips of the thick filaments. No protein binding has been detected in stretched or unstretched control fibers treated with either pre-immune serum (Fig. 5(a)), “adsorbed” serum from which immunoglobulins have been removed (Fig. 6(a)), or gamma globulin fractions prepared from non-immune serum. Two possibilities are suggested by these results. One is that the 360,966 Jf, protein is associated with thin filaments, but that antibody binding sites are accessible only outside the A-band. The other is that the high molecular weight protein is a structural component of connecting filaments. Several findings indicate that the latter is true. (1) In Ouchterlony doublediffusion experiments native honeybee thin filament proteins, dispersed in 8 Murea, do not form precipitin lines with antiserum to the 366,666 M, protein under conditions in which Z-disc preparations react with antibody. (2) Immunoglobulins can be shown to accumulate in the region between the Z-band and the ends of thick filaments in myofibrils from which thin filaments are missing (Fig. 7)t. 7 Unexpectedly, thin filaments disappeared in fibers incubated with unfractionated antiserum. They were unaffected, however, by pm-immune serum, antiserum adsorbed with isolated Z-discs, or gamma globulins purified from antiserum. We suspect that the loss of thin filaments may have resulted from an activation of proteases of the complement system.
J. D. SAIDE
(bl
FIG. 5. (a) Longitudinal section of a rest length control myofibril treated with pre-immune serum. (Control fibrils treated with pre-immune serum, gamma globulin purified from non-immune serum (46 mgiml) or antiserum adsorbed with isolated Z-discs are indistinguishable.) (b) Honeybee myofibril labeled with gamma globulins (46 mg/ml) purified from antiserum to the 36O,OOOM, protein. Magnification : 9000 x
(3) Honeybee leg muscle myofibrils, which have broad I-bands but no apparent connections between thick filaments and the Z-line (Candia Carnevali et al., 1980), are not labeled by antibody in indirect immunofluorescence studies (Fig. 4(f)). Taken together, these observations provide compelling evidence that the 360,000 M, component is a connecting filament protein. In light of these results it seemed likely that the protein would be found in the projections which have been observed to extend from the surfaces of isolated Zdiscs (Saide & Ullrick, 1974). In fact, when isolated Z-discs are incubated with gamma globulin fractions prepared from antiserum, antibody coats this surface
CONNECTING
FILAMENT
673
PROTEIN
(a)
1 IJm FIG. 6. (a) Longitudinal section of a stretched control honeybee myofibril treated with antiserum adsorbed with isolated Z-discs. (b) Slightly oblique section through a lightly stained stretched honeybee myofibril treated with gamma globulin (46 mg/ml) purified from antiserum to the 360,000 M, protein. Magnification : 10,000 x
material, effectively sandwiching the Z-disc backbone between layers of densely staining protein (Fig. 8(b)). As in labeled myofibrils, there is no evidence of antibody accumulation within the Z-disc backbone, and in many sections pale lines of reduced binding appear at the junction between the backbone and the projections (Fig. 8(b)). No labeling has been detected in control Z-discs, treated with non-immune gamma globulins (Fig. 8(a)). Such results indicate that isolated Z-disc surface projections are remnants of connecting filaments, rather than stubs of thin filaments as was suggested earlier (Saide & Ullrick, 1974). This conclusion is supported by gel densitometry studies in which protein with the molecular weight
674
J. D. SAIDE
FIG. 7. Longitudinal adsorbed with isolated filaments in the labeled
of actin isolated
sections of slightly stretched honeybee Z-discs (left), or unfractionated antiserum myofibril. Magnification : 19,600 x
has been found to account in dilute lactic acid.
for less than
myofibrils (right).
treated with antiserum Note the absence of thin
2% of the total
protein
in Z-discs
4. Discussion The presence of connections between thick filaments and the Z-band in insect fibrillar flight muscle is now well established. Connecting filaments were noted first in Diptera, by Auber & Couteaux (1962,1963) and later in Apis (Garamviilgyi, 1965,1966u,b,1969; Trombitas & Tigyi-Sebes, 1972,1974,1979; Saide & Ullrick, 1973) and Lethocerus muscle (Zebe et al., 1968; Reedy, 1971; White & Thorsen, 1973 ; Ullrick et al., 1977). In Lethocerus, the most convincing evidence is based on electron micrographs of fibers stretched under rigor conditions so that thin filaments are torn from the Z-band (Reedy, 1971). Traversing the regions between the Z-band and the broken ends of thin filaments are fine thread-like structures, which, in cross section, are found to occupy the same lattice positions as thick filaments in the A-band (White & Thorson, 1973). In oblique sections of stretched honeybee (Apis) flight muscle, filament counts in equal areas of the I-band and overlap zone of the A-band yield similar numbers (Saide & Ullrick, 1973). Moreover, longitudinal sections of stretched fibrils, treated to remove thin filaments, also reveal thick filament continuity with the Z-band (Trombitas & Tigyi-Sebes, 1972,1974).
CONNECTING
FILAMENT
675
PROTEIN
(a
O-5 urn FIG. 8. Isolated Z-discs, viewed on edge, treated with (a) gamma globulin fractionated from nonimmune rabbit serum (2 mg/ml) or (b) gamma globulin prepared from antiserum to the 360,000 ,Wr protein (2 mg/ml).
676
J.
D. SAIDE
Although it is generally agreed that the connecting material in stretched muscle is filamentous, there has been some dispute about its form in unstretched fibers (see Ashhurst, 1977). Pringle has proposed that the connecting substance is an unstructured gel, which becomes oriented into filaments only under axial stress (Pringle, 1974,1977). This view is based on Ashhurst’s report that in oblique sections through unstretched Hemiptera fibers, connecting filaments are absent from their expected lattice position in the I-band (Ashhurst, 1967). Although irregularly shaped structures, interpreted as connecting filaments, have been described in oblique sections of rest length Diptera (Auber & Couteaux, 1962,1963) and Hymenoptera muscle (Saide & Ullrick, 1973), Ashhurst suggests that these apparent filaments are more likely to be protrusions of Z-band material into the narrow I-band (Ashhurst, 1977). In the present study evidence indicates that the material linking thick filaments to the Z-line is filamentous even in the absence of stress effects. This conclusion is based on the appearance of isolated Z-disc surface projections which our results suggest are derived from this connecting substance. In the micrograph of Z-discs, treated with gamma globulin solutions, the projections are aggregated and are not shown to advantage. However, in untreated Z-discs in which the projections are well-preserved and favorably oriented, they are found to be filamentous (Plate VII, Saide & Ullrick, 1974). These structures are not as distinct or rod-like as thin or thick filaments. They have a fluffy quality, and they appear flexible. It may be that they do not remain rigidly oriented in the unstretched myofibril and consequently are sometimes lost from their appropriate lattice position in oblique sections. Our conclusion that unstretched connecting material is in the form of filaments is also supported by the observations of Trombitas & Tigyi-Sebes (1979). These authors have published micrographs of honeybee flight muscle thick filaments which arise from adjacent sarcomeres and are joined at their tapered ends by slender, threadlike connections. In this investigation immunostaining techniques have provided evidence that connecting filaments in flight muscles from representatives of two insect orders, Hymenoptera (Apis) and Hemiptera (Lethocerus), contain a 360,000 M, protein. The protein has been localized to a region between the lateral margins of the Z-band and the ends of the myosin-containing filaments. Although the component extends a short distance beyond the tapered portions of these filaments toward the M-line, it is not clear how it is integrated within the thick filament structure. We are also uncertain how the protein is associated with Z-band components. It is likely that the 360,000 M, protein does not actually penetrate the Z-band, since no antibody binding has been observed here. Although enhanced density at the Z-line might go undetected because of its intrinsic opacity, the absence of labeling is further suggested by the failure of antibody to accumulate at the outer surface (perimeter) of the Z-line. The frequent appearance of a narrow, unstained region at the I-Z junction of labeled myofibrils and isolated Z-discs also supports the view that the 360,000 M, protein is restricted to filaments lying outside the Z-band. The possibility cannot be ruled out, however, that this protein is in fact assembled within the Z-band lattice but is masked from antibody by associated proteins.
CONNECTING
FILAMENT
PROTEIN
677
We do not know whether the high molecular weight material is the only component in connecting filaments or whether it is complexed with one or more additional proteins. Bullard et al. (1977a,b) (see also Trombitas et al., 1977) reported that antibodies raised against insect paramyosin preparations stained the region between thick filaments and the Z-band. Since the antigenic species located in this area was not found to be paramyosin, it was assumed to be a contaminant in the paramyosin preparation. This material has not been identified, however. While it may be that the contaminating antigen is a protein distinct from the 360,000 M, material, the possibility exists that it is the same component or a breakdown product. We noted earlier that the high molecular weight protein is apparently degraded to lower molecular weight, immunologically similar polypeptides. The properties of the 360,000 M, protein and other proteins that may be associated with it in connecting filaments should be of interest since these structures have been shown to have unusual physical characteristics. In honeybee myofibrils, connecting filaments can be extended to well over ten times their rest length and can support stresses which cause thick filaments to pull apart (Garamviilgyi, 1965,1966a,b,1969). Their restoring force has been dramatically demonstrated in experiments in which relaxed muscle is stretched and then allowed to go into rigor. When such preparations are released, the recoil forces of the connecting filaments cause the sarcomeres to shorten, and thin filaments, prevented from sliding by rigor links, are found crumpled against the Z-band (Trombitas & Tigyi-Sebes, 1977). The highly compliant nature of connecting filaments suggested to us that the amino acid composition of the 360,000 M, protein might be similar to that of rubber-like proteins such as resilin or elastin (Seifter & Gallop, 1966). However, our results indicate that proline is no more than 7.5% of the total residues, that hydroxyproline is absent, and that hydrophobic residues are not in unusually high proportion. The amino acid composition, then, offers no clues to explain the exceptional extensibility of these structures. Wang el al. (1979) identified three extremely large polypeptides (titin) with molecular weights exceeding 400,000 in chicken skeletal myofibrils. Immunostaining studies demonstrated that they were widespread in vertebrate and invertebrate striated muscles and, under various conditions, distributed in myofibrils at the Z-bands, M-lines, A-I junctions, or throughout the A-band. Although the molecular weights of titin and the 360,000 M, protein are similar, we find that antibodies to the latter component bind exclusively in the I-band, and label only those fibers that have been demonstrated to have connecting filaments. These observations suggest that titin and the high molecular weight material are unrelated. To summarize, a 360,006 M, protein has been identified in gels of isolated Z-discs. With immunostaining techniques we have provided evidence that it is located in the connecting filaments, which join thick filaments to the Z-band in insect oscillatory flight’ muscle, and that it arises from isolated Z-disc surface projections. For purposes of reference, and because of its origin we suggest that this protein be called “projectin”.
6’78
J. D. SAIDE
I thank MS Margaret Phillips, MS Jean Maloney and MS I. Weiss for their excellent technical assistance, Dr David Sack for sectioning tissue blocks, Dr Carl Franzblau for making available his amino acid analyzer, Mr George Crombie for performing the analyses, and Dr Benjamin Kaminer for helpful suggestions during the preparation of this manuscript. This research was supported by grants from the Muscular Dystrophy Association and the National Institutes of Health (HL22985). The work was done during the tenure of an Established Investigatorship Award from the American Heart Association.
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Goldberg, A. & Lehman, W. (1978). Biochem. J. 171, 413-418. Guba, F., Garemviilgyi, N. & Ernst, E. (1960). Proc. 4th Int. Congr. Electron Microscopy, vol. 2, pp. 324-325, Springer-Verlag, Berlin. Jewell, B. R. & Ruegg, J. C. (1966). Proc. Roy. Sot. ser. B, 164, 428-459. Lowry, D. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 193, 265-275. Neville, D. M. (1971). J. BioZ. Chem. 246, 6328-6334. Ouchterlony, 0. (1953). Actu Puthol. Microbial. Scund. 32, 231-240. Pringle, J. W. S. (1974). Symp. BioZ. Hung. 17, 67-78. Pringle, J. W. S. (1977). In Insect FZight Muscle (Tregear, R. T., ed.), pp. 177-196, Elsevier, North Holland, Amsterdam. Pringle, J. W. S. (1978). Proc. Roy. Sot. ser. B, 201, 1077130. Reedy, M. K. (1971). In Contractility of Muscle Cells and Related Processes (Podolsky, R. J., ed.), pp. 229-246, Prentice-Hall, Englewood Cliffs, New Jersey. Saide, J. D. (1980). J. Cell BioZ. 87, 269a. Saide, J. D. L Ericson, S. G. (1979). Biophys. J. 25, 242a. Saide, J. D. & Ullrick, W. C. (1973). J. Mol. BioZ. 79, 329-337. Saide, J. D. & Ullrick, W. C. (1974). J. Mol. BioZ. 87, 671-683. Seifter, S. & Gallop, P. M. (1966). In The Proteins (Neurath, H., ed.) vol. 4, 2nd edit., pp. 153458, Academic Press, N.Y. and London. Sober, H. A. & Peterson, E. A. (1958). Fed, Proc. Fed. Amer. Sot. Exp. BioZ. 17, 1116-1126. Szent-Gyorgyi, A. G., Cohen, C. & Kendrick-Jones, .J. (1971). .I. Mol. BioZ. 56, 239-258.
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Trombitas, C. &, Tigyi-Sebes, A. (1977). In Insect Ftight Muscle (Tregear, R. T., ed.), pp. 7980, Elsevier, North Holland, Amsterdam. Trombitas, C., Tigyi-Sebes, A. & Pallai, G. (1977). In Insect FZigfr,t Muscle (Tregear, R. T., ed.), pp. 53356, Elsevier, North Holland, Amsterdam. Trombitas, K. & Tigyi-Sebes, A. (1972). Acta B&him. Biophys. Acud. Sci., Hung. 7, 193194. Trombitas. K. & Tigyi-Sebes, A. (1974). Acta B&him. Biophys. Acad. Sci., Hung. 9, 243253. Trombitas, K. & Tigyi-Sebes, A. (1979). Nature (London), 281, 319-320. Ullrick, W. C.. Toselli, P. A., Chase, D. & Dasse, K. (1977). J. Ultrastruct. Res. 69, 263-271. Wang, K., McClure. J. & Tu, A. (1979). Proc. Nut. ACT. Sci., U.S.A. 76, 3698-3702. White, D. C. S. & Thorson, J. (1973). Progr. Biophys. Mol. Biol. 27, 173-255. Wray, J. S. (1979). Nature (London), 289, 325-326. Zebe, E.. Meinrenkev. W. & Ruegg, I. C. (1968). 2. Zellforsch. 87. 603-621. Edited
by H. E. Huxley