Preliminary Biochemical Investigations of the Intermediate Filaments 1

Transcription

1 1970 by Academic Press, Inc. J. IJLTRASTRUCTURE RESEARCH 33, (1970) 399 Preliminary Biochemical Investigations of the Intermediate Filaments 1 JOHN E. RASH, ~ JERRY W. SHAY, z AND JOHN J. BIESELE Department of Zoology, The University of Texas at Austin, Austin, Texas Received July 22, 1969, and in revised form March 24, 1970 Preliminary biochemical investigations of differentiating cardiac muscle first revealed the occurrence of a third class of filaments, differing in appearance and reactivities from the thick and thin filaments of the sarcomere. These intermediate filaments were more resistant to extraction with neutral salts, fl-mercaptoethanol, and urea than were the thick filaments, thin filaments, or Z material. Combined with the data from the accompanying report, sufficient evidence is presented to discount virtually all previous models of myofibrillogenesis. In 1965, Heuson-Stiennon (11) first reported the general occurrence of a heterogeneous class of filaments in presarcomere myoblasts of developing striated muscle and indicated that the observed filaments were not identical to the sarcomeric thin filaments, as was (and still is) commonly assumed. In the accompanying report, visual identification of at least three classes of filaments is presented and is compared with the data from striated muscle presented by Heuson-Stiennon (11), Kelly (19), and Ishikawa et al. (16, 17). The present investigation, initiated in 1967 and completed in 1968 (22, 23), was originally conceived as a series of experiments to identify and characterize the presarcomere subunits and to determine whether the "free cytoplasmic thin filaments" were composed of actin (20), myosin (11), or both (10). As a direct result of this investigation, a third class of filaments differing significantly in composition and reactivities from the thick and thin filaments of cardiac muscle was first recognized (22, 23, 25) and is the subject of an accompanying report (26). No definitive information is presently available concerning the biochemical composition or reactivities of the newly described intermediate filaments of cardiac muscle. Data from possibly similar filaments recently described in de~veloping skeletal muscle z Supported by research and training grants 5-T01-GM-00337, GM , and FR and by Research Career Award 5-K6-CA (to J. J. B.), all of the NIH. Present address: Finney-Howell Cancer Research Laboratory, Johns Hopkins Medical Institutions, Baltimore, Maryland Present address: Department of Physiology and Cell Biology, University of Kansas. Lawrence, Kansas J. Ultrastructure ~esearch

2 400 RASH, SHAY, AND BIESELE (17--19) is correspondingly inadequate, although fragmentary but contradictory evidence has been presented (17, 20). Isolated filaments from muscle homogenates, considered to be thin filaments by Obinata et al. (20) but possessing the properties of the newly described intermediate filaments (20, 22), were shown by biochemical techniques to possess appreciable amounts of myosin-binding actin. However, heavy meromyosin labeling experiments by Ishikawa et ai. (17), which demonstrated specific attachment to thin filaments with no observed binding to intermediate filaments or other subcellular structures, are considered to be convincing evidence that the actin component observed by Obinata et al. (20) must reside in contaminating thin filaments necessarily present in the crude muscle homogenates. Because the following experiments led to the initial identification of the intermediate filaments in differentiating cardiac muscle (25, 26) and the identification of the peripheral cellular matrix as a probable source of presarcomeric thin filaments (19, 26), we submit our original data as a basis for additional inquiry. MATERIALS AND METHODS Hearts of embryonic chick from 38 hours to 19 days of incubation were excised, rinsed once in S/Srensen's phosphate buffer (Sabatini et al., 28), and placed in one or more of various extraction media (24, 30). Attempts were made to extract specific myofibrillar components with techniques reported to remove actin and/or thin filaments (24, 30), myosin and/or thick filaments (8, 9, 15, 22), and tropomyosin and/or Z bands, intercalated disks, and desmosomes (24). Electron microscope examination of the extracted tissues followed fixation and embedding procedures listed in the accompanying report (26). In order to reduce osmotic damage and to increase in situ preservation of solubilized components, 2½ % buffered glutaraldehyde was added gradually to the final extraction media. Electron microscope examination was completed at the University of Texas at Austin on a student training electron microscope (Siemens Elmiskop I) equipped with variable intermediate lens and double condenser. Resolution of these early micrographs was considered adequate for general cytomorphology but not for high magnification measurements. Thin filament extraction. In an effort to differentiate between chemically different but perhaps physically similar filaments, electron microscope examinations followed nonspecific extractions of actin (4, 30, 31) and of thin filaments (22, 23, 25) using one of several neutral salt extraction procedures. Embryonic hearts of stages 11 to 15 were excised and placed for 1 hour in the "actomyosin" extractant (2, 8, 9, 15, 30-32) composed of 0.6 M potassium iodide, 5 mm sodium thiosulfate, and 0.01 M /%mercaptoethanol solution, removed, and fixed in phosphate-buffered glutaraldehyde (26). Additional extractions were carried out with buffered KI, slowly diluted

3 PRELIMINARY BIOCHEMICAL INVESTIGATIONS OF THE INTERMEDIATE FILAMENTS 401 from 0.6 M to 0.1 M before fixation. In an attempt to reduce osmotic damage to the tissue and to increase the opacity of the almost transparent hearts, additional extractions 'were attempted following brief exposure (30 seconds) to buffered osmium tetroxide, a procedure reported (23, 25) to allow removal of the thin filaments while partially stabilizing the thick filaments, Z bands, and various other cytoplasmic structures. Although only 30 seconds of exposure to OsO4 was found to stabilize the Z bands [possibly indicating the presence of unsaturated carbon-carbon bonds characteristic of lipids (3)], longer periods of prefixation were employed before extraction, yielding data on the relative "fixability" of the thick, intermediate, and thin filaments. After rinsing in buffer, the slightly darkened tissues were placed in 0.6 M KI for 5-30 minutes, rinsed, and prepared for electron microscopy. Thick filament disruption. Because OsO~ is a strong oxidizing agent and was observed by us to react rapidly with sulfhydryl groups of/%mercaptoethanol as well as with unsaturated carbon compounds (lipids), we hoped to clarify the nature of the high osmiophilia of the Z bands, thick filaments, and intermediate filaments observed in the preceding extraction experiments. The high proportion of cysteine in the myosin filament (5, 7) prompted attempts to disrupt only the thick filaments by use of 0.01 M/3-mercaptoethanol (25) in 0.1 M S6rensen's buffer. After 15 minutes to 3 hours of exposure, the tissue was placed in buffered glutaraldehyde and prepared for electron microscopy. Z band,extraction. Disruption of Z bands, intercalated disks, and desmosomes without major disruption of thick or thin filaments was accomplished utilizing 3 M urea as previously reported by Rash et al. (24). Additional urea extractions were attempted!following stabilization of Z bands and thick filaments by brief prefixation with OsO4 (see above). RESULTS Attempt.s to biochemically identify specific myofibrillar components by differential extraction.of the various filaments and Z band substance revealed unexpected results. Initial extraction, by means of the Szent-Gy/Srgyi (30, 31) neutral salt method for the removal of "actomyosin," disrupted thick and thin filaments (Fig. 1 a) but not Z bands, intercalated disks, or desmosomes (Fig. 1 b), but the degree of tissue disruption and the lack of specificity indicated the necessity of other approaches. By slow'ly diluting the 0.6 M KI extraction medium to 0.1 M before fixation, a random redeposition of thick filaments was accomplished intra- and extracellularly (Fig. 2). [We wish to thank Dr. H. E. Huxley (12, 14) for assistance in interpreting this micrograph.] These filaments varied in diameter and length, but none were less than 150 A in diameter and all had lateral projections. Material of undetermined derivation formed "halos" around the thick filaments, but limited resolution restricted

4 402 RASH, SHAY, AND BIESELE detailed analysis. Thin filaments were attached to the partially disrupted Z bands but were not seen free in the cytoplasm or the extracellular matrix. A few isolated filaments (arrows), not then recognized as intermediate filaments, were observed, but it must be remembered [see accompanying report (26)] that few intermediate filaments normally remain in this older tissue. In order to increase cellular permeability to 0.6 M KI and to stabilize sulfhydryl groups and unsaturated carbon compounds, brief osmication preceded several extractions with neutral salt solutions. In very young tissue, the Z bands remain continuous with similarly dense material beneath the plasmalemma (Fig. 3 a), closely resembling sarcolemmal-z band attachments (29) in untreated myocytes (Fig. 3b). The sarcomeric thin filaments and the peripheral cellular matrix [now shown to be similar to presarcomere thin filaments (19, 26)] were removed with 0.6 M KI (Fig. 3a), but the thick filaments, and the Z material were little affected. Thus, preliminary evidence was obtained which indicated a possible similarity in composition between the thin filaments and the then unresolved peripheral filamentous ',matrix." Attempted disruption of disulfide bridges in myosin filaments (5) by 30 minutes' exposure to 0.01 M fi-mercaptoethanol in 0,1 M S/Srensen's buffer sequentially removed portions of the thick filaments corresponding to the A zones (Fig. 4). Despite major tissue damage, the Z bands, intercalated disks, desmosomes, thin filaments, and the now recognized intermediate filaments remained clearly recognizable. [The "beaded" appearance of thin filaments may represent attached myosin molecules (cf. 13, 27), but limited resolution did not permit definite conclusions.] In contrast to early assumptions by Heusson-Stiennon (11), the differential response of thick and intermediate filaments is assumed to indicate lack of homology. A previous report by Rash et al. (24) demonstrated the extraction of Z band material with 3 M urea but did not indicate the maintained presence of intermediate filaments, alteration in filament diameters (thin filaments measured 65 and 85 A and thick filaments A), or the stability of the intermediate filaments (see double arrow, FIo. 1. Early chick ventricle extracted with neutral salt solution (0.6 M KI and 0.6 M NaC1) designed to extract myosin (9, 15), but shown to have actin contamination (30). Z bands, intercalated disks and desmosomes remain, x and x ca Fro. 2. Random redeposition of thick filaments (M) after dilution of 0.6 M KI extraction medium. Thin filaments remain attached to Z bands. Several intermediate filaments (arrows) are present in this older cytoplasm, x FIG. 3. Continuity of early Z band (26 somite chick ventricle) with similar material attached to sarcolemma. Peripheral filamentous matrix and sarcomere thin filaments removed following brief exposure to OsO4 and to prolonged exposure to 0.6 M KI. Compare to Fig. 3b, from tissue fixed without extraction, x and x FIG. 4. Substantial disruption of thick filaments following 3 hours exposure to 0.01 M fl-mercaptoethanol in phosphate buffer. Intermediate filaments (IF) are little affected. Beaded appearance of thin filaments may be due to attachment of released myosin molecules. Note continuity of intercalated disk (ID) and Z band. x

5 o z m

6 404 RASH, SHAY, AND BIESELE Fig. 5). Under the conditions reported the Z material, and the intermediate filaments differ in resistance to extraction and, thus, possibly differ in composition. However, the dense tufts of material which remained near Z bands in the earlier report may not differ in composition from intermediate filament material and may be remnants of a portion of Z material. After thick filament stabilization by brief OsO4 prefixation (see above), thin filaments could be removed (Figs. 6 and 7) by exposing the tissues for 2-6 hours in 0.3 M to 1 M urea. At low magnification, nuclei are recognizable, as are numerous welldeveloped myofibrils, but details of sarcomere substructure are not resolvable. Higher magnifications (Figs. 7a and b) demonstrate almost total removal of the thin filaments from the A and I zones, with thick (130 A) filaments and Z bands of the sarcomere remaining clearly recognizable. Although the thick filaments have reduced diameters, it should be noted that osmium-fixed thick filaments normally are smaller than glutaraldehyde-fixed filaments (1, 6, 21). An additional class of "free cytoplasmic thin filaments" (23) now shown to be the larger (115 and 130 A) intermediate filaments resist prolonged extraction with urea and are not noticeably altered. It should be noted, however, that the thin filaments are extremely labile after brief exposure to osmium tetroxide (Rash, unpublished observation) and their removal during subsequent staining procedures may be attributed to nonspecific effects of the various buffers and stains employed. The figures from the various extraction procedures represent selected areas within the tissues and are not intended to convey the overall effects of the various reagents tested. However, the reproducibility of the various extraction procedures and the differential effects of the reagents on specific myofibrillar subunits first indicated to us the possibility of an additional class (or classes) of filaments in differentiating cardiac muscle. We thus suggest that these naively conceived procedures may serve as a useful base for additional inquiry. DISCUSSION In the accompanying paper (26), a third class of filaments was demonstrated in differentiating cardiac myoblasts and myocytes. These intermediate filaments were shown to be heterogeneous with respect to diameters, to be not physically equivalent to thick or thin filaments or intact Z band material, and to be reduced in number in late stages of myocyte differentiation. Although no enzymatic digestions have been FIG. 5. Disruption of Z bands, intercalated disks, and desmosomes with 3 M urea. Intermediate filaments and thin filaments (double-headed arrows) clearly recognizable, x FIG. 6. Low magnification of early bicellular layer of heart following 1 minute exposure to OsO4 and to 6 hours exposure to 0.3 M urea. x FIG. 7. High magnifications from same tissue. Thin filaments are removed, but thick filaments, Z bands, and intermediate filaments (double headed arrows) remain. Cross section (Fig. 7b) reveals extent of thin filament removal and

7 > E > > > --4

8 406 RASH, SHAY, AND BIESELE attempted, we have assumed a protein composition. In this report we have demonstrated limited biochemical evidence based on extraction experiments which support the physical data and which initially indicated the presence of the intermediate filaments (23). Under the conditions reported, intermediate filaments were first recognized due to their relatively high resistance to disruption by several different procedures. The lack of disruption with/~-mercaptoethanol presumably indicates the presence of very few disulfide bridges. This should be contrasted with the ease of disruption of the thick filaments and the relatively high proportion of cysteine residues in myosin. These data and the physical data from the accompanying report are considered sufficient evidence to discount the Heuson-Stiennon model (11) of thick filament formation by growth of intermediate filament-like precursors. The disruption of thin filaments by exposure to urea or neutral salt solutions following brief exposure to osmium tetroxide can be contrasted with the preservation of the larger intermediate filaments under the same conditions. The parallel disruption of the sarcomeric thin filaments, the peripheral thin filament matrix, and the smaller intermediate filaments may indicate a similarity in composition of these three subunits. Similar conclusions were obtained in the accompanying report, based on physical similarities and close proximity during very early myofibrillogenesis. Additionally, a mechanism of intermediate filament-thin interconversion, possibly in association with Z material deposition was considered likely. The disruption of Z material (Z bands, intercalated disks, and desmosomes) with 1 M to 3 M urea (23, 24) without disruption of intermediate filaments may indicate a fundamental difference in composition. However, tufts of dense material which remain near disrupted Z bands (cf. 24) are similar to tufts of dense material occasionally observed with attached intermediate filaments near forming Z bands and desmosomes. In possible support of the concept of filament interconversion is the observation of numerous larger thin filaments (85 ~) near Z bands following disruption of Z bands with urea. Similar 85 fi, filaments are occasionally observed "trapped" in intercalated disks (cf. Figs. 46 and 47 in ref. 5a). The possibility of a release of Z material precursors as the mediator of intermediate-thin filament interconversion following urea exposure would help to explain the similarity in deposition of Z bands, intercalated disks, and desmosomes, and the apparent obligatory association of various diameters of filaments during formation of the various Z structures. In summary, we have presented the results of several preliminary attempts to identify and characterize the presarcomere subunits present in myoblasts and early myocytes. The results indicate fundamentally different properties for Z bands, thick filaments, thin filaments, and the newly discovered intermediate filaments. However, insufficient data are presently available to permit any definitive statements concerning

9 PRELIMINARY BIOCHEMICAL INVESTIGATIONS OF THE INTERMEDIATE FILAMENTS 407 composition or reactivities of the intermediate filaments. We suggest that others attempt fluorescent labeling experiments (particularly fluorescent antitropomysin), enzymatic digestions, and specific radioactive amino acid labeling experiments, because formation, possible interconversion, and disappearance of the intermediate filaments may have important bearing on the formation and deposition of specific sarcomere subunits. REFERENCES 1. ALLEN, E. R. and PEPE, F. A., Amer. J. Anat. 116, 115 (1965). 2. BAILEY, K., Bioehem. J. 43, 271 (1948). 3. BURKL, W. and SCmECHL, H., J. Histoehem. Cytochem. 16, 157 (1968). 4. CORSI, A. and PERRY, S. V., Biochem. J. 68, 12 (1958). 5. DREIZEN, P., GERSHMAN, L. C., TROTTA, P. P. and SHRACHER, A., J. Gen. Physiol. 50 (2), 85 (1967). 5a. FAWCETT, D. W. and McNuTT, N. S., J. Cell Biol. 42, 1 (1969). 6. FISCHMAN, D. A., J. Cell Biol. 32, 557 (1967). 7. GIBBONS, I. R., Annu. Rev. Biochem. 37, 521 (1968). 8. HANSON, J. and HUXLEY, H. E., Biochim. Biophys. Acta 23, 250 (1957). 9. HASSELBACH, W. and SCHNEIDER, G., Biochem. Z. 321,461 (1951). 10. HAY, E. D., in DEHAAN, R. L. and URSPRUNG, H. (Eds.), Organogenesis, p. 315, Holt, New York (1965). 11. HEUSON-STEINNON, J. A., J. Microsc. (Paris) 4, 657 (1965). 12. HUXLEY, H. E., J. MoI. Biol. 7, 381 (1963) Proc. Royal Soc. Ser. B, 160, 442 (1964) personal communication (1968). 15. HUXLEY, H. E. and HANSON, J., Biochim. Biophys. Acta 23, 229 (1957). 16. ISHIKAWA, H., BISCHOFF, R. and HOETZER, H., J. Cell Biol. 38, 538 (1968) ibid. 43, 312 (1969). 18. KELLY, D. E., Anat. Rec. 160, 374 (1968) ibid. 163, 403 (1968). 20. OBINATA, R., YAMAMOTO, M. and MARUYAMA, K., Develop. Biol. 14, 192 (1966). 21. PRZYBYLSKI, R. L. and BLUMBERG, J. M., Lab. Invest. 15, 839 (1966). 22. RASH, J. E., A Third Class of Filaments in Early Cardiac Myogenesis, Ph. D. dissertation, The Univ. of Texas at Austin, RASH, J. E., SHAY, J. W. and BIESELE, J. J., J. CellBiol. 35, ll0a (1967) J. Ultrastruet. Res. 24, 181 (1968) J. Cell Biol. 43, l12a (1969) J. Ultrastruet. Res. 0, 000 (1970). 27. REEDY, M. K., HOLMES, K. C. and TREGEAR, R. T., Nature 207, 1276 (1965). 28. SABATINI, D. D., BENSCH, K. and BARRNETT, R. J., J. Cell Biol. 17, 19 (1963). 29. STEIN, R. J., RICHTER, W. R., ZUSSMAN, R. A. and BRYNJOLFSSON, G., d. Cell Biol. 29, 168 (1966). 30. SZENT-GY6RGYI, A. G., Arch. Bioehem. Biophys. 31, 97 (1951 a) J. Biol. Chem. 192, 361 (1951b). 32. VILLAFRANCA, G. W., SCHEINBLUM, T. S. and PHILPOTT, D. E., Biochim. Biophys. Acta 34, 147 (1959).