Classes of distinguishable 10 nm cytoplasmic filaments

the serum, which is part of the culture medium. One of these proteins is a2-macroglobulin. As the number of components detected is comparable in NHF dissociated with either EDTA or trypsin, an intracellular localization of these serum proteins is most likely. Immunofluorescence and electronmicroscopic immunochemical techniques will allow us to give a more precise answer to this question. This work wassupported by a grant from the Belgian Cancer Fund (ASLK) and by grant no. 3.0025.75 (FGWO).

References I. Fisher, H W, Puck, T T & Sate, G, Proc natl acad xi US 44 (1958) 4. 2. Lieberman, 1 & Ove, P, Biochim biophys acta 25 ( 1957) 449. 3. - J biol them 223 (1958) 637. 4. Hamburger. R N. Pious. D A & Mills, S E, Immunology 6 (1963) 439. 5 Tarone. G & Comoglio, P M, FEBS lett 67 (1976) 364. 6. Weiss, L, Exp cell res 17 (1959) 499. 7. - Ibid 17 (1959) 508. x. Taylor, A C, Exp cell res, suppl. X (1961) 154. 9. Martin, G R & Rubin, H, Exp cell res X5 (1974) 319. 10. Grinnell, F, Exp cell res 97 ( 1976) 265. Il. - Ibid 102 (1976) 51. I?. Gulp, L A, Terry, A H & Buniel, J F. Biochemistry 14 (1975) 406. 13. Orr, C W & Roseman, S. J membrane biol I (1969) 125. 14. Pessac, B, Alliot, F, Cornet, M & Girard, A, J cell physiol90 (1977) 23. 15. Kerbel, R S & Blakeslee, D, Immunology 3 1 (1976) 881. 16. Laurell, C B, Stand j clin lab invest 29, suppl. 124 (1972) 21. 17. Verbruggen, R, Clin them 21 (1975) 5. 18. Van der Schueren, B. Cassiman, J J & Van den Berghe, H, Cell tiss res I74 (1976) 499. 19. Verbruggen, R, Biochem j ISI (1975) 149. 20. Fasman, G D, Handbook of biochemistry and molecular biology. Proteins vol. 2, p. 491. CRC Press, Cleveland, Ohio (1976). 21. Weeke, B, Stand j immunol 2, suppl. I (1973) 47. 22. Andersen, M H & Krall, J, Stand j immtmol 4, suppl. 2 (1975) 163. 23. Bauer, K, Humangenetik 13 (1971) 49. Received May 18, 1977 Accepted July 19, 1977

Classes of distinguishable cytoplasmic filaments

10 nm

P. F. DAVISON,’ B.-S. HONG’ and P. COOKE,’ LDepartment oj’Fine Stnccture Research, Boston Biomedicul Reseurch Institute. Boston, MA 02114, und ‘Depurtment of Physiology, University of Connecticut, Furmington. CN 06032, USA Summury. The protein subunits from the 10 nm diameter filaments from chicken neurons and smooth muscle cells have similar mobilities on detergent-polyacrylamide gels but they are distinguished by thei] tryptic and chymotryptic peptide maps.

Electron microscopy has revealed as common constituents of eukaryotic cells at least three morphologically distinguishable classes of protein filaments: (1) microtubules, 24-26 nm diameter tubes that usually appear hollow; (2) microfilaments (4-6 nm diameter, frequently identified with actin filaments); and (3) a class intermediate in size. The latter measure approx. IO nm in diameter and have been variously called 100 A filaments, intermediate filaments, and, when they are found in neurons, glia or epidermal cells, neurofilaments, glial filaments, and tonofilaments. For brevity in this communication we will refer to this latter class as decefilaments (with reference to their 10 nm diameter). We have examined two types of decefilaments from chicken tissues by peptide mapping procedures and have demonstrated that they are chemically distinguishable. Therefore the decefilaments include at least two and probably more different types of organelles. Neurofilaments are perhaps the prototypical decefilaments; they have been isolated from certain invertebrates on the one hand and from mammals on the other, but these types differ in their protomer molecular weight and their solubility characteristics although all are insoluble in physiologic saline [l-3]. Tonofilaments [4] and the filaments isolated from smooth muscle also are insoluble at neutral pH except in f‘\p

Cdl

Rr.\

109 (1977)

472

Preliminary notes

-fi <,

jJ h

C D E

11

5

10

Fig. 1. Abscissa:

migration (cm); ordinate: OD. Densitometer traces from two Coomassie Bluestained 7.5% polyacrylamide gel slabs. The upper four that were run in tris-glycine-SDS with 2 M urea added to the running gel show the proteins in: (A) an extract from chicken smooth muscle; (B) a mixture of chicken brain tubuhn (partially resolved tubulins (Yand /3 are peaks 4 and 5) and brain actin; (C) actin from chicken striated muscle; (D) proteins extracted from chicken brain axons. The lower two traces show: (E) purified calf brain neurofilament; (F) chicken smooth muscle as in (A). The proteins in the second gel (in this case formulated without urea) were previously iodinated with chloramine-T; iodination leave the mobility unchanged (compare 2 and 3 with 2’ and 3’).

denaturants [S]. The distinction between mammalian neurofilaments and glial tilaments that was originally based on morphological studies [6] has more recently been questioned with the application of immunologic techniques to study the disposition of these filaments in cells [7]. We and others using the indirect immunofluorescence method have seen strong fluorescence in glial as well as neuronal cells in cryostat sections after reacting the cells with fluorescent antibody to calf neurofilament. Certain other classes of cells have also shown reaction with these antibodies, often giving rise to a perinuclear zone of fluorescence; the cells we have studied have included BHK20 (see also [8]), mouse 3T6, primary Exp Cd Res 109 (1977)

chick chorion cells and several strains of human astrocytomas provided by Dr Paul Kornblith. In the electron microscope all these cells are seen to contain decefilaments of similar morphology. In order to determine whether all decefilaments share a common primary structure we have as a first step compared by a peptide mapping procedure chicken neurofilament and smooth muscle filaments isolated from chicken gizzard muscle [5]; the latter has been one of the few tissues other than brain that has provided enough protein for analytical studies. Unfortunately we could not compare these proteins by immunological techniques because our antibody to mammalian neurofilament that reacts with other mammalian nerve extracts does not crossreact with chicken brain protein [9]. Methods The isolation of neurotilament from myelinated neurons and of decefilaments from smooth muscle have been described [2,5]; the preparation of iodinated peptide maps from proteins purified by polyacrylamide gel electrophoresis has also been reported [lo]. The autoradiographs obtained after two weeks exposure of the tryptic and chymotryptic maps prepared on thin layer silica gel plates were scanned with a Joyce-Loebl densitometer.

Results and discussion Filament-enriched fractions from selected tissue extracts were subjected to polyacrylamide gel electrophoresis. The densitometric traces from two gel slabs are illustrated in fig. 1. Samples E and F among others were run in a discontinuous buffer with added sodium dodecyl sulfate (SDS) [ 111; samples A-D were run similarly but the gels were prepared with 2 M urea added, Trace C is actin purified from chicken muscle by a procedure similar to that of Mommaerts & Parrish [12]. Trace E is purified calf neurofilament protein. Trace B is chick embryo brain tubulin prepared by the association-dissociation method of

Preliminary notes Shelanski et al. [13]; the (Yand p chains of tubulin (peaks 4 and 5) are separated in this urea-SDS gel whereas they usually comigrate in a simple SDS gel (Dr R. E. Fine, personal communication; see also [ 141). This preparation contains brain actin (peak 6) as a contaminant. Trace A is the decefilament-rich extract from chicken gizzard muscle [5] where the peak labeled 1 is probably myosin, peak 3 is inferred to be actin from its mobility and peak 2 is concluded to be the protomer of the decefilament. The latter protein migrates with tubulin in a simple, discontinuous, SDS gel and with /3tubulin in the urea-SDS gel that was used here. Trace D shows the proteins eluted by 2 M guanidine hydrochloride from a preparation of axonal segments that were obtained from 12 chicken brains by the selective flotation procedure devised by De Vries & Norton [15]. Peak 10 is inferred from its mobility to be brain actin. Two other peaks (8 and 9) bracket the position of calf neurofilament (peak 11); peak 8 was concluded to be the chicken neurofilament protein because it was the predominant peak among the denaturant-soluble proteins not only from the axon segments as shown here but also from whole chicken optic nerve (see also [ 141). The peak not numbered in fig. 1D was only a minor component in the optic nerve extract and is therefore unlikely to be the neurofilament. The protein in peak 9 was present in variable proportions in different preparations and was probably a degradation product from the neurofilament, like the satellite band we have seen in calf neurotilament preparations. Although it is not obvious from traces A and D, the chicken neurofilament protein (peak 8) migrated slightly but consistently faster than the muscle decefilament (peak 2) in both gel electrophoresis systems used in this study. The chicken neurofilament like

Fig. 2. Abscissa: R, value; ordinate:

473

OD.

Densitometertracts from autoradiographsof iodinatedpeptidemaps:(a) chymotryptic;(b) tryptic;

(A) chick neurotilament [8]; (B) calf neurofilament [ll]; (C) muscle decefilament (2’); (D) chick brain actin [lo]; (E) chick smooth muscle actin (3’); (F) chick striated muscle actin [7]. The numbers in parenthesis refer to the peaks numbered in fig. I.

those from rat and rabbit (not shown) migrates slightly slower than the calf neurofilament. In all cases the neurofilament proteins migrate between the tubulins and actin. For the experiments illustrated in this report the proteins in the chicken smooth muscle and axon extracts, purified calf neurofilament and chick-striated muscle actin were iodinated at trace levels with lz51 and subjected to electrophoresis [lo]. The stained gel resulting was scanned with a Joyce-Loebl densitometer. Traces of part of this gel are reproduced in fig. 1E and F, where F, which is the iodinated duplicate of the sample run in trace A is shown in order to demonstrate that no change in Exp Cell Res 109 (1977)

474

Preliminury notes

mobility results from trace iodination. The iodinated proteins in peaks 2’, 3’, 7, 8, 10 and I1 were extracted and then precipitated with acidified methanol. The proteins were digested with trypsin or chymotrypsin and the digests were subjected to chromatography in isopropanol-ammonia on thin layer plates. Autoradiographs from the plates on Kodak SB-5 X-ray film were scanned with a densitometer (fig. 2). The differences or similarities between the proteins must now be evaluated subjectively from the iodinated peptide maps. It is quite evident that the chymotryptic maps of gizzard and brain actin (fig. 2a, D and E) are very similar; the striated muscle actin (F) shows some differences. The same can be said (though with perhaps less confidence) of the tryptic maps of these actins (fig. 2 b, D, E, F). The maps offer support for the identification of peak 8 (fig. 1) with chicken neurofilament because there is a similarity between both tryptic and chymotryptic maps of this protein and those of calf neurofilament (fig. 2, A, B). Several features in both maps differentiate traces A and C. Thus the proteins of the smooth muscle decefilament (fig. 1, peak 2) and that from the chick brain neurofilament differ not only in electrophoretic mobility in the SDS gel (and therefore presumably in size) but also in peptide sequence. In separate experiments we have also prepared maps from (Y and p tubulins (fig. 1, peaks 4 and 5) and observed clear differences between their maps and those from the smooth muscle filament so the protein

Exp Cd Res 109 (1977)

is different from P-tubulin although it migrates in the same position under the electrophoretic conditions used here. We conclude that at least two distinguishable families of decefilament proteins exist in eukaryotes. From the fact that tonofilaments are built from protein subunits of various sizes [4] we also conclude that tonofilaments form a third class of decefilament. The functions subserved by these proteins remain to be determined. We wish to thank MS Randy Jones for excellent technical assistance and Dr Paul Kornblith who supplied the astrocyte cultures. This investigation was supported by grant NS 1 I122 from the NIH.

References 1. Shelanski, M L, Albert, S, DeVreis, G H & Norton, W T, Science 174 (1971) 243. 2. Davison, P F & Winslow, B, J neurobiol 5 (1974) 119. 3. Gilbert, D S, Newby, B J & Anderton, B H, Nature 256 (1975) 586. 4. Steinert, P M, Idler, W W & Zimmerman, S B, J mol biol 108 (1976) 547. 5. Cooke, P, J cell biol68 (1976) 539. 6. Wuerker, R B, Tissue and cell 2 (1970) I. 7. Yen, S-M, Dahl, D, Schachner, M & Shelanski, M L, Proc natl acad sci US 73 (1976) 529. 8. Blase, S H, Shelanski, M L & Chacko, S, Proc natl acad sci US 74 (1977) 662. 9. Davison, P F & Hong, B-S, Brain research. In press. IO. Davison. P F. Anal biochem 75 (1976) 129. I I. Laemmli, U K, Nature 227 (1970) 680. 12. Mommaerls. W F H M & Parrish. R G, J biol them 188 (1951) 545. 13. Shelanski, M L, Gaskin, F & Cantor, C R, Proc natl acad sci US 70 (1973) 765. 14. Davison, P F, Brain res 100 (1975) 73. 15. DeVries, G H & Norton, W T, Fed proc 30 (1971) 1142. Received June 29, 1977 Accepted July 22, 1977