J O U R N A L OF ULTRASTRUCTURE RESEARCH
61, 149-157 (1977)
Studies on the Isolation and Substructure of Mammalian Neurofilaments WILLIAM W. SCHLAEPFER Department of Pathology, Washington University School of Medicine, 660 South Euclid, St. Louis, Missouri 63110 Received March 14, 1977, and in revised form June 1, 1977 Intact neurofilaments were isolated from minced segments of desheathed r a t peripheral nerve by subjecting tissues to osmotic shock in media containing chelating agents and sedimenting heavier tissue components by centrifugation. Isolated neurofilaments were layered on carbon-Formvar-coated grids, fixed by flotation on 5% formalin, and visualized by negative s t a i n i n g with unbuffered uranyl acetate. Neurofilaments comprised the predominant constituent organelle of these preparations. Individual neurofilaments coursed as discrete u n b r a n c h i n g linear structures for distances of at least 10 t~m. Fresh or fixed neurofllaments appeared as compact cylindrical structures 80-110/~ in diameter; lateral sidearm projections were not evident. Exposure to 0.5 M urea prior to fixation altered neurofilaments and exposed substructural configurations. Urea-treated preparations revealed the presence of 20- to 25-4 protofilaments. Pairs of protofilaments appeared to be arranged in helical configurations as evidenced by profiles with herringbone substructural patterns. Accordingly, m a m m a l i a n neurofilaments appear to closely resemble similar organelles of invertebrate species.
Neurofilaments are a prominent constituent of central and peripheral neural tissues, comprising the major structural component in the axoplasm of large myelinated nerve fibers (14, 15, 45, 50). They may well represent the prototype of an intermediate-sized cytoplasmic filament which has been widely observed in nonneural tissues. Both neurofilaments (5, 6) and intermediate-sized filaments of nonneural tissues (27, 44) have been differentiated from the smaller actin filaments by their nonreactivity with heavy meromyosin. Furthermore, exposure of tissues to colchicine or vinca alkaloids produces a notable increase in the number of neurofilaments (4, 8, 47) as well as intermediatesized filaments of non-neural tissues (1,
2, 7, 11, 20, 22-24, 26). In spite of their abundance in neuritic processes, neurofilaments have remained a poorly understood organelle. The elucidation of neurofilament biochemistry has progressed very slowly. Neurofilament-enriched fractions of mammalian brain reveal an SDS-polyacrylamide gel electro-
phoretic pattern (9, 28, 43, 52) which is significantly disparate from electrophoretic profiles of neurofilament-rich peripheral nerves (21, 41) or from those obtained from pure axoplasmic samples of giant invertebrate axons (19, 25, 36). Furthermore, a biochemical marker for neurofilaments has not yet been found which would enable the recognition and localization of neurofilamentous protein within tissue subfractions. Studies of neurofilaments have been confined by their dependence upon transmission electron microscopy for the detection and identification of the intact organelle. Visualization of neurofilaments by negative staining techniques has generally been limited to direct examinations of axoplasmic samples extruded from invertebrate axons (19, 25, 34) or ectoplasmic samples microdissected from individual mammalian neurons (37). Neurofilamentenriched fractions isolated from mammalian brain tissue are generally subjected to a variety of chemical manipulations, and the resulting neurofilaments are 149
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
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poorly v i s u a l i z e d b y n e g a t i v e s t a i n (9). A c c o r d i n g l y , t h e p r e s e n c e of n e u r o f i l a ments in tissue fractions has usually been v e r i f i e d t h r o u g h e x a m i n a t i o n of p e l l e t e d m a t e r i a l by t r a n s m i s s i o n e l e c t r o n microscopy (12, 28, 43, 52). A new technique has been developed which has enabled the preservation and s e p a r a t i o n of m a m m a l i a n n e u r o f i l a m e n t s f r o m r a t p e r i p h e r a l n e r v e as w e l l as t h e i r visualization in negatively stained preparations. The present study documents this t e c h n i q u e a n d d e s c r i b e s s u b s t r u c t u r a l feat u r e s of i s o l a t e d m a m m a l i a n n e u r o f i l a m e n t s . F u r t h e r a p p l i c a t i o n of t h i s techn i q u e c a n be a n t i c i p a t e d . T h e a v a i l a b i l i t y of a n e u r o f i l a m e n t - r i c h t i s s u e f r a c t i o n w h o s e n e u r o f i l a m e n t c o n s t i t u e n c y c a n be v i s u a l l y m o n i t o r e d s h o u l d be p a r t i c u l a r l y useful in probing physical and chemical p r o p e r t i e s of n e u r o f i l a m e n t s . MATERIALS AND METHODS Major peripheral nerves from the hind and forelimbs of 300- to 350-g male albino rats were excised and placed in isotonic saline following cardiac perfusion of etherized animals with saline to clear the vasculature of blood. Nerves were desheathed with a pair of fine eye forceps under a dissecting microscope by applying gentle distad retraction to the perineurial sleeve while securing the endoneurial content. Each rat yielded approximately 300-400 rag (wet weight) of desheathed nerve, some weight increases occurring in nerves during exposure to isotonic saline. Desheathed nerves were combined with approximately 150 mg (wet weight) of anterior and posterior i~erve roots which had been dissected from the rat spinal cord and meninges and also placed in a isotonic saline solution. These tissues were subjected to osmotic shock by immersion and gentle agitation in a hypotonic medium consisting of ethylenedinitrilotetraacetic acid (EDTA, 2 mM), ethyleneglycol-bis(~-aminoethyl ether)-N,N'-tetraacetic acid (EGTA, 2 re_M), and a sodium phosphate buffer (pH 7.0, 1 mM). After a 2-hr interval of osmotic shock, the tissues revealed a two- to threefold increase in weight. These swollen nerve fascicles were then transversely minced into 0.2- to 0.4mm segments using No. 11 surgical blades and fine eye forceps, placed in a 5-ml cylindrical vial and agitated with a 5-mm magnetic stirrer for 1 hr, avoiding bubble formation. The resulting tissues of mushy consistency were placed in a 1-ml Sorvall conical glass centrifuge tube and spun in a Sorvall
RC2-B centrifuge at 15 000 rpm for 30 min at 1820°C. Approximately 100-200 t~l of slightly opalescent, neurofilament-rich supernatant was carefully decanted from 800-900 td of precipitate. Negatively stained preparations of neurofllaments were made directly from the supernatant or from supernatant fractions which were fixed for 2 hr by equal admixtures of supernatant with 5% formaldehyde which had been buffered to pH 7.0 with 0.1 M Na~HPQ. Five percent glutaraldehyde or a combination of 2% glutaraldehyde and 5% paraformaldehyde were used as subst{tutes for formaldehyde but did not enhance the quality of fixation. Some preparations were fixed in these solutions by flotation of carbon-Formvar-coated 400mesh grids onto which a thin wet film of supernatant had been layered. Some supernatant fractions were adulterated with urea (500 mM) in the presence of NaC1 (75 raM) and incubated under these conditions for 1 hr prior to the preparation of grids. All grids were washed in PBS prior to staining with I or 2% buffered uranyl acetate or 1% phosphotungstic acid buffered to pH 6.8 with KOH. Neurofilaments were examined and photographed at magnifications up to x 40 000, using a Siemens IA Elmskop electron microscope equipped with a 100-t~m condensor and 15-~m objective apertures and operated at 80 kV. The microscope was calibrated using a carbon replica of a diffraction grating. RESULTS L a r g e n u m b e r s of i n t a c t m a m m a l i a n neurofilaments were visualized by negative s t a i n i n g techniques following their separation from r a t peripheral nerve by o s m o t i c shock a n d d i f f e r e n t i a l c e n t r i f u g a tion. M a n y overlapping n e u r o f i l a m e n t s could be s e e n a t low m a g n i f i c a t i o n s , espec i a l l y i n t h i c k l y l a y e r e d p r e p a r a t i o n s (Fig. 1). N e u r o f i l a m e n t s composed t h e p r e d o m i n a n t o r g a n e l l e w h i c h could be r e c o g n i z e d i n n e g a t i v e l y s t a i n e d p r e p a r a t i o n s , although they were invariably admixed with scattered particulate and membranous profles. An occasional intact microtubule w a s i n f r e q u e n t l y observed. I n d i v i d u a l collagen fibrils were e n c o u n t e r e d very rarely i n some p r e p a r a t i o n s . T h e profiles of i n d i v i d u a l n e u r o f i l a m e n t s were seen with greatest clarity in preparations contrasted with unbuffered u r a n y l a c e t a t e . A s m a l l a c c u m u l a t i o n of t h i s e l e c t r o n - d e n s e s t a i n t e n d e d to r i m t h e
FIG. 1. Numerous overlapping neurofilaments comprise the predominant organelle isolated from rat peripheral nerve by centrifugation of osmotically shocked tissues. Accumulation of electron-dense stain along the lateral margin of the neurofilaments accounts for their visualization at low magnification. Preparation was negatively stained with uranyl acetate following fixation by flotation of grid on 5% formalin. × 5000. FIG. 2. Individual neurofilaments course as discrete cylindrical structures with relatively smooth external surfaces. The widths of neurofilamentous profiles generally vary from 80 to 110 /~, but thinner segments occur, often near the termination of individual neurofilaments. Fixed and stained as in Fig. 1. × 100 000. 151
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lateral margins of the neurofilaments, thereby accentuating their outlined image (Fig. 2). Neurofilaments were seen to course as discrete, unbranching linear structures, often with considerable curvature (Fig. 1). Occasionally, stain would become pooled in the space enclosed within encircling neurofilaments. Individual neurofilaments could be followed for measured distances of at least 10 t~m, and it is likely that they extended for considerably greater lengths. Overlapping of neurofilaments obscured the terminations and continuities of individual filaments, especially in micrographs of low magnification used to assess filament length. The negatively stained image ofindividual neurofilaments indicated that they have a basically cylindrical structure (Fig. 2). The lateral margins of intact neurofilaments were straight and parallel, reflecting a relatively smooth surface. Lateral sidearm projections were not seen. This characteristic image was seen in preparations from unfixed supernatants as well as from fractions fixed in situ or on grids with various aldehyde fixatives. Irregularities in neurofilamentous profiles were more evident in unfixed preparations and were believed to be due to variable states of resistance to preparative artifacts among constituent neurofilaments. Disrupted neurofilaments were evidenced by linear arrays of irregular fragmented globules. These changes may have arisen from multiple small axial varicosities which could be seen along occasional filaments. A distance of 80-110 /~ generally separated the parallel lateral borders of intact and clearly marginated segments of neurofilaments. Some neurofilaments revealed contiguous segments of smaller caliber which were often but not always situated near the terminus of individual neurofilaments. These thinner segments of neurofilaments had less accumulated electron-dense stain along their lateral margins and, accordingly, were less clearly visualized. While some neurofilaments re-
vealed a tapered, single-stranded terminus, most neurofilaments terminated abruptly without disclosing substructural components. Subunit components within the cylindrical core of the neurofilament were not clearly manifested ir~ negatively stained preparations made directly from neurofilament-rich supernatant fractions. Faint linear or granular patterns could be discerned along segments of many neurofilamerits, but they were often nonrepetitive or resembled too closely similar patterns which were present in the background of the preparation. Furthermore, some granularity along the axial core of neurofilament could not be clearly distinguished from early artifactual changes. Substructural features of neurofilaments were evidenced in negatively stained preparations of neurofilaments which had been exposed to urea prior to fixation. This treatment led to a reduction in the numbers of intact neurofilaments which could be visualized by negative staining. Most of the residual, ureatreated neurofilaments appeared unchanged, but some revealed distinctive alterations. The most prevalent urea-induced change in neurofilaments was manifested by a n expansion in width of the neurofilament profile accompanied by the appearance of a substructural pattern within the dilated axial core (Figs. 3 and 4). The urea-enlarged neurofilaments measured up to 260/~ in width and showed a herringbone substructural pattern in which the parallel arrays which coursed in one direction were often predominant (Figs. 3 and 4). This pattern was suggestive of a helical or spiral arrangement of linear subunits. The crisscrossing linear subunits of the urea-treated neurofilaments measured 20-25/k in width. Assuming a spiral configuration, the subunits appeared to rise at a 28 +_ 4° angle from the horizontal plane with a pitch of 40-50 /~ in expanded neurofilaments with 150to 180-/~ diameters. Urea-induced enlarge-
Fro. 3. Enlarged neurofilament (160/~ in diameter) and nonenlarged filament (upper right) following a 1-hr incubation in urea (500 mM) and NaC1 (75 raM). The enlarged neurofilament reveals a herringbone substructure indicative of a helical a r r a n g m e n t of constituent protofilaments. Fixed and stained as in Fig. 1. x 250 000. FIG. 4. Herringbone substructural pattern in neurofilament enlarged to 210/~ in diameter following a 1-hr incubation in urea (500 raM) and NaC1 (75 raM). Crisscrossing protofilaments measure 20-30/~ in width. Fixed and stained as in Fig. 1. x 335 000. Fro. 5. Splaying of neurofilamentous terminus into paired filamentous subunits following a 1-hr exposure to urea (500 m/l//) and NaC1 (75 raM). Fixed and stained as in Fig. 1. × 275 000. Fro. 6. Neurofilamentous profile revealing multiple parellel protofilamentous components following a 1-hr incubation in urea (500 raM) and NaC1 (75 mM). Intact neurofilament is present a t lower left. Fixed and stained as in Fig. 1. x 100 000. 153
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ments of greater magnitude were associated with a corresponding increase in the size of the pitch, as illustrated in Fig. 4 showing a neurofilament with diameter and subunit pitches of 210 and 80 A, respectively. A less frequently encountered alteration of neurofilaments which followed their exposure to urea was manifested as a splaying of the profile into subunit components (Figs. 5 and 6). On very rare occasions, a neurofilamentous profile was seen which appeared to divide into paired subunit components (Fig. 5). More often, profiles of multiple parallel protofilaments could be seen (Fig. 6), without transition to a larger filamentous structure being evidenced. The profiles of subunit protofilaments measured 20-30/~ in width. DISCUSSION The use of osmotic shock to separate neurofilaments from peripheral nerve was prompted by previous findings of this laboratory (40). These studies demonstrated a marked depletion of neurofilaments among minced segments of rat peripheral nerve following their exposure to low ionic strength buffer. This loss of neurofilaments was associated with an increasing liberation of protein into incubation media of decreasing ionic strength. However, examination of nerve extracts obtained from osmotically shocked nerve disclosed an instability of neurofilamentous structures when present in dilute concentrations in media of low ionic strength (41). Accordingly, the present study has modified the treatment of peripheral nerve by osmotic shock in order to maintain the structural integrity of neurofilaments during their separation and isolation. The most critical procedural modifications include a prolonged immersion of desheathed peripheral nerve (or intact nerve roots) in hypotonic solution, the inclusion of cationic chelators in this incubation medium, and the reduction of tissue dilutions during
subsequent mincing and centrifugation of tissues. Negatively stained profles of neurofilaments of r a t peripheral nerve closely resembled the appearance of neurofilament profiles of invertebrate giant axons (19, 25) as well as those from mammalian brain tissues (9, 37). Similar profiles have been identified as intermediate-sized filaments of non-neural tissues (27). Actin filaments are of smaller caliber (39), while microtubules are larger and are distinguished by longitudinal subdivisions (13). Negatively stained images of isolated intact neurofilaments reveal a relatively smooth external surface on the neurofilament. In sectioned tissue, neurofilaments are surrounded by irregular wispy sidearm projections (49, 51). In tissues impregnated with heavy metals, both neurofilaments and their lateral projections have been visualized as part of the threedimensional cytoplasmic network (32--35, 37). The absence of lateral appendages on isolated neurofilaments is indicative of a weak association between the axial filament and sidearm appendage which may not survive isolation procedures. It is also possible that sidearm appendages represent axoplasmic material which becomes adherent to neurofilaments during in vivo fixation and processing for ultrastructural examination. Irregular globules which were randomly scattered amid invertebrate neurofilaments were considered to represent detached neurofilamentous sidearms (19). Similar globules were also noted to be admixed among peripheral nerve neurofilaments but could not be differentiated from fragments of disrupted neurofilaments (41). Furthermore, antibodies reactive against surface components in the axial core of neurofilaments also reacted with elements on the surface of these irregular globules (41). Freshly isolated, intact neurofilaments from peripheral nerve displayed a compact cylindrical structure. Substructural fea-
SUBSTRUCTURE OF ISOLATED NEUROFILAMENTS tures of neurofilaments were not seen, except when neurofilaments were examined after exposure to urea. It is likely that the denaturing effects of urea weakened cohesive bonds, thereby allowing the neurofilament to expand, exposing the subunit pattern to the axial core. The relatively infrequent occurrence of altered neurofilaments during their disruption by urea would suggest that the urea-enlarged neurofilament represents an unstable configuration during disassociation of subunits. The lack of transitions between compact and expanded neurofilaments would also indicate that urea-induced neurofilament expansion is also a very transient event. The alterations of neurofilaments induced by urea revealed the presence of 20to 25-A protofilaments within mammalian neurofilaments. Furthermore, these protofilaments appear to be arranged in helical configurations. However, the precise configuration of protofilamentous arrangement within neurofilaments remains less certain. Geometric analyses of the ureainduced, herringbone neurofilamentous profiles as representative of helical structures would suggest that a pair of protofilaments is incorporated into each helical spiral. The measured pitch of 40-50 A within neurofilamentous helices 150-180 /~ in diameter would account for approximately half the anticipated distance (8096 /~) which would occur between corresponding loops of an individual protofilamentous spiral with a 28° angle rise from the horizontal. Accordingly, a pair of intertwining protofilaments could account for the observed profiles of urea-altered neurofilamentous helical structures. The occasionally observed splitting of neurofilamentous profiles into paired subunits and the more frequently encountered multiplicity of parallel protofilaments suggest additional complexity of the substructural arrangement of neurofilaments from rat nerve. In fact, the structural configuration of these mammalian neurofila-
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ments may closely resemble the pattern of assembly which has been observed in invertebrate neurofilaments. Negatively stained preparations of neurofilaments from the giant axons of the seaworm, Myxicola infundibulum, have revealed a superhelical pattern (coiled coils) in which the parent neurofilament is composed of a pair of spiraling 40- to 55-A subunits, each in turn consisting of two 20- to 25-A protofilaments (30). Circular dichroism of purified neurofilaments from Myxicola axoplasm indicates that neurofilaments contain more than 60% a-helix (31). X-ray diffraction analyses of neurofilament-rich Myxicola axoplasm have demonstrated a prominent 5.2-A meridional arc (10), indicative of a coiled-coil a-helical structure (38), which suggests that the superhelix is a prominent structural configuration within axoplasmic protein constituents. The underlying helical substructure of neurofilaments may also be partly responsible for larger spiral configurations within axoplasmic structure. Individual neurofilaments are believed to spiral in relationship to neighboring neurofi][aments as they course longitudinally within the axon (50). The superimposition of small and larger twisting structural patterns has been described as characteristic of both squid (34, 35) and Myxicola (1618) axoplasm. A spiraling arrangement of neurofilamentous subunits could account for the central translucency often noted in transversely sectioned neurofilaments (3, 219, 49-51). However, it would seem unlikely that protofilaments could be visualized :in their helical configurations since the pitch of the helix (40-50/~) is considerably less than the thickness of the sectioned tissue. Nevertheless, it is possible that preparative procedures could alter neurofilaments, thus enabling the visualization of their protofilamentous components. The presence of a spiraling substructure in mammalian neurofilaments may be evidenced in sectioned tissues of certain dis-
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eased conditions. ~Twisted tubules" are k n o w n to a c c u m u l a t e i n t h e p e r i k a r y a a n d n e u r i t i c processes of h u m a n c e r e b r a l cort i c a l n e u r o n s i n specific d e g e n e r a t i v e states (42, 46, 48). These argyrophilic s t r u c t u r e s a p p e a r as a p a i r of i n t e r t w i n i n g f i l a m e n t s w i t h c o m b i n e d d i a m e t e r of 200 w h i c h n a r r o w s to 100 /~ a t s u c c e s s i v e 800-/~ i n t e r v a l s . I t is q u i t e c o n c e i v a b l e that these twisted filamentous structures a r i s e f r o m a l t e r a t i o n s of n e u r o f i l a m e n t s , t h u s e x p o s i n g t h e i n h e r e n t s p i r a l i n g conf i g u r a t i o n of t h i s o r g a n e l l e . T h e h y p o t h e t ical n e u r o f i l a m e n t a l t e r a t i o n i n t h e s e degenerating neurons might not involve m u c h m o r e t h a n a l o o s e n i n g b e t w e e n composite s u b u n i t s , n o t u n l i k e t h e u r e a - i n d u c e d e n l a r g e m e n t s of n e u r o f i l a m e n t s demonstrated in the present study. This work was supported by Grant No. NS 08620 and Research Career Development Award No. NS 70037 from the National Institutes of Health. REFERENCES 1. BHISEY, A. N., AND FREED, J. J., Exp. Cell Res. 64, 419 (1971). 2. BLOSE, S. H., AND CHACKO, S., J. Cell Biol. 70, 459 (1976). 3. BLUMCKE,S., AND NIEDORF,H. R.,Beitr. Pathol. Anat. Allg. Pathol. 130, 133 (1964). 4. BUNGE, R., AND BUNGE, M . , A n a t . Rec. 160, 323 (1968). 5. BURTON, P. R., AND KIRKLAND, W. L., Nature New Biol. 239, 244 (1972). 6. CHANG, C. M., AND GOLDMAN, R. D., J. Cell Biol. 57, 867 (1973). 7. CROOP, J., AND HOLTZER, H., J. Cell Biol. 65, 271 (1975). 8. DANIELS, M. P., J. Cell Biol. 58, 463 (1973). 9. DAVISON,P. F., AND WINSLOW,B., J. Neurobiol. 5, 119 (1974). 10. DAY, W. A., AND GILBERT, D. S., Biochim. Biophys. Acta 285, 503 (1972). 11. DEBRABANDER, M., AERTS, F., VANDEVEIRE, R., AND BORGERS, M., Nature (London) 253, 119 (1975). 12. DEVRIES, G. H., ENG, L. F., LEWIS, D. L., AND HADFIELD,M. G., Biochim. Biophys. Acta 439, 133 (1976). 13. ERICKSON, H. P., J. Supramol. Struct. 2, 393 (1974). 14. FRIEDE,R. L., MIYAGHISHI,T., AND HU, K. H., J. Anat. 108, 365 (1971). 15. FRIEDE, R. L., AND SAMORAJSKI,T., Anat. Rec.
167, 379 (1970). 16. GILBERT, D. S., J. Physiol. (London) 222, 44P (1972). 17. GILBERT,D. S., Nature New Biol. 237, 195 (1972). 18. GILBERT, D. S., J. Physiol. (London) 253, 257 (1975). 19. GILBERT,D. S., NEWBY, B. J., AND ANDERTON, B. H., Nature (London) 256, 586 (1975). 20. GOLDMAN,R. D., ANDKNIPE, D. M., Cold Spring Harbor Syrup. Quant. Biol. 37, 523 (1972). 21. HOFFMAN,P. N., AND LASEK, R. J., J. Cell Biol. 66, 351 (1975). 22. HOLMES, K. V., AND CHOPPIN, P. W., J. Cell Biol. 39, 526 (1968). 23. HOLTROP,M. E., RAISZ,L. G., AND SIMMONS, H. A., J. Cell Biol. 60, 346 (1974). 24. HOLTZER, H., CROOP, J., DIENSTMAN, S., ISHIKAWA, H., AND SOMLYO, A. P., Proc. Nat. Acad. Sci. USA 72, 513 (1975). 25. HUNEEUS, F. C., AND DAVISON, P. F., J. Mol. Biol. 52, 415 (1970). 26. ISHIKAWA,H., BISCHOFF, R., AND HOLTZER, H., J. Cell Biol. 38, 538 (1968). 27. ISHIKAWA,H., BISCHOFF, a., A N D HOLTZER, H., J. Cell Biol. 43, 312 (1969). 28. IQBAL,K., WISNIEWSKI,H. M., GRUNDKE-IQBAL, I., KORTHALS,J. K., AND TERRY, R. D., J. Histochem. Cytochem. 23, 563 (1975). 29. KADOTA,T., AND KADOTA,K., J. Electron Microsc. 21, 218 (1972). 30. KRISHNAN, N., AND LASEK, R. J., J. Cell Biol. 67, 226a (1975). 31. LASEK,R. J., ANDHOFFMAN,P. N., in GOLDMAN, R., POLLARD,T., AND ROSENBAUM,J. (Eds.), Cell Motility, pp. 1021-1049. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 32. LEBEux, Y. J., Z. Zellforsch. Mikrosk. Anat. 143, 239 (1973). 33. LEWIS, J. C., AND BURTON,P. R., J. Cell Biol. 59, A193 (1973). 34. METUZALS,J., J. Cell Biol. 43, 480 (1969). 35. METUZALS,J., AND IZZARD, C. S., J. Cell Biol. 43, 456 (1969). 36. METUZALS,J., AND MUSHYNSKI, W. E., Biol. Bull. 147, 491 (1974). 37. METUZALS,J., AND MUSHYNSKI,W. E., J. Cell Biol. 61, 701 (1974). 38. PAULING,L., AND COREY, R. B., Nature (London) 171, 59 (1953). 39. POLLARD,T. D., AND WEII-IING,R. R., CRC Crit. Rev. Biochem. 2, 1 (1974). 40. SCHLAEPFER,W. W., J. Ultrastruct. Res. 36, 367 (1971). 41. SCHLAEPFER,W. W., J. Cell Biol. 74, 226 (1977). 42. SCHOCHET,S. S., LAMBERT,P. W., AND LINDENBERG, R., Acta Neuropathol. 11,330 (1968). 43. SHELANSKI,M. L., ALBERT,S., DEVRIES, G. H., AND NORTON, W. T., Science 174, 1242 (1971).
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Studies on the Isolation and Substructure of Mammalian Neurofilaments WILLIAM W. SCHLAEPFER Department of Pathology, Washington University School of Medicine, 660 South Euclid, St. Louis, Missouri 63110 Received March 14, 1977, and in revised form June 1, 1977 Intact neurofilaments were isolated from minced segments of desheathed r a t peripheral nerve by subjecting tissues to osmotic shock in media containing chelating agents and sedimenting heavier tissue components by centrifugation. Isolated neurofilaments were layered on carbon-Formvar-coated grids, fixed by flotation on 5% formalin, and visualized by negative s t a i n i n g with unbuffered uranyl acetate. Neurofilaments comprised the predominant constituent organelle of these preparations. Individual neurofilaments coursed as discrete u n b r a n c h i n g linear structures for distances of at least 10 t~m. Fresh or fixed neurofllaments appeared as compact cylindrical structures 80-110/~ in diameter; lateral sidearm projections were not evident. Exposure to 0.5 M urea prior to fixation altered neurofilaments and exposed substructural configurations. Urea-treated preparations revealed the presence of 20- to 25-4 protofilaments. Pairs of protofilaments appeared to be arranged in helical configurations as evidenced by profiles with herringbone substructural patterns. Accordingly, m a m m a l i a n neurofilaments appear to closely resemble similar organelles of invertebrate species.
Neurofilaments are a prominent constituent of central and peripheral neural tissues, comprising the major structural component in the axoplasm of large myelinated nerve fibers (14, 15, 45, 50). They may well represent the prototype of an intermediate-sized cytoplasmic filament which has been widely observed in nonneural tissues. Both neurofilaments (5, 6) and intermediate-sized filaments of nonneural tissues (27, 44) have been differentiated from the smaller actin filaments by their nonreactivity with heavy meromyosin. Furthermore, exposure of tissues to colchicine or vinca alkaloids produces a notable increase in the number of neurofilaments (4, 8, 47) as well as intermediatesized filaments of non-neural tissues (1,
2, 7, 11, 20, 22-24, 26). In spite of their abundance in neuritic processes, neurofilaments have remained a poorly understood organelle. The elucidation of neurofilament biochemistry has progressed very slowly. Neurofilament-enriched fractions of mammalian brain reveal an SDS-polyacrylamide gel electro-
phoretic pattern (9, 28, 43, 52) which is significantly disparate from electrophoretic profiles of neurofilament-rich peripheral nerves (21, 41) or from those obtained from pure axoplasmic samples of giant invertebrate axons (19, 25, 36). Furthermore, a biochemical marker for neurofilaments has not yet been found which would enable the recognition and localization of neurofilamentous protein within tissue subfractions. Studies of neurofilaments have been confined by their dependence upon transmission electron microscopy for the detection and identification of the intact organelle. Visualization of neurofilaments by negative staining techniques has generally been limited to direct examinations of axoplasmic samples extruded from invertebrate axons (19, 25, 34) or ectoplasmic samples microdissected from individual mammalian neurons (37). Neurofilamentenriched fractions isolated from mammalian brain tissue are generally subjected to a variety of chemical manipulations, and the resulting neurofilaments are 149
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poorly v i s u a l i z e d b y n e g a t i v e s t a i n (9). A c c o r d i n g l y , t h e p r e s e n c e of n e u r o f i l a ments in tissue fractions has usually been v e r i f i e d t h r o u g h e x a m i n a t i o n of p e l l e t e d m a t e r i a l by t r a n s m i s s i o n e l e c t r o n microscopy (12, 28, 43, 52). A new technique has been developed which has enabled the preservation and s e p a r a t i o n of m a m m a l i a n n e u r o f i l a m e n t s f r o m r a t p e r i p h e r a l n e r v e as w e l l as t h e i r visualization in negatively stained preparations. The present study documents this t e c h n i q u e a n d d e s c r i b e s s u b s t r u c t u r a l feat u r e s of i s o l a t e d m a m m a l i a n n e u r o f i l a m e n t s . F u r t h e r a p p l i c a t i o n of t h i s techn i q u e c a n be a n t i c i p a t e d . T h e a v a i l a b i l i t y of a n e u r o f i l a m e n t - r i c h t i s s u e f r a c t i o n w h o s e n e u r o f i l a m e n t c o n s t i t u e n c y c a n be v i s u a l l y m o n i t o r e d s h o u l d be p a r t i c u l a r l y useful in probing physical and chemical p r o p e r t i e s of n e u r o f i l a m e n t s . MATERIALS AND METHODS Major peripheral nerves from the hind and forelimbs of 300- to 350-g male albino rats were excised and placed in isotonic saline following cardiac perfusion of etherized animals with saline to clear the vasculature of blood. Nerves were desheathed with a pair of fine eye forceps under a dissecting microscope by applying gentle distad retraction to the perineurial sleeve while securing the endoneurial content. Each rat yielded approximately 300-400 rag (wet weight) of desheathed nerve, some weight increases occurring in nerves during exposure to isotonic saline. Desheathed nerves were combined with approximately 150 mg (wet weight) of anterior and posterior i~erve roots which had been dissected from the rat spinal cord and meninges and also placed in a isotonic saline solution. These tissues were subjected to osmotic shock by immersion and gentle agitation in a hypotonic medium consisting of ethylenedinitrilotetraacetic acid (EDTA, 2 mM), ethyleneglycol-bis(~-aminoethyl ether)-N,N'-tetraacetic acid (EGTA, 2 re_M), and a sodium phosphate buffer (pH 7.0, 1 mM). After a 2-hr interval of osmotic shock, the tissues revealed a two- to threefold increase in weight. These swollen nerve fascicles were then transversely minced into 0.2- to 0.4mm segments using No. 11 surgical blades and fine eye forceps, placed in a 5-ml cylindrical vial and agitated with a 5-mm magnetic stirrer for 1 hr, avoiding bubble formation. The resulting tissues of mushy consistency were placed in a 1-ml Sorvall conical glass centrifuge tube and spun in a Sorvall
RC2-B centrifuge at 15 000 rpm for 30 min at 1820°C. Approximately 100-200 t~l of slightly opalescent, neurofilament-rich supernatant was carefully decanted from 800-900 td of precipitate. Negatively stained preparations of neurofllaments were made directly from the supernatant or from supernatant fractions which were fixed for 2 hr by equal admixtures of supernatant with 5% formaldehyde which had been buffered to pH 7.0 with 0.1 M Na~HPQ. Five percent glutaraldehyde or a combination of 2% glutaraldehyde and 5% paraformaldehyde were used as subst{tutes for formaldehyde but did not enhance the quality of fixation. Some preparations were fixed in these solutions by flotation of carbon-Formvar-coated 400mesh grids onto which a thin wet film of supernatant had been layered. Some supernatant fractions were adulterated with urea (500 mM) in the presence of NaC1 (75 raM) and incubated under these conditions for 1 hr prior to the preparation of grids. All grids were washed in PBS prior to staining with I or 2% buffered uranyl acetate or 1% phosphotungstic acid buffered to pH 6.8 with KOH. Neurofilaments were examined and photographed at magnifications up to x 40 000, using a Siemens IA Elmskop electron microscope equipped with a 100-t~m condensor and 15-~m objective apertures and operated at 80 kV. The microscope was calibrated using a carbon replica of a diffraction grating. RESULTS L a r g e n u m b e r s of i n t a c t m a m m a l i a n neurofilaments were visualized by negative s t a i n i n g techniques following their separation from r a t peripheral nerve by o s m o t i c shock a n d d i f f e r e n t i a l c e n t r i f u g a tion. M a n y overlapping n e u r o f i l a m e n t s could be s e e n a t low m a g n i f i c a t i o n s , espec i a l l y i n t h i c k l y l a y e r e d p r e p a r a t i o n s (Fig. 1). N e u r o f i l a m e n t s composed t h e p r e d o m i n a n t o r g a n e l l e w h i c h could be r e c o g n i z e d i n n e g a t i v e l y s t a i n e d p r e p a r a t i o n s , although they were invariably admixed with scattered particulate and membranous profles. An occasional intact microtubule w a s i n f r e q u e n t l y observed. I n d i v i d u a l collagen fibrils were e n c o u n t e r e d very rarely i n some p r e p a r a t i o n s . T h e profiles of i n d i v i d u a l n e u r o f i l a m e n t s were seen with greatest clarity in preparations contrasted with unbuffered u r a n y l a c e t a t e . A s m a l l a c c u m u l a t i o n of t h i s e l e c t r o n - d e n s e s t a i n t e n d e d to r i m t h e
FIG. 1. Numerous overlapping neurofilaments comprise the predominant organelle isolated from rat peripheral nerve by centrifugation of osmotically shocked tissues. Accumulation of electron-dense stain along the lateral margin of the neurofilaments accounts for their visualization at low magnification. Preparation was negatively stained with uranyl acetate following fixation by flotation of grid on 5% formalin. × 5000. FIG. 2. Individual neurofilaments course as discrete cylindrical structures with relatively smooth external surfaces. The widths of neurofilamentous profiles generally vary from 80 to 110 /~, but thinner segments occur, often near the termination of individual neurofilaments. Fixed and stained as in Fig. 1. × 100 000. 151
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WILLIAM W. SCHLAEPFER
lateral margins of the neurofilaments, thereby accentuating their outlined image (Fig. 2). Neurofilaments were seen to course as discrete, unbranching linear structures, often with considerable curvature (Fig. 1). Occasionally, stain would become pooled in the space enclosed within encircling neurofilaments. Individual neurofilaments could be followed for measured distances of at least 10 t~m, and it is likely that they extended for considerably greater lengths. Overlapping of neurofilaments obscured the terminations and continuities of individual filaments, especially in micrographs of low magnification used to assess filament length. The negatively stained image ofindividual neurofilaments indicated that they have a basically cylindrical structure (Fig. 2). The lateral margins of intact neurofilaments were straight and parallel, reflecting a relatively smooth surface. Lateral sidearm projections were not seen. This characteristic image was seen in preparations from unfixed supernatants as well as from fractions fixed in situ or on grids with various aldehyde fixatives. Irregularities in neurofilamentous profiles were more evident in unfixed preparations and were believed to be due to variable states of resistance to preparative artifacts among constituent neurofilaments. Disrupted neurofilaments were evidenced by linear arrays of irregular fragmented globules. These changes may have arisen from multiple small axial varicosities which could be seen along occasional filaments. A distance of 80-110 /~ generally separated the parallel lateral borders of intact and clearly marginated segments of neurofilaments. Some neurofilaments revealed contiguous segments of smaller caliber which were often but not always situated near the terminus of individual neurofilaments. These thinner segments of neurofilaments had less accumulated electron-dense stain along their lateral margins and, accordingly, were less clearly visualized. While some neurofilaments re-
vealed a tapered, single-stranded terminus, most neurofilaments terminated abruptly without disclosing substructural components. Subunit components within the cylindrical core of the neurofilament were not clearly manifested ir~ negatively stained preparations made directly from neurofilament-rich supernatant fractions. Faint linear or granular patterns could be discerned along segments of many neurofilamerits, but they were often nonrepetitive or resembled too closely similar patterns which were present in the background of the preparation. Furthermore, some granularity along the axial core of neurofilament could not be clearly distinguished from early artifactual changes. Substructural features of neurofilaments were evidenced in negatively stained preparations of neurofilaments which had been exposed to urea prior to fixation. This treatment led to a reduction in the numbers of intact neurofilaments which could be visualized by negative staining. Most of the residual, ureatreated neurofilaments appeared unchanged, but some revealed distinctive alterations. The most prevalent urea-induced change in neurofilaments was manifested by a n expansion in width of the neurofilament profile accompanied by the appearance of a substructural pattern within the dilated axial core (Figs. 3 and 4). The urea-enlarged neurofilaments measured up to 260/~ in width and showed a herringbone substructural pattern in which the parallel arrays which coursed in one direction were often predominant (Figs. 3 and 4). This pattern was suggestive of a helical or spiral arrangement of linear subunits. The crisscrossing linear subunits of the urea-treated neurofilaments measured 20-25/k in width. Assuming a spiral configuration, the subunits appeared to rise at a 28 +_ 4° angle from the horizontal plane with a pitch of 40-50 /~ in expanded neurofilaments with 150to 180-/~ diameters. Urea-induced enlarge-
Fro. 3. Enlarged neurofilament (160/~ in diameter) and nonenlarged filament (upper right) following a 1-hr incubation in urea (500 mM) and NaC1 (75 raM). The enlarged neurofilament reveals a herringbone substructure indicative of a helical a r r a n g m e n t of constituent protofilaments. Fixed and stained as in Fig. 1. x 250 000. FIG. 4. Herringbone substructural pattern in neurofilament enlarged to 210/~ in diameter following a 1-hr incubation in urea (500 raM) and NaC1 (75 raM). Crisscrossing protofilaments measure 20-30/~ in width. Fixed and stained as in Fig. 1. x 335 000. Fro. 5. Splaying of neurofilamentous terminus into paired filamentous subunits following a 1-hr exposure to urea (500 m/l//) and NaC1 (75 raM). Fixed and stained as in Fig. 1. × 275 000. Fro. 6. Neurofilamentous profile revealing multiple parellel protofilamentous components following a 1-hr incubation in urea (500 raM) and NaC1 (75 mM). Intact neurofilament is present a t lower left. Fixed and stained as in Fig. 1. x 100 000. 153
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ments of greater magnitude were associated with a corresponding increase in the size of the pitch, as illustrated in Fig. 4 showing a neurofilament with diameter and subunit pitches of 210 and 80 A, respectively. A less frequently encountered alteration of neurofilaments which followed their exposure to urea was manifested as a splaying of the profile into subunit components (Figs. 5 and 6). On very rare occasions, a neurofilamentous profile was seen which appeared to divide into paired subunit components (Fig. 5). More often, profiles of multiple parallel protofilaments could be seen (Fig. 6), without transition to a larger filamentous structure being evidenced. The profiles of subunit protofilaments measured 20-30/~ in width. DISCUSSION The use of osmotic shock to separate neurofilaments from peripheral nerve was prompted by previous findings of this laboratory (40). These studies demonstrated a marked depletion of neurofilaments among minced segments of rat peripheral nerve following their exposure to low ionic strength buffer. This loss of neurofilaments was associated with an increasing liberation of protein into incubation media of decreasing ionic strength. However, examination of nerve extracts obtained from osmotically shocked nerve disclosed an instability of neurofilamentous structures when present in dilute concentrations in media of low ionic strength (41). Accordingly, the present study has modified the treatment of peripheral nerve by osmotic shock in order to maintain the structural integrity of neurofilaments during their separation and isolation. The most critical procedural modifications include a prolonged immersion of desheathed peripheral nerve (or intact nerve roots) in hypotonic solution, the inclusion of cationic chelators in this incubation medium, and the reduction of tissue dilutions during
subsequent mincing and centrifugation of tissues. Negatively stained profles of neurofilaments of r a t peripheral nerve closely resembled the appearance of neurofilament profiles of invertebrate giant axons (19, 25) as well as those from mammalian brain tissues (9, 37). Similar profiles have been identified as intermediate-sized filaments of non-neural tissues (27). Actin filaments are of smaller caliber (39), while microtubules are larger and are distinguished by longitudinal subdivisions (13). Negatively stained images of isolated intact neurofilaments reveal a relatively smooth external surface on the neurofilament. In sectioned tissue, neurofilaments are surrounded by irregular wispy sidearm projections (49, 51). In tissues impregnated with heavy metals, both neurofilaments and their lateral projections have been visualized as part of the threedimensional cytoplasmic network (32--35, 37). The absence of lateral appendages on isolated neurofilaments is indicative of a weak association between the axial filament and sidearm appendage which may not survive isolation procedures. It is also possible that sidearm appendages represent axoplasmic material which becomes adherent to neurofilaments during in vivo fixation and processing for ultrastructural examination. Irregular globules which were randomly scattered amid invertebrate neurofilaments were considered to represent detached neurofilamentous sidearms (19). Similar globules were also noted to be admixed among peripheral nerve neurofilaments but could not be differentiated from fragments of disrupted neurofilaments (41). Furthermore, antibodies reactive against surface components in the axial core of neurofilaments also reacted with elements on the surface of these irregular globules (41). Freshly isolated, intact neurofilaments from peripheral nerve displayed a compact cylindrical structure. Substructural fea-
SUBSTRUCTURE OF ISOLATED NEUROFILAMENTS tures of neurofilaments were not seen, except when neurofilaments were examined after exposure to urea. It is likely that the denaturing effects of urea weakened cohesive bonds, thereby allowing the neurofilament to expand, exposing the subunit pattern to the axial core. The relatively infrequent occurrence of altered neurofilaments during their disruption by urea would suggest that the urea-enlarged neurofilament represents an unstable configuration during disassociation of subunits. The lack of transitions between compact and expanded neurofilaments would also indicate that urea-induced neurofilament expansion is also a very transient event. The alterations of neurofilaments induced by urea revealed the presence of 20to 25-A protofilaments within mammalian neurofilaments. Furthermore, these protofilaments appear to be arranged in helical configurations. However, the precise configuration of protofilamentous arrangement within neurofilaments remains less certain. Geometric analyses of the ureainduced, herringbone neurofilamentous profiles as representative of helical structures would suggest that a pair of protofilaments is incorporated into each helical spiral. The measured pitch of 40-50 A within neurofilamentous helices 150-180 /~ in diameter would account for approximately half the anticipated distance (8096 /~) which would occur between corresponding loops of an individual protofilamentous spiral with a 28° angle rise from the horizontal. Accordingly, a pair of intertwining protofilaments could account for the observed profiles of urea-altered neurofilamentous helical structures. The occasionally observed splitting of neurofilamentous profiles into paired subunits and the more frequently encountered multiplicity of parallel protofilaments suggest additional complexity of the substructural arrangement of neurofilaments from rat nerve. In fact, the structural configuration of these mammalian neurofila-
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ments may closely resemble the pattern of assembly which has been observed in invertebrate neurofilaments. Negatively stained preparations of neurofilaments from the giant axons of the seaworm, Myxicola infundibulum, have revealed a superhelical pattern (coiled coils) in which the parent neurofilament is composed of a pair of spiraling 40- to 55-A subunits, each in turn consisting of two 20- to 25-A protofilaments (30). Circular dichroism of purified neurofilaments from Myxicola axoplasm indicates that neurofilaments contain more than 60% a-helix (31). X-ray diffraction analyses of neurofilament-rich Myxicola axoplasm have demonstrated a prominent 5.2-A meridional arc (10), indicative of a coiled-coil a-helical structure (38), which suggests that the superhelix is a prominent structural configuration within axoplasmic protein constituents. The underlying helical substructure of neurofilaments may also be partly responsible for larger spiral configurations within axoplasmic structure. Individual neurofilaments are believed to spiral in relationship to neighboring neurofi][aments as they course longitudinally within the axon (50). The superimposition of small and larger twisting structural patterns has been described as characteristic of both squid (34, 35) and Myxicola (1618) axoplasm. A spiraling arrangement of neurofilamentous subunits could account for the central translucency often noted in transversely sectioned neurofilaments (3, 219, 49-51). However, it would seem unlikely that protofilaments could be visualized :in their helical configurations since the pitch of the helix (40-50/~) is considerably less than the thickness of the sectioned tissue. Nevertheless, it is possible that preparative procedures could alter neurofilaments, thus enabling the visualization of their protofilamentous components. The presence of a spiraling substructure in mammalian neurofilaments may be evidenced in sectioned tissues of certain dis-
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WILLIAM W. SCHLAEPFER
eased conditions. ~Twisted tubules" are k n o w n to a c c u m u l a t e i n t h e p e r i k a r y a a n d n e u r i t i c processes of h u m a n c e r e b r a l cort i c a l n e u r o n s i n specific d e g e n e r a t i v e states (42, 46, 48). These argyrophilic s t r u c t u r e s a p p e a r as a p a i r of i n t e r t w i n i n g f i l a m e n t s w i t h c o m b i n e d d i a m e t e r of 200 w h i c h n a r r o w s to 100 /~ a t s u c c e s s i v e 800-/~ i n t e r v a l s . I t is q u i t e c o n c e i v a b l e that these twisted filamentous structures a r i s e f r o m a l t e r a t i o n s of n e u r o f i l a m e n t s , t h u s e x p o s i n g t h e i n h e r e n t s p i r a l i n g conf i g u r a t i o n of t h i s o r g a n e l l e . T h e h y p o t h e t ical n e u r o f i l a m e n t a l t e r a t i o n i n t h e s e degenerating neurons might not involve m u c h m o r e t h a n a l o o s e n i n g b e t w e e n composite s u b u n i t s , n o t u n l i k e t h e u r e a - i n d u c e d e n l a r g e m e n t s of n e u r o f i l a m e n t s demonstrated in the present study. This work was supported by Grant No. NS 08620 and Research Career Development Award No. NS 70037 from the National Institutes of Health. REFERENCES 1. BHISEY, A. N., AND FREED, J. J., Exp. Cell Res. 64, 419 (1971). 2. BLOSE, S. H., AND CHACKO, S., J. Cell Biol. 70, 459 (1976). 3. BLUMCKE,S., AND NIEDORF,H. R.,Beitr. Pathol. Anat. Allg. Pathol. 130, 133 (1964). 4. BUNGE, R., AND BUNGE, M . , A n a t . Rec. 160, 323 (1968). 5. BURTON, P. R., AND KIRKLAND, W. L., Nature New Biol. 239, 244 (1972). 6. CHANG, C. M., AND GOLDMAN, R. D., J. Cell Biol. 57, 867 (1973). 7. CROOP, J., AND HOLTZER, H., J. Cell Biol. 65, 271 (1975). 8. DANIELS, M. P., J. Cell Biol. 58, 463 (1973). 9. DAVISON,P. F., AND WINSLOW,B., J. Neurobiol. 5, 119 (1974). 10. DAY, W. A., AND GILBERT, D. S., Biochim. Biophys. Acta 285, 503 (1972). 11. DEBRABANDER, M., AERTS, F., VANDEVEIRE, R., AND BORGERS, M., Nature (London) 253, 119 (1975). 12. DEVRIES, G. H., ENG, L. F., LEWIS, D. L., AND HADFIELD,M. G., Biochim. Biophys. Acta 439, 133 (1976). 13. ERICKSON, H. P., J. Supramol. Struct. 2, 393 (1974). 14. FRIEDE,R. L., MIYAGHISHI,T., AND HU, K. H., J. Anat. 108, 365 (1971). 15. FRIEDE, R. L., AND SAMORAJSKI,T., Anat. Rec.
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