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X-ray diffraction pattern of axoplasm
BIOCHIMICA ET BIOPHYSICAACTA
503
BBA Report BBA 31150 X-Ray diffraction pattern of axoplasm
W.A. DAY and D.S. GILBERT M.R. C Neurobiology Unit, 26/29 Drury Lane, London, W.C. 2 and Department of Zoology and Comparative Physiology, Queen Mary College, Mile End Road, London, E.1 (GreatBritain)
(Received October 12th, 1972)
SUMMARY Axoplasm is a dilute gel of fibrous proteins, which, in the past, has resisted attempts at characterization by X-ray diffraction. We have succeeded in producing samples sufficiently concentrated and ordered to give a diffraction pattern. Dried, stretched strands of whole axoplasm from the Myxicola giant axon give a fibre diagram resembling that of the k - m - e - f class of a proteins.
There have been numerous attempts to obtain an X-ray diffraction pattern from the fibrous proteins of nerve cells, but none succeeded in obtaining a pattern which defined the molecular organisation of the proteins x ,2. Recently, a technique for quickly extracting large amounts of pure axoplasm from the giant axon of the fan worm, Myxicola infundibulum, was discovered by one of us, and light microscopy showed that the fibrous proteins were nearly as well oriented in the extracted axoplasm as they are in vivo 3 ,4. A search for an X-ray diffraction pattern commenced. Efforts to obtain a pattern from fresh material failed, so strands of fresh, whole axoplasm were draped horizontally between two rods in a desiccator, in order to concentrate the protein by drying. These essentially lipid-free dried strands, when cut into about 20, 2-cm long sticks and bundled tightly together in the X-ray beam, gave a faint diagram. Three factors improved the contrast of the patterns: (a) more sticks in the beam, (b) high humidity during exposure to X-rays, and (c) stretching the axoplasm. Axoplasmic strands were stretched by lead weights (approx. 0.25 g), suspended from the centres of the strands while they dried. Axoplasm withstood this mechanical abuse better if 2 - 3 ml of 0.5 M EDTA, pH 7, were dripped down the freshly extracted axoplasm, followed by 2 - 3 ml of distilled water. The first rinse removed Ca 2+, which entered the gel during Biochim. Biophys. Aeta, 285 (1972) 503-506
504
BBA REPORT
Fig. 1. Wide-angleX-ray diffraction pattern of axoplasm. Axoplasm from 5 worms was rinsed with EDTA and water, then stretched with lead weights in a desiccator (CaC12) for several days. The dried axoplasm was cut into 47 sticks, which were wedged into a 1-mm glass capillary. The sticks were then wetted with approximately their own volume of distilled water. The capillary was sealed, dusted with calcite to give a ring of 3.035 A calibration spots, and oriented vertically in a toroid camera for 50 h to give this pattern. Cu Kc~radiation was used, from a Hilger-Watts f'me-focusX-ray generator operated at 50 kV. extraction and caused it to disintegrate; the second rinse prevented salt crystal formation during drying. The wide-angle X-ray pattern of axoplasm (Fig. 1) is characteristic of the k e r a t i n m y o s i n - e p i d e r m i n - f l b r i n o g e n( k - m - e - f ) class of a proteins: there is a diffuse ring centred at 3.9 + 0.4 A (mean + S.D. of 4 specimens), meridional arcs at 5.2 + 0.1 A, and large equatorial spots centred at 10.2 + 0.4 A,. The 5.2 A, arcs are commonly attributed to reflections from coiled a-helical coils s . In addition, axoplasm has consistently shown meridional arcs at 25.6 + 0.9 A. Weaker meridional arcs (not visible in Fig." 1) have also Biochim. Biophys. Acta, 285 (1972) 503-506
BBA REPORT
505
Fig. 2. Neurofilaments in a longitudinal section ofMyxicola axoplasm. Freshly extracted axoplasm was fixed in 2% glutaraldehyde, postfixed in 1% buffered osmic acid, and embedded in Taab araldite. Long axis of the axoplasm is vertical. Stretching reduces the helical swerving of the filaments.
Biochim. 8iophys. Acta, 285 (1972) 5 0 3 - 5 0 6
506
BBA REPORT
been seen at 18.4 + 0.8 A. Low-angle patterns have shown only diffuse equatorial streaks extending outwards to wide equatorial arcs near 60 A. In electron micrographs Myxicola axoplasm appears packed with typical neurofdaments, oriented predominantly parallel to the length of the axon (Fig. 2, see also ref. 6, and Gilbert, D.S., in preparation). On this basis, we provisionally identify the ot diagram with the neurofilament proteins, and conclude that a large proportion of these proteins exist as coiled o~-helical coils 7,s. Presence of a helices has been confirmed by preliminary measurements of circular dichroism of homogenized, dialyzed Myxicola axoplasm (Richards, T. and Gratzer, W., personal communication). Although these data are insufficient to establish neurofilament structure, the fibre diagram suggests that neurofilaments are composed of fibrous subunits rather than globular subunits, as is currently supposed'. Many molecular models of fibrous subunits (or protofilaments) have been developed to account for the wide-angle pattern of o~-keratins , which resembles that of axoplasm. Protofilaments are thought to consist of a few a helices twisted together s . The number of protofilaments per neurofilament may vary, since there is a wide range of reported neurof'dament diameters 2'6, and a variable number of dark spots comprising neurofilament cross-sections9 . Meridional reflections of axoplasm near 18 A and 25 h indicate considerable long-range order along the length of the filaments; whereas the 60 A equatorial reflection may arise from regularity of spacing between parallel filaments. At the light microscopic level, squid and Myxicola axoplasm have been shown to have a compound helical structure 3,4 Gilbert 4 suggested a mechanism by which this structure could arise, based on the assumption that the fibrous proteins of axoplasm formed helices of molecular dimensions. Our data strongly support this assumption, and may therefore be related to the molecular origins of nerve cell shapes. We thank M.H.F. Wilkins for valuable assistance and encouragement, N. Webb and J. Pacy for technical advice and help, Z. Gabor for photography, and B.B. Boycott and R.W. Piddington for helpful discussions. REFERENCES 1 2 3 4 5 6 7 8
Schmitt, F.O. (1968)Neurosci. Res. Prog. Bull. 6, 119-144 Huneeus, F.C. and Davison, P.F. (1970) Z Mol. Biol. 52, 415-428 Gilbert, D.S. (1972) Nature New Biol. 237,195-198 Gilbert, D.S. (1972) £ PhysioL 222, 44-45 P Pauling, L. and Corey, R.B. (1953) Nature 171, 59-61 Schmitt, F.O. (1950)J. Exp. Zool. 113,499-512 Elliott, A. (1967) in Fibrous Proteins (Crewther, W.G., ed.), pp. 115-123, Butterworths, Sydney Fraser, R.D.B., MacRae, T.P., Millwaxd,G.R., Parry, D.A.D., Suzuki, E. and TuUoch,P.A. (1971) Applied Polymer Symp. No. 18, pp. 65-83, Wiley, New York 9 Wuerker,R.B. and Palay, S.L. (1969) Tissue Cell, 1,387-402
8iochim. Biophys. Acta, 285 (1972) 503-506
503
BBA Report BBA 31150 X-Ray diffraction pattern of axoplasm
W.A. DAY and D.S. GILBERT M.R. C Neurobiology Unit, 26/29 Drury Lane, London, W.C. 2 and Department of Zoology and Comparative Physiology, Queen Mary College, Mile End Road, London, E.1 (GreatBritain)
(Received October 12th, 1972)
SUMMARY Axoplasm is a dilute gel of fibrous proteins, which, in the past, has resisted attempts at characterization by X-ray diffraction. We have succeeded in producing samples sufficiently concentrated and ordered to give a diffraction pattern. Dried, stretched strands of whole axoplasm from the Myxicola giant axon give a fibre diagram resembling that of the k - m - e - f class of a proteins.
There have been numerous attempts to obtain an X-ray diffraction pattern from the fibrous proteins of nerve cells, but none succeeded in obtaining a pattern which defined the molecular organisation of the proteins x ,2. Recently, a technique for quickly extracting large amounts of pure axoplasm from the giant axon of the fan worm, Myxicola infundibulum, was discovered by one of us, and light microscopy showed that the fibrous proteins were nearly as well oriented in the extracted axoplasm as they are in vivo 3 ,4. A search for an X-ray diffraction pattern commenced. Efforts to obtain a pattern from fresh material failed, so strands of fresh, whole axoplasm were draped horizontally between two rods in a desiccator, in order to concentrate the protein by drying. These essentially lipid-free dried strands, when cut into about 20, 2-cm long sticks and bundled tightly together in the X-ray beam, gave a faint diagram. Three factors improved the contrast of the patterns: (a) more sticks in the beam, (b) high humidity during exposure to X-rays, and (c) stretching the axoplasm. Axoplasmic strands were stretched by lead weights (approx. 0.25 g), suspended from the centres of the strands while they dried. Axoplasm withstood this mechanical abuse better if 2 - 3 ml of 0.5 M EDTA, pH 7, were dripped down the freshly extracted axoplasm, followed by 2 - 3 ml of distilled water. The first rinse removed Ca 2+, which entered the gel during Biochim. Biophys. Aeta, 285 (1972) 503-506
504
BBA REPORT
Fig. 1. Wide-angleX-ray diffraction pattern of axoplasm. Axoplasm from 5 worms was rinsed with EDTA and water, then stretched with lead weights in a desiccator (CaC12) for several days. The dried axoplasm was cut into 47 sticks, which were wedged into a 1-mm glass capillary. The sticks were then wetted with approximately their own volume of distilled water. The capillary was sealed, dusted with calcite to give a ring of 3.035 A calibration spots, and oriented vertically in a toroid camera for 50 h to give this pattern. Cu Kc~radiation was used, from a Hilger-Watts f'me-focusX-ray generator operated at 50 kV. extraction and caused it to disintegrate; the second rinse prevented salt crystal formation during drying. The wide-angle X-ray pattern of axoplasm (Fig. 1) is characteristic of the k e r a t i n m y o s i n - e p i d e r m i n - f l b r i n o g e n( k - m - e - f ) class of a proteins: there is a diffuse ring centred at 3.9 + 0.4 A (mean + S.D. of 4 specimens), meridional arcs at 5.2 + 0.1 A, and large equatorial spots centred at 10.2 + 0.4 A,. The 5.2 A, arcs are commonly attributed to reflections from coiled a-helical coils s . In addition, axoplasm has consistently shown meridional arcs at 25.6 + 0.9 A. Weaker meridional arcs (not visible in Fig." 1) have also Biochim. Biophys. Acta, 285 (1972) 503-506
BBA REPORT
505
Fig. 2. Neurofilaments in a longitudinal section ofMyxicola axoplasm. Freshly extracted axoplasm was fixed in 2% glutaraldehyde, postfixed in 1% buffered osmic acid, and embedded in Taab araldite. Long axis of the axoplasm is vertical. Stretching reduces the helical swerving of the filaments.
Biochim. 8iophys. Acta, 285 (1972) 5 0 3 - 5 0 6
506
BBA REPORT
been seen at 18.4 + 0.8 A. Low-angle patterns have shown only diffuse equatorial streaks extending outwards to wide equatorial arcs near 60 A. In electron micrographs Myxicola axoplasm appears packed with typical neurofdaments, oriented predominantly parallel to the length of the axon (Fig. 2, see also ref. 6, and Gilbert, D.S., in preparation). On this basis, we provisionally identify the ot diagram with the neurofilament proteins, and conclude that a large proportion of these proteins exist as coiled o~-helical coils 7,s. Presence of a helices has been confirmed by preliminary measurements of circular dichroism of homogenized, dialyzed Myxicola axoplasm (Richards, T. and Gratzer, W., personal communication). Although these data are insufficient to establish neurofilament structure, the fibre diagram suggests that neurofilaments are composed of fibrous subunits rather than globular subunits, as is currently supposed'. Many molecular models of fibrous subunits (or protofilaments) have been developed to account for the wide-angle pattern of o~-keratins , which resembles that of axoplasm. Protofilaments are thought to consist of a few a helices twisted together s . The number of protofilaments per neurofilament may vary, since there is a wide range of reported neurof'dament diameters 2'6, and a variable number of dark spots comprising neurofilament cross-sections9 . Meridional reflections of axoplasm near 18 A and 25 h indicate considerable long-range order along the length of the filaments; whereas the 60 A equatorial reflection may arise from regularity of spacing between parallel filaments. At the light microscopic level, squid and Myxicola axoplasm have been shown to have a compound helical structure 3,4 Gilbert 4 suggested a mechanism by which this structure could arise, based on the assumption that the fibrous proteins of axoplasm formed helices of molecular dimensions. Our data strongly support this assumption, and may therefore be related to the molecular origins of nerve cell shapes. We thank M.H.F. Wilkins for valuable assistance and encouragement, N. Webb and J. Pacy for technical advice and help, Z. Gabor for photography, and B.B. Boycott and R.W. Piddington for helpful discussions. REFERENCES 1 2 3 4 5 6 7 8
Schmitt, F.O. (1968)Neurosci. Res. Prog. Bull. 6, 119-144 Huneeus, F.C. and Davison, P.F. (1970) Z Mol. Biol. 52, 415-428 Gilbert, D.S. (1972) Nature New Biol. 237,195-198 Gilbert, D.S. (1972) £ PhysioL 222, 44-45 P Pauling, L. and Corey, R.B. (1953) Nature 171, 59-61 Schmitt, F.O. (1950)J. Exp. Zool. 113,499-512 Elliott, A. (1967) in Fibrous Proteins (Crewther, W.G., ed.), pp. 115-123, Butterworths, Sydney Fraser, R.D.B., MacRae, T.P., Millwaxd,G.R., Parry, D.A.D., Suzuki, E. and TuUoch,P.A. (1971) Applied Polymer Symp. No. 18, pp. 65-83, Wiley, New York 9 Wuerker,R.B. and Palay, S.L. (1969) Tissue Cell, 1,387-402
8iochim. Biophys. Acta, 285 (1972) 503-506