Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 5006-5010, October 1978
Cell Biology
Differences in a and (3 polypeptide chains of tubulin resolved by electron microscopy with image reconstruction (microtubules/zinc-tubulin sheets/optical diffraction)
RICHARD H. CREPEAU, BRUCE MCEWEN, AND STUART J. EDELSTEIN Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
Communicated by G. G. Hammes, July 17, 1978
ABSTRACT Electron microscopic techniques have been used to reveal two classes of subunits of tubulin in ordered arrays. Presumably the two classes correspond to the a and 13 polypeptide chains of tubulin that have been distinguished by chemical criteria. The two types of subunits alternate along individual protofilaments in microtubules, microtubule-precursor sheets, and extended zinc-tubulin sheets. The resolution of the two types of polypeptide chains is achieved by improved negative staining methods which produce micrographs with layer lines at 28 A-' and 84 A-1 in optical or computed transforms, in addition to the layer lines at 21 A-1 and 42 A-1 described previously [Crepeau, R. H., McEwen, B., Dykes, G. & Edelstein, S. J. (1977)J Mol. BioL 116,301-3151. In microtubules or microtubule-precursor sheets, adjacent protofilaments are staggered by about 10 A, but parallel, in the sense that the a-13 vector points in the same direction for all of the protofilaments of the microtubule. However, for the sheets assembled in the presence of zinc, adjacent protofilaments are staggered by about 21 A and oriented in an antiparallel arrangement with alterate protofilaments related by a 2-fold screw axis. The antiparallel alignment of the protofilaments in the zinc-tubulin sheets accounts for their planarity (no tubular structures are found in the presence of moderate concentrations of zinc), since the intrinsic curvature found with parallel alignment of protofilaments in the absence of zinc would be cancelled by the antiparallel arrangement. Tubulin, the principal component of microtubules, is a molecule with a molecular weight of 110,000 (1). It can be dissociated into a and 1 polypeptide chains which have similar molecular weights (about 55,000) but differ in electrophoretic mobility, amino acid sequence, and extent of phosphorylation (2-4). In terms of the structure of microtubules, morphological units spaced about 40 A along the microtubule axis are observed by electron microscopy and x-ray diffraction (5-9) corresponding in size to about 55,000 molecular weight or the size of a single a or 1 polypeptide chain. In principle, microtubules with 13 strands of protofilaments could be composed of (i) a and ,chains alternating along protofilaments; (ii) some other regular pattern, such as a2 and 12 homodimers alternating along protofilaments; or (iii) a random arrangement of a and 13 polypeptide chains. Pattern i has been favored on the basis of crosslinking data, which support a heterodimer (a13) structure for tubulin (10), and observations on flagellar microtubules, which give an 80 A-i reflection in transforms suggestive of a pairing of a and 13 chains as would be expected for a heterodimer (7). However, the situation is still ambiguous, since the crosslinking data also reveal appreciable amounts of homodimers (10) and the 80 A reflection could also arise from pairings of polypeptide chains in arrangements other than heterodimers. Direct evidence for pattern i, such as the observation of distinct structures alternating along protofilaments, would settle this issue, and such evidence is presented here. Our evidence for an alternating pattern of a and 13 chains is The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
based on negatively stained electron micrographs subjected to image reconstruction by optical diffraction and computed Fourier transform methods (11, 12). Earlier work with this approach by Erickson (6) and Amos and Klug (7) revealed the helical parameters of tubulin arrays but little information on the structure of the morphological units. Recently, we described methods for extending the resolution of the analysis beyond the previous limit of a 42 A-1 layer line to a 21 A-1 layer line, which revealed a distinct polarity to the individual morphological units (13). In addition, a novel form of tubulin assembly obtained in the presence of zinc (14) was characterized, and also gave reciprocal space maxima on 42 A-1 and 21 A-1 layer lines (13). These zinc-tubulin structures are extended sheets with up to 50 protofilaments compared to up to 13 protofilaments seen for the sheets formed in the absence of zinc that are precursors of microtubules (6, 13, 15). The zinc-tubulin sheets also differ from the microtubule precursor sheets in their lattice parameters. With new procedures for preparing the samples for electron microscopy, we have now observed two additional reciprocal space layer lines, at 28 A-1 and 84 A-1, which provide the data needed to resolve differences in alternate morphological units along individual protofilaments. MATERIALS AND METHODS Tubulin Preparation. Microtubules were purified from pig brains by alternating cycles of depolymerization and polymerization (16-19) as described (13). Zinc-induced tubulin sheets were grown in 0.25 mM ZnCl2/0. 1 M 2(N-morpholino)ethanesulfonic acid, pH 6.4/1 mM GTP/1 mM 2-mercaptoethanol/0.25 mM MgCl2 by allowing the solution to incubate at room temperature for approximately 1 hr. The room temperature incubation produced slightly better quality sheets than the 35°C previously used. Electron Microscopy. All samples examined in the electron microscope in this work were prepared along the lines of the mica flotation technique of Horne and Ronchetti (20). The room temperature incubation of the tubulin in the presence of zinc was halted after the presence of large quantities of sizeable sheets were detected on normal microscope grids and one drop of material was applied to a freshly cleaved mica sheet, approximately 2 X 2 cm, followed immediately with a single drop of 0.5% uranyl acetate. The mica sheet was then tilted with tweezers to ensure coverage of all the available surface. Excess material was drained by holding the edge of the mica to a piece of filter paper and setting it aside to dry. The sample-coated mica was then placed in a vacuum evaporator and a very thin carbon support film was evaporated onto the sample. This carbon-coated sample was then floated on a second solution of 0.5% uranyl acetate negative stain and picked up with holey grids on filter paper. To aid in the flotation, we sharpened the mica to a point as described by Horne and Ronchetti (20) and used a micrometer driver stage attached to tweezers to support the mica while the tip was immersed in the second stain solution very slowly until the carbon broke free of the mica. The carbon 5006
Cell Biology: Crepeau et at. was most easily floated starting at the pointed edge of the mica. The small hole size available on holey grids provided adequate support for the very thin carbon films used in this experiment. Samples were examined with a Philips EM 301 electron microscope under minimal electron dose conditions (13, 21). Optical Diffraction. The plates were initially examined in a vertical optical diffractometer (built by Lansing Instruments, Ithaca, NY), in order to select the sheets and areas most suitable for the (more lengthy) computer analysis. Computer Processing. The computer system used in this work is built around a Data General Nova 1200 computer with 32,000 words of memory. Peripheral equipment consists of a Syntex densitometer for digitization of the photographic plates, a fast Floating Point Systems Inc. hardware floating point processor designed for use with Data General FORTRAN 4, two disk drives and a magnetic tape drive for program and data storage, respectively, and a high-speed printer for the full Fourier transform output. Operation of this system is controlled through a Tektronix graphics terminal and associated hard copy unit. The graphics terminal provided a reasonable quality output device for viewing reconstructions and interactively controlling operation of the system. Programs for two-dimensional image reconstruction were based on the methods of DeRosier and Klug (11) as modified by G. Dykes for our laboratory computer system. In order to take advantage of the large number of high quality micrographs obtained, we developed a method for averaging the information available from many tubulin sheets. A program was written to determine the orientation of the sheet on the carbon support film by examination of the strongest reflections in each transform relative to those of a reference sheet. Then, by a modification of the transform, all sheets were adjusted into equivalent orientation with the reference sheet. The phase origin of each sheet was also varied in a systematic manner to achieve maximum agreement with the phases present in the reference sheet. To determine an average transform we scaled the amplitudes of each sheet in order that the most intense reflection (the main equatorial reflection) would have a value of 100 and averaged. If a given sheet failed to have a certain reflection significantly above the noise level, the sheet was not included in the averaging process for that reflection. The amplitudes and phases from each sheet for a specific reflection were treated as vectors and summed by vector addition. This sum, when divided by the number of reflections, gave the average amplitude and phase. The process was applied to the microtubule-precursor sheets obtained in the absence of zinc as well as the zinc-tubulin sheets. The average phases and amplitudes were then used for the reconstructions, which were displayed on the Tektronix 4014 screen by a contour program allowing variable magnification and selection of contour line number and range. The figures presented here of the reconstructions were photographed directly from the screen.
RESULTS The additional reciprocal space maxima that permitted resolution of a-ed differences were first observed with the zinctubulin sheets. When zinc-tubulin sheets were prepared for electron microscopic examination by flotation from mica, sheets with the same general appearance described earlier (11) were obtained. A typical sheet is presented in Fig. 1 left. However, examination of transforms of the sheets prepared with the mica technique revealed two important advantages. First, the sheets exhibited the high resolution maxima (on the 21 A-1 layer line) more consistently than the sheets produced without the mica
Proc. Nati. Acad. Sci. USA 75 (1978)
5007
step. Second, two additional layer lines were observed, at 28 A-' and 84 - that were absent in transforms of sheets prepared without the mica step. However, not all of the maxima were observed on all of the transforms. Therefore, computations were carried out to average the results of many sheets (a total of nine were taken through the complete analysis), so that the presence or absence of certain maxima could be placed on a firm statistical foundation. For the nine zinc-tubulin sheets, maxima at 38 positions of the transforms were averaged. The resulting average phases and amplitudes are presented in Fig. 2 upper. The phases were very close to the values predicted for a structure with a 2-fold screw axis (plgl), with an average deviation of 100 between the observed and predicted phases. Since this deviation is well within the experimental error of the analysis, confirmation of the screw axis was assumed, and in the reconstruction presented (Fig. 3 left), the values of phases and amplitudes were set to correspond exactly to the screw axis. (Reconstructions with the experimentally determined phases were essentially indistinguishable from those with the adjusted phases.) The reconstruction of the zinc-tubulin sheets presented in Fig. 3 left clearly shows two types of morphological units alternating along the length of the protofilaments. In the figure the two units labeled 1 and 2 can be recognized in alternate strands as inverted by the operation of a 2-fold screw axis. These two types of morphological units most likely correspond to the a and (3 chains of tubulin, but it is not possible with the data available to deduce which type (1 or 2) corresponds to a and which to (3. In addition, it is not possible to assign exact boundaries to the two types of morphological units. When micrographs giving the transform maxima on the two new layer lines (28 A-' and 84 A-1) were obtained for the zinc-tubulin sheets, efforts were directed at determining if the same layer lines could be observed for the narrower microtubule-precursor sheets formed in the absence of zinc (5, 11, 13), and an electron micrograph for a typical sheet produced by the mica technique is presented in Fig. 1 right. Evidence for the two new layer lines was obtained for such sheets, although the maxima were generally less strong than for the zinc sheets. Therefore, more transforms were averaged (a total of 11) than for the zinc sheets, and the resulting average phases and amplitudes are presented in Fig. 2 lower. A reconstruction of a microtubule-precursor sheet based on these average phases and amplitudes is presented in Fig. 3 right. As in the zinc-tubulin sheets, two distinct morphological units (labeled 1 and 2) are seen to alternate along the length of the protofilaments. In contrast to the zinc-tubulin sheets, alternate protofilaments in the microtubule-precursor sheets are aligned in a strictly parallel fashion with a hypothetical a-,8 vector pointing in the same direction for each microfilament in a given microtubule or precursor sheet. As in the zinc-tubulin sheets, we conclude that the two types of morphological units correspond to a and (3 polypeptide chains, but we have no way of assigning one type of morphological unit explicity to either a or (3. Similarly, we cannot define the exact boundaries of the units. In addition, at this level of resolution, it is not clear whether a correlation can be made between one type of morphological unit in the zinctubulin sheets and one type of morphological unit in the microtubule-precursor sheets. Such a correlation may be obscured, particularly in these two-dimensional projections, by differences in orientation or conformation that are responsible for the different lattices of the two types of sheets. When intact microtubules were examined in the electron microscope after preparation with the mica technique, transforms of the micrographs gave maxima similar to those of Fig. 2 lower (although each maximum was present with its pair arising from the opposite side of the tubule). Therefore we conclude that the
5008
Cell Biology: Crepeau et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
FIG. 1. (Left) Tubuflin sheet formed in the presence of zinc. (Right) Tubulin sheet formed under normal assembly conditions (no zinc). In both cases the sheets Were prepared with the mica flotation method and stained with uranyl acetate. The holey grids used were prepared after the method of Pease (22) except that the following modification developed by C. Akey was used: the 0.5% parlodion in n-amyl acetate was applied directly to the grids on a glass microscope slide, with the slide maintained at an angle of 30 45°, thereby eliminating a flotation step. After steaming, these grids could then be examined in a light microscope for quality prior to the baking step. The micrographs were recorded with an electron microscope magnification of 70,000, with an electron dosage of approximately 45 electrons/A2. Data were recorded on Kodak electron image plates, developed for high sensitivity with Kodak D-19 for 12 min.
a-d differences observed for the microtubule precursor sheets also apply to intact microtubules. DISCUSSION The results presented here provide evidence that microtubules and related structures are composed of protofilaments with distinct alternating morphological units which presumably correspond to the a and fl polypeptide chains. While the two classes of units differ significantly in their appearance in reconstructed two-dimensional projections (Fig. 3), these differences could be due to distinct conformations for the two types of polypeptide chains, some positive staining effects related to amino acid differences at homologous positions for two types of polypeptide chains with very similar conformation, or different orientations for the a and f3 subunits. These alternatives may be distinguished by examination of unstained structures along the lines of the low-dosage technique of Unwin and Henderson (21) and initiated by us for the zinc sheets (13). In the initial examination of zinc-tubulin sheets with unstained techniques, differences in alternating morphological units of the type reported here were not observed (13), but it appears that resolution of these differences requires the added stabilization afforded by the mica procedure. Therefore, we are now investigating the structure revealed when the unstained approach is combined with the mica procedure. While the distinct alternating morphological units are observed for both extended zinc-tubulin sheets and narrower microtubule-precursor sheets, it is not yet possible to assign the two types of morphological units to a or (3, nor has it been possible to identify one of the morphological units seen in the
zinc tubulin sheets with one of the units from the microtubule-precursor sheets. In terms of establishing which morphological unit is a and which is (3, identification may be possible through the use of the tubulin polypeptide chain-specific antibodies that have recently become available (23). In terms of correlating the two types of morphological units between zinc-tubulin sheets and microtubule-precursor sheets, data from three-dimensional reconstructions may provide sufficient information for this purpose, although at the present time the three-dimensional structure has been determined only for the sheets formed in the presence of zinc (unpublished data). Such studies will also permit evaluation of whether the distinct features of the inner and outer surfaces of the microtubule wall observed by Mandelkow et al. (9), principally on the basis of x-ray diffraction studies, will also be apparent in negatively stained images from the electron microscope. Since the results presented here deal exclusively with unchromatographed tubulin, future studies will also be required to assess the structural contributions of the high molecular weight proteins (24-26). A significant feature of the reconstruction of the microtubule-precursor sheets is that subunits of the same type are in register between adjacent protofilaments. Thus the arrangement corresponds to the pattern proposed by Amos and Klug (7) for the B tubule of outer doublets, rather than the staggered pattern proposed by these investigators for the A tubule and postulated for brain microtubules and precursor sheets by Erickson (6). A microtubule formed from sheets with the B-type lattice presented in Fig. 3 right would be incompatible with a 13-protofilament, 3-start helical structure, but would be
Cell Biology: Crepeau et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
5009
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FIG. 2. Average phases and amplitudes for transforms of tubulin sheets. (Upper) Transforms of sheets formed in the presence of zinc. (Lower) Transforms of sheets formed under normal conditions (no zinc). The spacing of the lattice lines is given in terms of the corresponding real space values. At each reciprocal lattice location corresponding to an observed maximum, the average amplitude is presented, followed by the average phase. The amplitudes were scaled by setting the strongest reflection to 100: the (2,0) maximum for the zinc sheets in the upper panel and the (1,0) maximum for the normal sheets in the lower. Below the amplitude and phase at each reciprocal lattice location, the number of sheets used to obtain the average is given in parentheses. For each sheet examined (9 zinc-tubulin sheets and 11 microtubule precursor sheets), maxima were included in the averaging only if their phase was within 450 of the average. For the zinc-tubulin sheets in the upper panel, the presence of a 2-fold screw axis was indicated by the close agreement of A (amplitude) and 0 (phase) with the relationship A,@ (h,k) = A,-0 + rh (-h,k). For the special case of h = 0, the relationship reduced to A,0 (O,k) = A,-0 (O,k) such that all reflections on the meridian must have 0 00 or 1800. Further, for k = 0, the relationship becomes A,0 (h,O) = A,-0 + wh (-h,O) = A,-(-0 + wrh) (h,O) by Friedel's law or A,@ (h,O) = A,@- wh (h,O). For h even, this is an identity, but for h odd it can only mean A = 0 such that systematic absences will be found at points (h,O) where h is odd. All of these relationships are found for the zinc sheets, confirming the presence of a 2-fold screw axis orthogonal to the protofilament axis. For the sheets formed in the absence of zinc, the angle (a) of the h reciprocal lattice lines with respect to the meridian exhibited variations from sheet to sheet in the range 6°-10°. The angle in the figure is drawn arbitrarily, using the upper value from this range. With the sheets prepared by the mica method this range of angles was somewhat below the values reported previously for sheets examined without the mica step (6, 13).
Cell Biology: Crepeau et al.
5010
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Proc. Natl. Acad. Sci. USA 75 (1978)
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Cell Biology
Differences in a and (3 polypeptide chains of tubulin resolved by electron microscopy with image reconstruction (microtubules/zinc-tubulin sheets/optical diffraction)
RICHARD H. CREPEAU, BRUCE MCEWEN, AND STUART J. EDELSTEIN Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
Communicated by G. G. Hammes, July 17, 1978
ABSTRACT Electron microscopic techniques have been used to reveal two classes of subunits of tubulin in ordered arrays. Presumably the two classes correspond to the a and 13 polypeptide chains of tubulin that have been distinguished by chemical criteria. The two types of subunits alternate along individual protofilaments in microtubules, microtubule-precursor sheets, and extended zinc-tubulin sheets. The resolution of the two types of polypeptide chains is achieved by improved negative staining methods which produce micrographs with layer lines at 28 A-' and 84 A-1 in optical or computed transforms, in addition to the layer lines at 21 A-1 and 42 A-1 described previously [Crepeau, R. H., McEwen, B., Dykes, G. & Edelstein, S. J. (1977)J Mol. BioL 116,301-3151. In microtubules or microtubule-precursor sheets, adjacent protofilaments are staggered by about 10 A, but parallel, in the sense that the a-13 vector points in the same direction for all of the protofilaments of the microtubule. However, for the sheets assembled in the presence of zinc, adjacent protofilaments are staggered by about 21 A and oriented in an antiparallel arrangement with alterate protofilaments related by a 2-fold screw axis. The antiparallel alignment of the protofilaments in the zinc-tubulin sheets accounts for their planarity (no tubular structures are found in the presence of moderate concentrations of zinc), since the intrinsic curvature found with parallel alignment of protofilaments in the absence of zinc would be cancelled by the antiparallel arrangement. Tubulin, the principal component of microtubules, is a molecule with a molecular weight of 110,000 (1). It can be dissociated into a and 1 polypeptide chains which have similar molecular weights (about 55,000) but differ in electrophoretic mobility, amino acid sequence, and extent of phosphorylation (2-4). In terms of the structure of microtubules, morphological units spaced about 40 A along the microtubule axis are observed by electron microscopy and x-ray diffraction (5-9) corresponding in size to about 55,000 molecular weight or the size of a single a or 1 polypeptide chain. In principle, microtubules with 13 strands of protofilaments could be composed of (i) a and ,chains alternating along protofilaments; (ii) some other regular pattern, such as a2 and 12 homodimers alternating along protofilaments; or (iii) a random arrangement of a and 13 polypeptide chains. Pattern i has been favored on the basis of crosslinking data, which support a heterodimer (a13) structure for tubulin (10), and observations on flagellar microtubules, which give an 80 A-i reflection in transforms suggestive of a pairing of a and 13 chains as would be expected for a heterodimer (7). However, the situation is still ambiguous, since the crosslinking data also reveal appreciable amounts of homodimers (10) and the 80 A reflection could also arise from pairings of polypeptide chains in arrangements other than heterodimers. Direct evidence for pattern i, such as the observation of distinct structures alternating along protofilaments, would settle this issue, and such evidence is presented here. Our evidence for an alternating pattern of a and 13 chains is The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
based on negatively stained electron micrographs subjected to image reconstruction by optical diffraction and computed Fourier transform methods (11, 12). Earlier work with this approach by Erickson (6) and Amos and Klug (7) revealed the helical parameters of tubulin arrays but little information on the structure of the morphological units. Recently, we described methods for extending the resolution of the analysis beyond the previous limit of a 42 A-1 layer line to a 21 A-1 layer line, which revealed a distinct polarity to the individual morphological units (13). In addition, a novel form of tubulin assembly obtained in the presence of zinc (14) was characterized, and also gave reciprocal space maxima on 42 A-1 and 21 A-1 layer lines (13). These zinc-tubulin structures are extended sheets with up to 50 protofilaments compared to up to 13 protofilaments seen for the sheets formed in the absence of zinc that are precursors of microtubules (6, 13, 15). The zinc-tubulin sheets also differ from the microtubule precursor sheets in their lattice parameters. With new procedures for preparing the samples for electron microscopy, we have now observed two additional reciprocal space layer lines, at 28 A-1 and 84 A-1, which provide the data needed to resolve differences in alternate morphological units along individual protofilaments. MATERIALS AND METHODS Tubulin Preparation. Microtubules were purified from pig brains by alternating cycles of depolymerization and polymerization (16-19) as described (13). Zinc-induced tubulin sheets were grown in 0.25 mM ZnCl2/0. 1 M 2(N-morpholino)ethanesulfonic acid, pH 6.4/1 mM GTP/1 mM 2-mercaptoethanol/0.25 mM MgCl2 by allowing the solution to incubate at room temperature for approximately 1 hr. The room temperature incubation produced slightly better quality sheets than the 35°C previously used. Electron Microscopy. All samples examined in the electron microscope in this work were prepared along the lines of the mica flotation technique of Horne and Ronchetti (20). The room temperature incubation of the tubulin in the presence of zinc was halted after the presence of large quantities of sizeable sheets were detected on normal microscope grids and one drop of material was applied to a freshly cleaved mica sheet, approximately 2 X 2 cm, followed immediately with a single drop of 0.5% uranyl acetate. The mica sheet was then tilted with tweezers to ensure coverage of all the available surface. Excess material was drained by holding the edge of the mica to a piece of filter paper and setting it aside to dry. The sample-coated mica was then placed in a vacuum evaporator and a very thin carbon support film was evaporated onto the sample. This carbon-coated sample was then floated on a second solution of 0.5% uranyl acetate negative stain and picked up with holey grids on filter paper. To aid in the flotation, we sharpened the mica to a point as described by Horne and Ronchetti (20) and used a micrometer driver stage attached to tweezers to support the mica while the tip was immersed in the second stain solution very slowly until the carbon broke free of the mica. The carbon 5006
Cell Biology: Crepeau et at. was most easily floated starting at the pointed edge of the mica. The small hole size available on holey grids provided adequate support for the very thin carbon films used in this experiment. Samples were examined with a Philips EM 301 electron microscope under minimal electron dose conditions (13, 21). Optical Diffraction. The plates were initially examined in a vertical optical diffractometer (built by Lansing Instruments, Ithaca, NY), in order to select the sheets and areas most suitable for the (more lengthy) computer analysis. Computer Processing. The computer system used in this work is built around a Data General Nova 1200 computer with 32,000 words of memory. Peripheral equipment consists of a Syntex densitometer for digitization of the photographic plates, a fast Floating Point Systems Inc. hardware floating point processor designed for use with Data General FORTRAN 4, two disk drives and a magnetic tape drive for program and data storage, respectively, and a high-speed printer for the full Fourier transform output. Operation of this system is controlled through a Tektronix graphics terminal and associated hard copy unit. The graphics terminal provided a reasonable quality output device for viewing reconstructions and interactively controlling operation of the system. Programs for two-dimensional image reconstruction were based on the methods of DeRosier and Klug (11) as modified by G. Dykes for our laboratory computer system. In order to take advantage of the large number of high quality micrographs obtained, we developed a method for averaging the information available from many tubulin sheets. A program was written to determine the orientation of the sheet on the carbon support film by examination of the strongest reflections in each transform relative to those of a reference sheet. Then, by a modification of the transform, all sheets were adjusted into equivalent orientation with the reference sheet. The phase origin of each sheet was also varied in a systematic manner to achieve maximum agreement with the phases present in the reference sheet. To determine an average transform we scaled the amplitudes of each sheet in order that the most intense reflection (the main equatorial reflection) would have a value of 100 and averaged. If a given sheet failed to have a certain reflection significantly above the noise level, the sheet was not included in the averaging process for that reflection. The amplitudes and phases from each sheet for a specific reflection were treated as vectors and summed by vector addition. This sum, when divided by the number of reflections, gave the average amplitude and phase. The process was applied to the microtubule-precursor sheets obtained in the absence of zinc as well as the zinc-tubulin sheets. The average phases and amplitudes were then used for the reconstructions, which were displayed on the Tektronix 4014 screen by a contour program allowing variable magnification and selection of contour line number and range. The figures presented here of the reconstructions were photographed directly from the screen.
RESULTS The additional reciprocal space maxima that permitted resolution of a-ed differences were first observed with the zinctubulin sheets. When zinc-tubulin sheets were prepared for electron microscopic examination by flotation from mica, sheets with the same general appearance described earlier (11) were obtained. A typical sheet is presented in Fig. 1 left. However, examination of transforms of the sheets prepared with the mica technique revealed two important advantages. First, the sheets exhibited the high resolution maxima (on the 21 A-1 layer line) more consistently than the sheets produced without the mica
Proc. Nati. Acad. Sci. USA 75 (1978)
5007
step. Second, two additional layer lines were observed, at 28 A-' and 84 - that were absent in transforms of sheets prepared without the mica step. However, not all of the maxima were observed on all of the transforms. Therefore, computations were carried out to average the results of many sheets (a total of nine were taken through the complete analysis), so that the presence or absence of certain maxima could be placed on a firm statistical foundation. For the nine zinc-tubulin sheets, maxima at 38 positions of the transforms were averaged. The resulting average phases and amplitudes are presented in Fig. 2 upper. The phases were very close to the values predicted for a structure with a 2-fold screw axis (plgl), with an average deviation of 100 between the observed and predicted phases. Since this deviation is well within the experimental error of the analysis, confirmation of the screw axis was assumed, and in the reconstruction presented (Fig. 3 left), the values of phases and amplitudes were set to correspond exactly to the screw axis. (Reconstructions with the experimentally determined phases were essentially indistinguishable from those with the adjusted phases.) The reconstruction of the zinc-tubulin sheets presented in Fig. 3 left clearly shows two types of morphological units alternating along the length of the protofilaments. In the figure the two units labeled 1 and 2 can be recognized in alternate strands as inverted by the operation of a 2-fold screw axis. These two types of morphological units most likely correspond to the a and (3 chains of tubulin, but it is not possible with the data available to deduce which type (1 or 2) corresponds to a and which to (3. In addition, it is not possible to assign exact boundaries to the two types of morphological units. When micrographs giving the transform maxima on the two new layer lines (28 A-' and 84 A-1) were obtained for the zinc-tubulin sheets, efforts were directed at determining if the same layer lines could be observed for the narrower microtubule-precursor sheets formed in the absence of zinc (5, 11, 13), and an electron micrograph for a typical sheet produced by the mica technique is presented in Fig. 1 right. Evidence for the two new layer lines was obtained for such sheets, although the maxima were generally less strong than for the zinc sheets. Therefore, more transforms were averaged (a total of 11) than for the zinc sheets, and the resulting average phases and amplitudes are presented in Fig. 2 lower. A reconstruction of a microtubule-precursor sheet based on these average phases and amplitudes is presented in Fig. 3 right. As in the zinc-tubulin sheets, two distinct morphological units (labeled 1 and 2) are seen to alternate along the length of the protofilaments. In contrast to the zinc-tubulin sheets, alternate protofilaments in the microtubule-precursor sheets are aligned in a strictly parallel fashion with a hypothetical a-,8 vector pointing in the same direction for each microfilament in a given microtubule or precursor sheet. As in the zinc-tubulin sheets, we conclude that the two types of morphological units correspond to a and (3 polypeptide chains, but we have no way of assigning one type of morphological unit explicity to either a or (3. Similarly, we cannot define the exact boundaries of the units. In addition, at this level of resolution, it is not clear whether a correlation can be made between one type of morphological unit in the zinctubulin sheets and one type of morphological unit in the microtubule-precursor sheets. Such a correlation may be obscured, particularly in these two-dimensional projections, by differences in orientation or conformation that are responsible for the different lattices of the two types of sheets. When intact microtubules were examined in the electron microscope after preparation with the mica technique, transforms of the micrographs gave maxima similar to those of Fig. 2 lower (although each maximum was present with its pair arising from the opposite side of the tubule). Therefore we conclude that the
5008
Cell Biology: Crepeau et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
FIG. 1. (Left) Tubuflin sheet formed in the presence of zinc. (Right) Tubulin sheet formed under normal assembly conditions (no zinc). In both cases the sheets Were prepared with the mica flotation method and stained with uranyl acetate. The holey grids used were prepared after the method of Pease (22) except that the following modification developed by C. Akey was used: the 0.5% parlodion in n-amyl acetate was applied directly to the grids on a glass microscope slide, with the slide maintained at an angle of 30 45°, thereby eliminating a flotation step. After steaming, these grids could then be examined in a light microscope for quality prior to the baking step. The micrographs were recorded with an electron microscope magnification of 70,000, with an electron dosage of approximately 45 electrons/A2. Data were recorded on Kodak electron image plates, developed for high sensitivity with Kodak D-19 for 12 min.
a-d differences observed for the microtubule precursor sheets also apply to intact microtubules. DISCUSSION The results presented here provide evidence that microtubules and related structures are composed of protofilaments with distinct alternating morphological units which presumably correspond to the a and fl polypeptide chains. While the two classes of units differ significantly in their appearance in reconstructed two-dimensional projections (Fig. 3), these differences could be due to distinct conformations for the two types of polypeptide chains, some positive staining effects related to amino acid differences at homologous positions for two types of polypeptide chains with very similar conformation, or different orientations for the a and f3 subunits. These alternatives may be distinguished by examination of unstained structures along the lines of the low-dosage technique of Unwin and Henderson (21) and initiated by us for the zinc sheets (13). In the initial examination of zinc-tubulin sheets with unstained techniques, differences in alternating morphological units of the type reported here were not observed (13), but it appears that resolution of these differences requires the added stabilization afforded by the mica procedure. Therefore, we are now investigating the structure revealed when the unstained approach is combined with the mica procedure. While the distinct alternating morphological units are observed for both extended zinc-tubulin sheets and narrower microtubule-precursor sheets, it is not yet possible to assign the two types of morphological units to a or (3, nor has it been possible to identify one of the morphological units seen in the
zinc tubulin sheets with one of the units from the microtubule-precursor sheets. In terms of establishing which morphological unit is a and which is (3, identification may be possible through the use of the tubulin polypeptide chain-specific antibodies that have recently become available (23). In terms of correlating the two types of morphological units between zinc-tubulin sheets and microtubule-precursor sheets, data from three-dimensional reconstructions may provide sufficient information for this purpose, although at the present time the three-dimensional structure has been determined only for the sheets formed in the presence of zinc (unpublished data). Such studies will also permit evaluation of whether the distinct features of the inner and outer surfaces of the microtubule wall observed by Mandelkow et al. (9), principally on the basis of x-ray diffraction studies, will also be apparent in negatively stained images from the electron microscope. Since the results presented here deal exclusively with unchromatographed tubulin, future studies will also be required to assess the structural contributions of the high molecular weight proteins (24-26). A significant feature of the reconstruction of the microtubule-precursor sheets is that subunits of the same type are in register between adjacent protofilaments. Thus the arrangement corresponds to the pattern proposed by Amos and Klug (7) for the B tubule of outer doublets, rather than the staggered pattern proposed by these investigators for the A tubule and postulated for brain microtubules and precursor sheets by Erickson (6). A microtubule formed from sheets with the B-type lattice presented in Fig. 3 right would be incompatible with a 13-protofilament, 3-start helical structure, but would be
Cell Biology: Crepeau et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
5009
h -6
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FIG. 2. Average phases and amplitudes for transforms of tubulin sheets. (Upper) Transforms of sheets formed in the presence of zinc. (Lower) Transforms of sheets formed under normal conditions (no zinc). The spacing of the lattice lines is given in terms of the corresponding real space values. At each reciprocal lattice location corresponding to an observed maximum, the average amplitude is presented, followed by the average phase. The amplitudes were scaled by setting the strongest reflection to 100: the (2,0) maximum for the zinc sheets in the upper panel and the (1,0) maximum for the normal sheets in the lower. Below the amplitude and phase at each reciprocal lattice location, the number of sheets used to obtain the average is given in parentheses. For each sheet examined (9 zinc-tubulin sheets and 11 microtubule precursor sheets), maxima were included in the averaging only if their phase was within 450 of the average. For the zinc-tubulin sheets in the upper panel, the presence of a 2-fold screw axis was indicated by the close agreement of A (amplitude) and 0 (phase) with the relationship A,@ (h,k) = A,-0 + rh (-h,k). For the special case of h = 0, the relationship reduced to A,0 (O,k) = A,-0 (O,k) such that all reflections on the meridian must have 0 00 or 1800. Further, for k = 0, the relationship becomes A,0 (h,O) = A,-0 + wh (-h,O) = A,-(-0 + wrh) (h,O) by Friedel's law or A,@ (h,O) = A,@- wh (h,O). For h even, this is an identity, but for h odd it can only mean A = 0 such that systematic absences will be found at points (h,O) where h is odd. All of these relationships are found for the zinc sheets, confirming the presence of a 2-fold screw axis orthogonal to the protofilament axis. For the sheets formed in the absence of zinc, the angle (a) of the h reciprocal lattice lines with respect to the meridian exhibited variations from sheet to sheet in the range 6°-10°. The angle in the figure is drawn arbitrarily, using the upper value from this range. With the sheets prepared by the mica method this range of angles was somewhat below the values reported previously for sheets examined without the mica step (6, 13).
Cell Biology: Crepeau et al.
5010
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Proc. Natl. Acad. Sci. USA 75 (1978)
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