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X-ray diffraction from microtubules
J. Mob. Biol. (1971) 59, 375-380
LETTERS TO TEE EDITOR
X-ray Diffraction
from Microtubules
X-ray diffraction patterns from sperm-tail microtubules indicate that subunits with a 40 to 50 A packing diameter form filaments, alternately half-staggered, parallel to the tubule axis. A 12- or a 13-stranded structure fits best with the X-ray diagram. The strongest bonding is that between units within a longitudinal filament; the weaker lateral interactions are disrupted by drying. Microtubules are the structural elements of many motile systems. In particular, cilia and flagella have a “9 + 2” arrangement of these tubules, which appear in the nine “outer fibers” as connected pairs of hollow cylinders, each about 240 A in diameter (Gibbons t Grimstone, 1960). Electron microscopic studies of the negatively stained, intact structure reveal subunits corresponding to the globular protein of molecular weight 60,000 obtained from disaggregated microtubules (Renaud, Rowe & Gibbons, 1968; Shelanski & Taylor, 1968; Stephens, 1968). Analysis of optical diffraction patterns of electron micrographs from Trichonympha flagella indicates a helical arrangement of subunits in the surface lattice of the microtubules, with a cell 40 b, x 50 d (Grimstone & Klug, 1966). In this paper we describe an X-ray diffraction study of hydrated microtubules. Previous X-ray work on unpurified cilia and sperm tails reported diffractions from the membrane lipid phase only (Pautard, 1952; Silvester, 1964). Our preparations of gels of sperm-tail outer fibers are weakly diffracting, and the diagram is not detailed. Nevertheless, the patterns reveal a surface lattice somewhat different from the one seen in the electron microscope, and we can place limits on possible symmetries for the structure. The simplest model, is a 12- or a 13-stranded array, with neighboring strands approximately half-staggered. Sperm tails from Strongylocentrotus droebachiensis were prepared by the method of Stephens, Renaud & Gibbons (1967). Three different procedures were used to remove the membranes: treatment with digitonin (Gibbons, 1965), washing with 1% Triton X-1000 (Stephens & Linck, 1969), and extraction with 50% glycerol for two days at 0°C. (The latter procedures were introduced after we observed that removal of membranes with digitonin produced a digitonin-cholesterol complex that contaminated subsequent “purified” samples. Strong diffraction from this complex was observed at 5.1 d and 6.1 A and in the 50 A region. These spacings have been erroneously attributed (Forslind, Swanbeck & Mohri, 1968) to diffraction from the microtubules themselves.) Purification of outer fibers then proceeded by dialysis (Stephens et al., 1967). For some experiments, A-subfibers were prepared by thermal depolymerization of the B-subfibers at low ionic strength (Stephens, 1970). Suspensions of outer fibers (about 0.5%) in 10 mM-Tris (pH 8), 10e4 M-EDTAwere centrifuged at 40,OOOrev./min for 24 to 48 hours to produce a birefringent pellet. The gels were sealed in l-mm quartz capillaries. Diffraction patterns were obtained both at ambient temperature and with cooling, using a focusing monochromator and CuKa, radiation from a semimicrofocus X-ray tube. Specimen-to-film distances were 5 to 7.5 cm, and a heliumfilled chamber was placed in the beam path. In some experiments, the specimen after X-ray exposure was fixed in glut,araldehyde, post-fixed with osmium tetroxide and 25 375
376
C. COHEN,
S. C. HARRISON
AND
R. E. STEPHENS
embedded in Araldite. The material was sectioned transverse to the specimen axis, stained with uranyl acetate and lead citrat’e and examined in a Phillips 300 electron microscope. The most prominent feature of the X-ray diagram obtained from hydrated microtubules (Plate I(a)) is a set of near-meridional layer-lines with a spacing of 40 A. The fist layer-line shows a strong off-meridional reflection and a much weaker second maximum. The only diffraction seen on the second layer-line is a relatively widely arced but apparently meridional reflection. The third and fourth layer-lines both show weak meriodional diffraction; on the third layer-line, off-meridional components are also evident. On particularly strong pictures, a faint set of meridional reflections is observed lying midway between the layer lines of the main repeat indicating a larger period of 80 A. The equator of the pattern (Plate II) shows a very intense reflection at 170 A and three other maxima at 99, 69 and 53 8. The very diffuse diffraction centered at about 10 A is accentuated in the equatorial direction. In the discussion following we consider only the major features of the pattern and assume that it is possible to interpret the diagram on the basis of a single diffracting system. The off-meridional reflection on the first layer-line and the meridional (or nearmeridional) reflection on the second indicate that any point of the microtubule surface lattice lies near the center of an approximately orthogonal cell defined by its four neighbours. That is, regardless of the determination of the exact orders, the presence of a low-order Bessel function on the second layer-line and a higher-order on the first indicates that units in adjacent rows are displaced axially by about 20 A-that is, by about half the axial repeat. The local packing relations of the structure units are defined by the unit cell shown in Figure l(a). The equator of the diagram shows that the diffracting structure is a cylindrical shell with a mean radius of about 110 A. The positions of the first three equatorial maxima and the intervening zeros are accounted for by a zero-order Bessel function. The intensity profile has the rapid fall-off expected for a thick-walled cylinder. (Note that the relatively intense equatorial reflection at 53 a would not arise from a uniform shell, and its position tells us the symmetry of the structure in projection-see below.) The size of the cylinder deduced here fits well with the results of electron microscopy. The wall of the microtubules has been observed to extend from a radius of about 80 A to about 120 A (Plate II(c); see also Gibbons & Grimstone, 1960; Porter, 1966; Grimstone & Klug, 1966). Having defined the unit cell and the size of the tubule, can we deduce the symmetry of the structure? To do this we require the orders of the Bessel functions contributing to the first and second layer-lines and to the equator. The radius of the diffracting structure and the positions of the intensity maxima together place limits on these orders. The position of the center of the first layer-line reflection is 0*0140 8- l from the meridian (measured along the layer-line). The corresponding direct-space radii for 6th-, 7th- and &h-order Bessel functions are 85, 98 and 110 d; the radii for other orders fall outside the permitted range. The weak second peak on this layer-line can best be interpreted as the second maximum of a 6th-order Bessel function, but in itself does not provide sufficient evidence to make a choice. The equatorial diffraction at 53 d can be used to restrict further the possible symmetries. A centered structure giving rise to an nth-order Bessel function on the first layer-line has in projection 2n-fold symmetry. It thus produces a Bnth-order Bessel function on the equator. The observed diameter of the microtubule limits the
I’LATE I. (a) X-ray tiiffractivn pattern from wet gels of A tubules from sperm-tail outer fibers. Four layer-lines of the 40 A repeat are clearly visible. Diffuse scatter from the monochromato~ produres the horizontal streak ant1 obscures the equatorial reflections. featuws aw a strong (b) X-ray diffraction pattern from air-dried outer fibers. The principal -IO A rneridiod reflection and a diEuse rcluatorid cliffractiorl st, ,zt)out, 50 A. A brYd ring. vhsxactrrist~ic of proteins, is also present at shout IO .A. [ rrrrin.rg
p
:;;/:
PLATE II. (a) The equator of the A tubule diffraction pattern. Four broad reflections may be seen, although in this photograph the first maximum is obscured. The vertical streaks are diffuse scatter from the monochromator. (b) Logarithmic plot of diffracted intensity as a function of distance along the equator. The data wrre obtained from densitometer traces of the film shown in (a) and of weaker exposures. Arrows in(licetr the positions of zeros and maxima in the transform of a cylindrical shell of radius 110 A. (c) Electron micrograph of a transverse section through the specimen of (a). Section stained with uranyl acetate and lead citrate ( x 90,WO).
LETTERS
(b)
TO
THE
377
EDITOR
(cl
FIG. 1. (a) The unit cell of the nlicrotubule surface lattice. A possible choice for a low-resolution primitive cell is shown (dashed lines) indicating the structure-unit packing distances, a and b. At the mean radius of the tubule wall, a = 40 A and b = 53 A (for a l2-stranded structure). (b) Tho surface lattice of a structure with a 6-fold rotation axis and the unit cell shown in (a). Three unit cells are outlined. Dashed lines show the principal directions of grooves in the microtubule wall: longitudinal on the outside and oblique on the inside. (c) The surface lattice of a structure with 13 units in two turns of the primitive helix. Two turns of this helix are shown as dotted lines.
possible even-order equatorial Bessel-function orders to 10, 12 and 14. From the first layer-line, n = 6 or 7, and the assignment of a twelfth order to the 53 A equatorial reflection (i.e. n = 6) is more consistent with the radius of the microtubule (for n = 6, R = 116 8; for n = 7, R = 134 A). This argument therefore favors a structure with a 6-fold rotation axis. The rotation would generate from the unit cell shown in Figure l(a) a 12-stranded structure with successive strands half-staggered parallel to the tubule axis (Fig. l(b)). The preceding argument assumes a meridional reflection on the second layer-line. Our best photographs show considerable disorientation, but measurements indicate that this reflection may not in fact be truly meridional. In this case a structure with 13 units in two turns of the basic helix would be consistent with the diagram (Fig. l(c)). (The second layer-line would then have a J1, the fist layer-line would have both J, and J,, and the equator would have a J,,.) Such a structure would appear as thirteen filaments parallel to the fiber axis. It is important to note that the local packing relations are very similar in both the 12- and 13-strand structures. The units cells of these surface lattices define the dimensions of the structure unit. The packing distances a and b in Figure l(a) are approximately 40 to 50 A. The molecular weight of outer-fiber protein subunits is about 60,000 (Stephens, 1968), which corresponds to a sphere about 50 i% in diameter. The structure unit must therefore
Equator
0.0
1
(0.0246)t
2 3 4
0.0503 & 0.0005 0.0742 & 0.0005 0.0980 & 0.0005 f Calculated
Zero-order term Zero-order term Zoro-ardor tarm Higher-order term E’irst off-meridional Second off-meridional (Meridional) (Meridional) (Meridional) from third
and fourth
U~UO59* O~OUU5 04101
0.0145 0419u 0.0140 04250 1 O.UUl
layer-lines.
consist of one molecule. There is also evidence from studies in solution that microtubule protein forms a stable dimer (Renaud et aE., 1968; Shelanski & Taylor, 1968). A tubule with dimeric structure units could have local perpendicular 2-fold axes and the observed 80 d repeat. A striking feature of electron micrographs of outer fibers is longitudinal striation arising from filaments which appear to run parallel to the axis of the microtubule. One-sided images of outer fibers reveal up to six such filaments implying a minimum of 12 strands in the structure. The exact number of strands in outer fibers has not been established by electron microscopy, but both 12 and 13 filaments have been reported in microtubules from various sources. Further aspects of the microtubule surface are suggested by the X-ray diagrams. The radius of 85 A inferred from the first layer-line (assuming n = 6) is significantly smaller than that of 116 A inferred from the 53 A equatorial reflection; similar differences are found for other possible symmetries. This difference implies different radial locations for the corresponding diffraction features and indicates the particular shape of the structure unit. It appears that the feature giving rise to the equatorial diffraction is near the outside of the microtubule (r = 116 A), while that giving rise to the first layer-line lies near the inner surface of the tubule wall (T = 85 8). One might picture that the protein subunits of the microtubule are so tapered at both the inside and outside that strong electron density variations occur at these boundaries. The walls of the microtubule would then appear grooved, longitudinally on the outside and obliquely on the inside. The strongest bonding in the microtubule is that between subunits within a longitudinal filament (cf. Grimstone & Klug, 1966). Electron micrographs often show fraying of outer fibers into individual strands (cf. Pease, 1963). An X-ray diffraction photograph of a dried microtubule sample is shown in Plate I(b). A similar pattern has previously been observed by Klug (personal communication). The pattern has a strong meridional reflection at 40 A, rather than the off-meridional diffraction seen with native specimens. The lateral coherence of the surface lattice of the microtubule is therefore destroyed by drying and the structure diffracts as simple linear arrays of protein subunits.
LETTERS
TO
THE
E:DlTOlE
379
Grimstone & Klug (1966) have studied the outer-fiber surface lattice by an optical diffraction analysis of electron micrographs of negatively stained preparations. The optical diffraction pattern shows near-meridional reflections on a 40 A layer-line but no higher-order meridional spacings. The surface lattice corresponding to this pattern can be described as a helix of small pitch: adjacent filaments are displaced by about l/S to l/4 of the 40 A axial repeat (Grimstone & Klug, 1966). In contrast, our X-ray diagram from hydrat’ed microtubules has a meridional (or near-meridional) 20 A spacing and off-meridional reflections farther out on the 40 A layer-line than in the optical diffraction pattern These features suggest half-staggering of adjacent strands. The surface lattice seen in the electron microscope would not give the observed near-meridional X-ray reflection at 20 A. A transition from the hydrated structure to t#hat seen in negatively stained fibers would require a displacement of neighboring strands, altering the weak lateral bonds but preserving t’he stronger axial interactions. (In air-dried preparations, no regular lateral bonding remains.) These results imply that there are different ordered states of bhe microtubule surface lattice produced by different environmental conditions. There are important aspect’s of the outer-fiber structure that we have not dealt wit,11in this analysis. The outer fiber consists of two connected tubules, termed A and B subfibers. In the electron microscope, it appears that the subfibers are closely similar in structure (Grimstone & Klug, 1966) but that only the A tubule bears a double row of arms (Gibbons & Grimstone, 1960). There is no evidence in our X-ray patterns for more than one diffracting structure. Moreover, X-ray photographs of gels of isolated A subfibers are indistinguishable from patterns obtained with whole outer fibers. The B subfiber is more labile than the A (Stephens, 1970). Electron micrographs of X-ray specimens kept at room t,emperature during exposure show for the most part A tubules rather than doublets. It is possible that there were insufficient intact doublets present in our specimens to show distinct diffraction from the B tubules. Or it may be that the two subfibers have the same surface lattice at low resolution. The arms on the A subfiber are positioned with a repeat of about 170 to 200 A (Gibbons, quoted in Grimstone & Klug, 1966), so that there cannot be strict equivalence of the subunits in the surface array. Perturbations that may be rclat#ed to the disposition of the arms have been detected by electron microscopy: Grimstone & Klug (1966) discovered periodicities of 90 and 160 A, possible orders of a 480 A repeat. We have found evidence in hydrated outer fibers for the 80 ~1 repcat. The structural basis of these perturbations should be defined by higherresolution Y-ray studies. XII t,he previous considerations have concerned static aspects of the microtubule struct’ure. It is lmowr~ that the arms (“dynein”) have enzymic activity and split ;YI’l’ (Gibbons, 1965). That these arms move, although not demonstrated, seems most. plausible. It is not known, however, mhcther the microtubule surface lattice untlergoes rearraugemcnt in different physiological states. We may, in effect, have described one of several structural states for the microtubule. The other states that have been observed show that the lateral bonds are not conserved. A longitudinal dislocation could therefore readily occur, with structural integrity maintained by the conserved axial interactions. The microtubule may thus have the bonding properties characteristic of protein assemblies in other motile systems (Caspar & Cohen, 1969).
380
C. COHEN,
S. C. HARRISON
AND
R. E. STEI’HENS
We thank Dr W. Longley for taking some of the early X-ray photographs; and we thank Dr D. L. D. Caspar for valuable discussion. This work was supported by Public Health Service Grants AM02633 to one of us (C.C.) and GM15500 to another author (R. E. S.). One of us (S. C. H.) is a Junior Fellow in the Society of Fellows, Harvard University. Children’s Cancer Research Foundation 35 Binney Street Boston, Mass. 02115, U.S.A.
CAROLYN COHEN STEPHEN C. HARRISON
R. E. STEPHENS
Department of Biology Brandeis University Waltham, Mass. 02154, U.S.A. Received 7 January
1970, and in revised form 14 September
1970
REFERENCES Caspar, D. L. D. & Cohen, C. (1969). In Symmetry and Function of Biological Systems at the MacromoZecuZar Level, ed. by A. Engstrom & B. Strandberg, p. 393. Stockholm: Almquist & Wiskell. Forslind, B., Swanbeck, G. & Mohri, H. (1968). Exp. Cell Res. 53, 678. Gibbons, I. R. (1965). Arch. Biol., Liege, 76, 317. Gibbons, I. R. & Grimstone, A. V. (1960). J. Bbphye. Biochem. Cytol. 7, 697. Grimstone, A. V. & Klug, A. (1966). J. CeU Xci. 1, 351. Pautard, F. G. (1952). In Spermatozoan Motility, ed. by D. W. Bishop, p. 189. Washington, D.C.: American Association for the Advancement of Science. Pease, D. C. (1963). J. Cell BioZ. 18, 313. Porter, K. (1966). In Principlea of BiomoZecda~ Organization, ed. by 0. E. W. Wostenholme & M. O’Connor, p. 308. Boston: Little, Brown & Co. Renaud, F. L., Rowe, A. J. & Gibbons, I. R. (1968). J. Cell BioZ. 36, 79. Shelsnski, M. t Taylor, E. W. (1968). J. Cell BioZ. 38, 304. Silvester, N. R. (1964). J. Mol. Biol. 8, 11. Stephens, R. E. (1968). J. Mol. Biol. 32, 277. Stephens, R. E. (1970). J. Mo2. Biol. 47, 353. Stephens, R. E. & Linck, R. W. (1969). J. Mol. BioZ. 40, 497. Stephens, R. E., Renaud, F. L. t Gibbons, I. R. (1967). Science, 156, 1606.
LETTERS TO TEE EDITOR
X-ray Diffraction
from Microtubules
X-ray diffraction patterns from sperm-tail microtubules indicate that subunits with a 40 to 50 A packing diameter form filaments, alternately half-staggered, parallel to the tubule axis. A 12- or a 13-stranded structure fits best with the X-ray diagram. The strongest bonding is that between units within a longitudinal filament; the weaker lateral interactions are disrupted by drying. Microtubules are the structural elements of many motile systems. In particular, cilia and flagella have a “9 + 2” arrangement of these tubules, which appear in the nine “outer fibers” as connected pairs of hollow cylinders, each about 240 A in diameter (Gibbons t Grimstone, 1960). Electron microscopic studies of the negatively stained, intact structure reveal subunits corresponding to the globular protein of molecular weight 60,000 obtained from disaggregated microtubules (Renaud, Rowe & Gibbons, 1968; Shelanski & Taylor, 1968; Stephens, 1968). Analysis of optical diffraction patterns of electron micrographs from Trichonympha flagella indicates a helical arrangement of subunits in the surface lattice of the microtubules, with a cell 40 b, x 50 d (Grimstone & Klug, 1966). In this paper we describe an X-ray diffraction study of hydrated microtubules. Previous X-ray work on unpurified cilia and sperm tails reported diffractions from the membrane lipid phase only (Pautard, 1952; Silvester, 1964). Our preparations of gels of sperm-tail outer fibers are weakly diffracting, and the diagram is not detailed. Nevertheless, the patterns reveal a surface lattice somewhat different from the one seen in the electron microscope, and we can place limits on possible symmetries for the structure. The simplest model, is a 12- or a 13-stranded array, with neighboring strands approximately half-staggered. Sperm tails from Strongylocentrotus droebachiensis were prepared by the method of Stephens, Renaud & Gibbons (1967). Three different procedures were used to remove the membranes: treatment with digitonin (Gibbons, 1965), washing with 1% Triton X-1000 (Stephens & Linck, 1969), and extraction with 50% glycerol for two days at 0°C. (The latter procedures were introduced after we observed that removal of membranes with digitonin produced a digitonin-cholesterol complex that contaminated subsequent “purified” samples. Strong diffraction from this complex was observed at 5.1 d and 6.1 A and in the 50 A region. These spacings have been erroneously attributed (Forslind, Swanbeck & Mohri, 1968) to diffraction from the microtubules themselves.) Purification of outer fibers then proceeded by dialysis (Stephens et al., 1967). For some experiments, A-subfibers were prepared by thermal depolymerization of the B-subfibers at low ionic strength (Stephens, 1970). Suspensions of outer fibers (about 0.5%) in 10 mM-Tris (pH 8), 10e4 M-EDTAwere centrifuged at 40,OOOrev./min for 24 to 48 hours to produce a birefringent pellet. The gels were sealed in l-mm quartz capillaries. Diffraction patterns were obtained both at ambient temperature and with cooling, using a focusing monochromator and CuKa, radiation from a semimicrofocus X-ray tube. Specimen-to-film distances were 5 to 7.5 cm, and a heliumfilled chamber was placed in the beam path. In some experiments, the specimen after X-ray exposure was fixed in glut,araldehyde, post-fixed with osmium tetroxide and 25 375
376
C. COHEN,
S. C. HARRISON
AND
R. E. STEPHENS
embedded in Araldite. The material was sectioned transverse to the specimen axis, stained with uranyl acetate and lead citrat’e and examined in a Phillips 300 electron microscope. The most prominent feature of the X-ray diagram obtained from hydrated microtubules (Plate I(a)) is a set of near-meridional layer-lines with a spacing of 40 A. The fist layer-line shows a strong off-meridional reflection and a much weaker second maximum. The only diffraction seen on the second layer-line is a relatively widely arced but apparently meridional reflection. The third and fourth layer-lines both show weak meriodional diffraction; on the third layer-line, off-meridional components are also evident. On particularly strong pictures, a faint set of meridional reflections is observed lying midway between the layer lines of the main repeat indicating a larger period of 80 A. The equator of the pattern (Plate II) shows a very intense reflection at 170 A and three other maxima at 99, 69 and 53 8. The very diffuse diffraction centered at about 10 A is accentuated in the equatorial direction. In the discussion following we consider only the major features of the pattern and assume that it is possible to interpret the diagram on the basis of a single diffracting system. The off-meridional reflection on the first layer-line and the meridional (or nearmeridional) reflection on the second indicate that any point of the microtubule surface lattice lies near the center of an approximately orthogonal cell defined by its four neighbours. That is, regardless of the determination of the exact orders, the presence of a low-order Bessel function on the second layer-line and a higher-order on the first indicates that units in adjacent rows are displaced axially by about 20 A-that is, by about half the axial repeat. The local packing relations of the structure units are defined by the unit cell shown in Figure l(a). The equator of the diagram shows that the diffracting structure is a cylindrical shell with a mean radius of about 110 A. The positions of the first three equatorial maxima and the intervening zeros are accounted for by a zero-order Bessel function. The intensity profile has the rapid fall-off expected for a thick-walled cylinder. (Note that the relatively intense equatorial reflection at 53 a would not arise from a uniform shell, and its position tells us the symmetry of the structure in projection-see below.) The size of the cylinder deduced here fits well with the results of electron microscopy. The wall of the microtubules has been observed to extend from a radius of about 80 A to about 120 A (Plate II(c); see also Gibbons & Grimstone, 1960; Porter, 1966; Grimstone & Klug, 1966). Having defined the unit cell and the size of the tubule, can we deduce the symmetry of the structure? To do this we require the orders of the Bessel functions contributing to the first and second layer-lines and to the equator. The radius of the diffracting structure and the positions of the intensity maxima together place limits on these orders. The position of the center of the first layer-line reflection is 0*0140 8- l from the meridian (measured along the layer-line). The corresponding direct-space radii for 6th-, 7th- and &h-order Bessel functions are 85, 98 and 110 d; the radii for other orders fall outside the permitted range. The weak second peak on this layer-line can best be interpreted as the second maximum of a 6th-order Bessel function, but in itself does not provide sufficient evidence to make a choice. The equatorial diffraction at 53 d can be used to restrict further the possible symmetries. A centered structure giving rise to an nth-order Bessel function on the first layer-line has in projection 2n-fold symmetry. It thus produces a Bnth-order Bessel function on the equator. The observed diameter of the microtubule limits the
I’LATE I. (a) X-ray tiiffractivn pattern from wet gels of A tubules from sperm-tail outer fibers. Four layer-lines of the 40 A repeat are clearly visible. Diffuse scatter from the monochromato~ produres the horizontal streak ant1 obscures the equatorial reflections. featuws aw a strong (b) X-ray diffraction pattern from air-dried outer fibers. The principal -IO A rneridiod reflection and a diEuse rcluatorid cliffractiorl st, ,zt)out, 50 A. A brYd ring. vhsxactrrist~ic of proteins, is also present at shout IO .A. [ rrrrin.rg
p
:;;/:
PLATE II. (a) The equator of the A tubule diffraction pattern. Four broad reflections may be seen, although in this photograph the first maximum is obscured. The vertical streaks are diffuse scatter from the monochromator. (b) Logarithmic plot of diffracted intensity as a function of distance along the equator. The data wrre obtained from densitometer traces of the film shown in (a) and of weaker exposures. Arrows in(licetr the positions of zeros and maxima in the transform of a cylindrical shell of radius 110 A. (c) Electron micrograph of a transverse section through the specimen of (a). Section stained with uranyl acetate and lead citrate ( x 90,WO).
LETTERS
(b)
TO
THE
377
EDITOR
(cl
FIG. 1. (a) The unit cell of the nlicrotubule surface lattice. A possible choice for a low-resolution primitive cell is shown (dashed lines) indicating the structure-unit packing distances, a and b. At the mean radius of the tubule wall, a = 40 A and b = 53 A (for a l2-stranded structure). (b) Tho surface lattice of a structure with a 6-fold rotation axis and the unit cell shown in (a). Three unit cells are outlined. Dashed lines show the principal directions of grooves in the microtubule wall: longitudinal on the outside and oblique on the inside. (c) The surface lattice of a structure with 13 units in two turns of the primitive helix. Two turns of this helix are shown as dotted lines.
possible even-order equatorial Bessel-function orders to 10, 12 and 14. From the first layer-line, n = 6 or 7, and the assignment of a twelfth order to the 53 A equatorial reflection (i.e. n = 6) is more consistent with the radius of the microtubule (for n = 6, R = 116 8; for n = 7, R = 134 A). This argument therefore favors a structure with a 6-fold rotation axis. The rotation would generate from the unit cell shown in Figure l(a) a 12-stranded structure with successive strands half-staggered parallel to the tubule axis (Fig. l(b)). The preceding argument assumes a meridional reflection on the second layer-line. Our best photographs show considerable disorientation, but measurements indicate that this reflection may not in fact be truly meridional. In this case a structure with 13 units in two turns of the basic helix would be consistent with the diagram (Fig. l(c)). (The second layer-line would then have a J1, the fist layer-line would have both J, and J,, and the equator would have a J,,.) Such a structure would appear as thirteen filaments parallel to the fiber axis. It is important to note that the local packing relations are very similar in both the 12- and 13-strand structures. The units cells of these surface lattices define the dimensions of the structure unit. The packing distances a and b in Figure l(a) are approximately 40 to 50 A. The molecular weight of outer-fiber protein subunits is about 60,000 (Stephens, 1968), which corresponds to a sphere about 50 i% in diameter. The structure unit must therefore
Equator
0.0
1
(0.0246)t
2 3 4
0.0503 & 0.0005 0.0742 & 0.0005 0.0980 & 0.0005 f Calculated
Zero-order term Zero-order term Zoro-ardor tarm Higher-order term E’irst off-meridional Second off-meridional (Meridional) (Meridional) (Meridional) from third
and fourth
U~UO59* O~OUU5 04101
0.0145 0419u 0.0140 04250 1 O.UUl
layer-lines.
consist of one molecule. There is also evidence from studies in solution that microtubule protein forms a stable dimer (Renaud et aE., 1968; Shelanski & Taylor, 1968). A tubule with dimeric structure units could have local perpendicular 2-fold axes and the observed 80 d repeat. A striking feature of electron micrographs of outer fibers is longitudinal striation arising from filaments which appear to run parallel to the axis of the microtubule. One-sided images of outer fibers reveal up to six such filaments implying a minimum of 12 strands in the structure. The exact number of strands in outer fibers has not been established by electron microscopy, but both 12 and 13 filaments have been reported in microtubules from various sources. Further aspects of the microtubule surface are suggested by the X-ray diagrams. The radius of 85 A inferred from the first layer-line (assuming n = 6) is significantly smaller than that of 116 A inferred from the 53 A equatorial reflection; similar differences are found for other possible symmetries. This difference implies different radial locations for the corresponding diffraction features and indicates the particular shape of the structure unit. It appears that the feature giving rise to the equatorial diffraction is near the outside of the microtubule (r = 116 A), while that giving rise to the first layer-line lies near the inner surface of the tubule wall (T = 85 8). One might picture that the protein subunits of the microtubule are so tapered at both the inside and outside that strong electron density variations occur at these boundaries. The walls of the microtubule would then appear grooved, longitudinally on the outside and obliquely on the inside. The strongest bonding in the microtubule is that between subunits within a longitudinal filament (cf. Grimstone & Klug, 1966). Electron micrographs often show fraying of outer fibers into individual strands (cf. Pease, 1963). An X-ray diffraction photograph of a dried microtubule sample is shown in Plate I(b). A similar pattern has previously been observed by Klug (personal communication). The pattern has a strong meridional reflection at 40 A, rather than the off-meridional diffraction seen with native specimens. The lateral coherence of the surface lattice of the microtubule is therefore destroyed by drying and the structure diffracts as simple linear arrays of protein subunits.
LETTERS
TO
THE
E:DlTOlE
379
Grimstone & Klug (1966) have studied the outer-fiber surface lattice by an optical diffraction analysis of electron micrographs of negatively stained preparations. The optical diffraction pattern shows near-meridional reflections on a 40 A layer-line but no higher-order meridional spacings. The surface lattice corresponding to this pattern can be described as a helix of small pitch: adjacent filaments are displaced by about l/S to l/4 of the 40 A axial repeat (Grimstone & Klug, 1966). In contrast, our X-ray diagram from hydrat’ed microtubules has a meridional (or near-meridional) 20 A spacing and off-meridional reflections farther out on the 40 A layer-line than in the optical diffraction pattern These features suggest half-staggering of adjacent strands. The surface lattice seen in the electron microscope would not give the observed near-meridional X-ray reflection at 20 A. A transition from the hydrated structure to t#hat seen in negatively stained fibers would require a displacement of neighboring strands, altering the weak lateral bonds but preserving t’he stronger axial interactions. (In air-dried preparations, no regular lateral bonding remains.) These results imply that there are different ordered states of bhe microtubule surface lattice produced by different environmental conditions. There are important aspect’s of the outer-fiber structure that we have not dealt wit,11in this analysis. The outer fiber consists of two connected tubules, termed A and B subfibers. In the electron microscope, it appears that the subfibers are closely similar in structure (Grimstone & Klug, 1966) but that only the A tubule bears a double row of arms (Gibbons & Grimstone, 1960). There is no evidence in our X-ray patterns for more than one diffracting structure. Moreover, X-ray photographs of gels of isolated A subfibers are indistinguishable from patterns obtained with whole outer fibers. The B subfiber is more labile than the A (Stephens, 1970). Electron micrographs of X-ray specimens kept at room t,emperature during exposure show for the most part A tubules rather than doublets. It is possible that there were insufficient intact doublets present in our specimens to show distinct diffraction from the B tubules. Or it may be that the two subfibers have the same surface lattice at low resolution. The arms on the A subfiber are positioned with a repeat of about 170 to 200 A (Gibbons, quoted in Grimstone & Klug, 1966), so that there cannot be strict equivalence of the subunits in the surface array. Perturbations that may be rclat#ed to the disposition of the arms have been detected by electron microscopy: Grimstone & Klug (1966) discovered periodicities of 90 and 160 A, possible orders of a 480 A repeat. We have found evidence in hydrated outer fibers for the 80 ~1 repcat. The structural basis of these perturbations should be defined by higherresolution Y-ray studies. XII t,he previous considerations have concerned static aspects of the microtubule struct’ure. It is lmowr~ that the arms (“dynein”) have enzymic activity and split ;YI’l’ (Gibbons, 1965). That these arms move, although not demonstrated, seems most. plausible. It is not known, however, mhcther the microtubule surface lattice untlergoes rearraugemcnt in different physiological states. We may, in effect, have described one of several structural states for the microtubule. The other states that have been observed show that the lateral bonds are not conserved. A longitudinal dislocation could therefore readily occur, with structural integrity maintained by the conserved axial interactions. The microtubule may thus have the bonding properties characteristic of protein assemblies in other motile systems (Caspar & Cohen, 1969).
380
C. COHEN,
S. C. HARRISON
AND
R. E. STEI’HENS
We thank Dr W. Longley for taking some of the early X-ray photographs; and we thank Dr D. L. D. Caspar for valuable discussion. This work was supported by Public Health Service Grants AM02633 to one of us (C.C.) and GM15500 to another author (R. E. S.). One of us (S. C. H.) is a Junior Fellow in the Society of Fellows, Harvard University. Children’s Cancer Research Foundation 35 Binney Street Boston, Mass. 02115, U.S.A.
CAROLYN COHEN STEPHEN C. HARRISON
R. E. STEPHENS
Department of Biology Brandeis University Waltham, Mass. 02154, U.S.A. Received 7 January
1970, and in revised form 14 September
1970
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