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Ultrastructure of the crystalline inclusion induced by tobacco etch virus visualized by freeze-etching
Printed in Sweden Copyright © 1974 by Academic Press, Inc. All rights of reproduction in any form reserved
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Ultrastructure of the Crystolline Inclusion Induced by Tobacco Etch Virus Visuolized by Freeze-Etching J. G. McDONALD and ERNEST HIEBERT Plant Pathology Department, Plant Virus Laboratory, University of Florida, Gainesville, Florida 32611 Received November 19, 1973 Structural subunits of the crystalline inclusion induced by tobacco etch virus were clearly resolved when inclusions were examined both in situ and in isolated preparations by freeze-etch electron microscopy. Data supported the contention that the crystal substructure is organized into laminae and that these laminae assemble into crystals by stacking vertically. The substructure of the laminae, revealed by examining isolated inclusions, appeared to consist of a single layer of subunits packed in rectangular array. When inclusions were cross-fractured in situ, subunits were evident in rectangular and semihexagonal arrays. Based on these observations a model is proposed for the arrangement of subunits in the crystal. The formation of intracellular inclusions, whose typical three dimensional morphology has been described as that of a truncate four-sided pyramid (3, 5), has been associated by various workers with infection by tobacco etch virus (TEV) (5, 8, 10, 11). These inclusions may be found in both the nucleus and the cytoplasm (5), but have been observed more frequently in the nucleus, and there is presumptive evidence, based on studies of the early events in the infection process (5), that this is their site of origin. Some workers who have not observed these inclusions in the cytoplasm have simply designated them as intranuclear inclusions. However, as it is clear that these crystalline inclusions may also occur in the cytoplasm, and so as to avoid confusion with the cytoplasmic cylindrical inclusions also induced by TEV (1), we will refer to them as TEV-induced crystalline inclusions (TEV-CrI). The substructure of TEV-CrI has not been extensively investigated. Substructural data have been obtained from thin sectioning (5) and negative staining (2, 3) studies, but, as yet, no model has been proposed for the arrangement of structural subunits. These subunits are most likely proteinaceous. Exhaustive chemical analysis has not been completed but protein is clearly the major, if not sole, inclusion component (3, 11, 12). Freeze-etch electron microscopy was previously found to be useful for substructural studies of proteinaceous cylindrical inclusions induced by several viruses in the
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p o t a t o virus Y g r o u p (TEV, p o t a t o virus Y, a n d t u r n i p mosaic) (6). These inclusions were e x a m i n e d b o t h in situ a n d in isolated form. W e r e p o r t here a similar investigation of the substructure of T E V - C r I . MATERIALS AND METHODS
Source of TEV-infected tissue. TEV (ATCC No. PV-69) was maintained in Datura stramonium L. by mechanical transmission, Source of isolated TEV-CrL Inclusions were isolated by purification method II of Knuhtsen et al. (3). Freshly isolated inclusions in 5 raM, p H 7.0, phosphate with 5 m M 2-mercaptoethanol, 0.01 M sodium chloride and 0.04 M sodium sulfite were prepared immediately for freeze-etching. Freeze-etching. Techniques for the preparation of leaf tissue and isolated inclusions for freeze-etching were those described earlier (6). The lower epidermis was removed from selected leaf tissue, and the tissue was immersed, exposed surface face down, in a cryoprotectant (30% glycerol in 0.02 M, p H 7.5, potassium phosphate buffer). The immersed tissue was then cut into l-ram squares, and exposed briefly to a mild vacuum. After an imbibition period of 2-4 hours at room temperature, single pieces were mounted on 3-ram gold disks, and frozen in liquid Freon. Single drops of isolated inclusions (without cryoprotectant) were frozen in a similar manner on 3-ram scratched copper disks. Freeze-etching was performed on a Balzers BA 360 M instrument (7). A standard etching time of 30 seconds at -100°C was used in all experiments. Replication was performed by platinum-carbon shadowing followed by carbon reinforcement. Replicas of leaf tissue were cleaned by flotation for 1 hour on 50% commercial bleach (Clorox), followed by flotation for 1.5 hours on 70% sulfuric acid, and finally by flotation overnight on 10% commercial bleach. Replicas of isolated inclusions were cleaned by flotation for 1 hour on 10% commercial bleach. Cleaned replicas were picked up on 200- or 300-mesh grids from distilled water. Negative staining. TEV-CrI were extracted in 1% ammonium molybdate from TEVinfected D. stramonium by the leaf chopping method (9). Extracts were mounted on Formvar carbon-coated 70 × 300-mesh grids. Electron microscopy. Material was examined with a Philips 200 electron microscope. During the course of this work the magnification of the electron micrograph image was periodically calibrated. Below a film (35 ram) magnification of 9 000 ×, calibration was made using a diffraction grating (54, 864 lines/inch), while calibration of higher magnifications was made using negatively stained (phosphotungstate) bovine liver catalase crystals [assuming a periodicity of 88 _+3 ~ (4)]. RESULTS
Freeze-etched isolated TEV-CrI Replicas of isolated inclusions revealed a variety of fracture face images. Structural subunits were m o s t clearly seen on fracture planes t h a t a p p e a r e d to be either the b a s a l or apical surface of the inclusion, or else some internal layer parallel to these surfaces. F r a c t u r e faces were frequently p a r t i a l l y o b s c u r e d b y r e m n a n t s of ice.
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Figure 1 reveals two distinct fracture face images. In one image, designated as image I, the subunits are clearly visible, while in the second image, designated as image II, the subunits are less apparent. In Fig. 1 the major fracture face (face A) shows image I, while various minor faces (faces B and C being the largest areas) show image II. These faces are interpreted as being structural layers lying parallel to the base of the truncated pyramid crystal, with faces B and C being a structural layer or layers beneath face A, that were revealed when portions of face A were removed. Face A shows structural subunits in rectangular array (indicated by arrows labeled P and Q) with a spacing of about 130 fit in direction P and about 135 fit in direction {2. The subunits appear to have a depression in the center, giving them the appearanc e of being doughnut-shaped. The platinum shadow striking these subunits leaves two spots on the subunit with relatively little platinum accumulation (in the center, and at the edge farthest from the platinum source). On the fractured qrystal face, these white dots form rectangular arrays (indicated by arrows labelled R and S) at an angle of 45 ° to the axis of the structural subunits, and have a spacing of about 100 A in direction R and 95 fit in direction S. Fracture faces B and C (image II) show these white dots prominently, and the arrays of dots on faces B and C are continuous with the array of dots on face A. The structural subunits on faces B and C (image II) are less apparent than those seen on face A (image I), but may be seen by viewing Fig. 1 at a low angle (in directions P and Q). The alignment of subunits on faces B and C appears to be continuous with the alignment of subunits on the adjoining portions of face A. Figure 2 also shows the surface of several layers of an inclusion. Fracture face A shows image I while faces B and C show image II. Small portions of faces B and C have been removed, revealing further faces also showing image II. Face C appears to lie above face B, but the position of face A relative to the other two faces is not clear. Faces B and C show structural subunits that are aligned parallel to the subUnits on face A. However, the alignment of subunits on faces B and C does not appear to be mutually continuous, but is offset by half a subunit in both directions. The spacing of subunits is about 145 A in direction P and about 140 fit in direction Q. This discrepancy, and those in the spacing of subunits in various directions in Fig. 1,
FIO. 1. Laminae of an isolated inclusion showing fracture images I (face A) and II (faces B and C) Subunits are in rectangular array and are spaced at 130 A in direction P and at 135/~ in direction Q. Also visible is a rectangular array of white dots oriented at 45° to the array of subunits, and spaced at 100/~ in direction R and at 95 A in direction S. In this and subsequent figures, the encircled arrows show direction of platinum shadow deposition, x 107 000. FIa. 2. Laminae of an isolated inclusion showing fracture images I (face A) and II (faces B and C). Subunits are spaced at 145 A in direction P and at 140 A in direction Q. x 97 000. FIG. 3. Laminae of an isolated inclusion showing fracture images I (faces A and B) and II (face C) Subunits are spaced at 145 ~ in directions P and Q. x 151 000.
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were probably due to tilting of the specimen out of the plane normal to the beam path in the electron microscope. A higher magnification view of fracture images I (faces A and B) and II (face C) is shown in Fig. 3. The spacing in both directions between the subunits is about 145 N, while the spacing between the white dots is about 100 A. Not all inclusion fractures showed both fracture face images. In Fig. 4 only image I is revealed (face A). This fracture face appears to have been shadowed at a low angle, with the result that only the centers and edges of doughnut-shaped subunits show accumulation of platinum. The white dot pattern seen in Figs. 1-3 is not evident. Fracture face B, presumably a cross-sectional fracture through the inclusion, reveals little detail. Figure 5 shows a high magnification view of image I, and again the white dot pattern is not evident. Freeze-etched T E V - C r I in situ
Replicas of freeze-etch fractures through TEV-infected tissue revealed abundant crystalline inclusions. In the tissue selected, most of the inclusions were observed in the cytoplasm rather than the nucleus. Inclusions were cross-sectionally fractured, revealing a variety of fracture face images (Figs. 6 and 7). Where structural subunits were clearly resolved they appeared in rectangular array (Fig. 8) and in two distinct semihexagonal (imperfectly hexagonal) arrays (Figs. 9 and 10). Clear resolution of subunits presumably resulted from fractures that tended to follow structural planes within the crystal. Conversely, where little substructure was discernible it was assumed that the fracture crossed structural planes obliquely. In Fig. 8 the subunits in this rectangular array are spaced at about 185 A on the horizontal axis (arrow labeled J) and at about 145 A on the vertical axis (arrow labeled K). Figure 9 shows a semihexagonal array where subunits are spaced at about 190 A on the horizontal axis (arrow labled L), at about 150 A on one diagonal axis (arrow labeled M), and at about 140 A on the other diagonal axis (arrow labeled N). Figure 10 shows a different kind of semihexagonal array where the subunits are less clearly resolved. Here the subunits are spaced at about 130 A on the horizontal axis (arrow labled T), at about 135 A on one diagonal axis (arrow labeled W), and at about 130 A on the other diagonal axis (arrow labeled U).
FIG. 4. Lamina of an isolated inclusion showing fracture image I (face A). An apparent cross-fracture of the inclusion is shown in fracture face B. × 87 000. FIG. 5. Higher magnification view of a lamina showing fracture image I. × 310 000. Fro. 6. Cross-fractured inclusions in situ in the cytoplasm adjacent to a chloroplast (CH). Various inclusion fracture face images are shown. × 51 000. FIG. 7. Cross-fractured inclusions in situ in the cytoplasm. Various inclusion fracture face images are shown, x 37 000.
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Negatively stained T E V - C r I
Crystalline inclusions were easily identified in crude extracts by their characteristic rectangular (often square) morphology, and were similar in gross and fine structure to those described by Knuhtsen et al. (3). The surface substructure of an inclusion is shown in Fig. 11 and consists of a symmetrical array of black dots that have an interval of about 105 bk in directions Z and V (indicated by arrows) and an interval of about 150 A in directions X and Y (indicated by arrows). Connecting the dots are dark striations that run parallel to the rectangular sides of the inclusion. These striations, intersecting at right angles, appear to delineate structural subunits spaced at about 105 A. In some areas the striations in both directions appear to alternate in intensity. Here these darker striations (indicated by the small arrows) appear to delineate structural subunits that are spaced at about 150/k. This latter structural subunit has a black dot in its center and at its four corners.
DISCUSSION In this study freeze-etching was found to be useful for studying the substructure of TEV-CrI. Structural subunits were resolved on inclusions both in situ and in isolated preparations. The dimensions of the subunits varied when inclusions were fractured in a similar fashion. Since tilt affects most replicas to some extent, it was assumed that the m a x i m u m measurements were the most accurate. It has previously been shown that TEV-CrI typically have the shape of a truncate four-sided pyramid (3, 5). That the bonds between the subunits on the horizontal axis are stronger than those on the vertical axis, is suggested by thin-sectioned inclusions, where frequently the inclusions appear partially dissociated on the horizontal axis (3, 5, 10). This gives the crystal a laminated appearance and suggests that the crystal is formed by vertical stacking of rectangular laminae. The substructure of these laminae was revealed when isolated inclusions were freeze-etched. Doughnut-shaped subunits in rectangular array were spaced at about 145 ~ in both directions. The appearance of the subunits was more distinct in fracture image I than in fracture image II. Where both fracture images were observed on the same inclusion, laminae showing image II appeared to lie beneath laminae showing image I, suggesting that the two images resulted from different processes. Possibly FIG. 8. Cross-fractured inclusion in situ showing a rectangular array of subunits spaced at 185 /~ in direction J and at 145 A in direction K. x 182 000. Fie. 9. Cross-fractured inclusion in situ showing a semihexagonal array of subunits spaced at 190 in direction L, at 150 A in direction M and at 140 A_in direction N. x 182 000. FIG. 10. Cross-fractured inclusion in situ showing a semihexagonal array of subunits spaced at 130 in direction T, at 135 ,~ in direction W, and at 130 ~ in direction U. x 182 000,
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image I resulted from fracturing the specimen, while image II resulted from subsequent etching. Where several laminae of the same inclusion were observed, the subunits on the different layers sometimes appeared to lie directly on top of each other (Fig. 1), while at other times the subunits appeared to be offset by half a subunit (Fig. 2). It is assumed that this difference did not result from shearing at the time of fracture; its significance will be discussed below. Various cross-sectional views of TEV-CrI were revealed when they were freezeetched in situ. Where subunits were clearly resolved, both rectangular and semihexagonal arrays were evident. A crystal model that is compatible with these fracture images is one where the crysta t substructure is organized into rectangular laminae composed of single layers of sabunits packed in rectangular array. These laminae are then vertically stacked so that the subunits on adjacent laminae are mutually offset by half a subunit in both directions. Consistent with this model are the results from freeze-etching isolated inclusions where subunits on different layers sometimes appeared perpendicular to one another (Fig. 1) and at other times were offset by half a subunit (Fig. 2). Arrays of subunits, similar to the rectangular and semihexagonal arrays shown in Figs. 8 and 9, would be produced by making crosssectional fractures of this model at an angle of 45 ° to the rows of subunits on each lamina. A rectangular array would be produced by an oblique fracture (at 45 ° to the base of the crystal), whereas a semihexagonal array would be produced by a perpendicular fracture. In this model, if it is assumed that the spacing of subunits on a lamina is 145 A, then theoretically the spacing of subunits in direction J (185 A) in Fig. 8, and direction L (190 ~ ) in Fig. 9 should be 206 A. However, this apparent discrepancy is within the limits of error that may result from specimen tilt, and from the difficulty in measuring subunit spacing accurately (for Figs. 8 and 9). Subunits on the horizontal axis in Fig. 10 are spaced much closer together than the subunits on the same axis in Figs. 8 and 9. However, an image similar to Fig. 10 (with similar subunit spacing) would be produced by an oblique fracture (at 45 ° to the base) of the model running parallel to the rows of subunits on each lamina. Results from freeze-etching did not provide an indication of the orientation of the rectangular array of structural subunits on a lamina with respect to the sides of the truncated pyramid crystal. Presumably, they are oriented either parallel, or at 45 °, to the sides. Negative staining studies may possibly provide this answer; however, the results presented here seem indecisive. Interpretation of the image produced by negatively staining a three-dimensional object is made difficult by the fact that more than one surface of the crystal may be stained. The image produced may therefore be a composite of several stained layers. If, however, the subunits delineated by the small arrows in Fig. 11 correspond to the structural units seen by freeze-etching, then the subunits on a lamina are oriented paralled to the edges of the crystal. The
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FIG. 1I. Inclusion in a tissue extract, negatively stained with 1% a m m o n i u m molybdate. Black dots are spaced at 105/~ in directions Z and V, and at 150 ~ in directions X and Y. x 175 000.
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black dots (indicating an accumulation of stain) seen at the centers of these subunits may correspond to the depressions seen on structural subunits when isolated inclusions were freeze-etched. Substructural information may also be obtained from thin-sectioning studies. Matsui and Yamaguchi (5) showed a cross-sectional view of a crystalline inclusion with striations running perpendicular to the base [see Fig. 8 of (5)]. The periodicity of these striations was not stated, but from the magnification estimate a periodicity of about 70 A may be calculated. The striated appearance of a protein crystal presumably results when rows of subunits are viewed in register. Sectioning of the proposed model at an angle of 45 ° to the rows of subunits on the laminae would result in the appearance of perpendicular rows of subunits, lying in register, and spaced at about 70 ~. Thus these data (5) are not inconsistent with proposed model. Further thin-sectioning studies may yield additional structural information, but, in general, this type of investigation is hampered by the necessity for subunits to be in register for them to be resolved. The authors are most grateful to Dr H. C. Aldrich for allowing us the use of his Balzers BA 360 M instrument, and for many helpful suggestions during the course of this work. We also thank Mrs R. C. Wase for technical assistance. This work was supported in part by Grant GB-32093 from the National Science Foundation. Submitted as Paper No. 5195 of the Florida Agricultural Experiment Station's Journal Series.
REFERENCES EDWARDSON, J. R., PURC~rULL, D. E. and CHRISTIE, R. G., Virology 34, 250 (1968). HITCHBORN,J. H. and HILLS, G. J., Virology 27, 528 (1965). KNUHXSEN,H., HIEB~RT,E. and PURCIFULL,D. E., Virology submitted (1974). LUFTIG, R., J. Ultrastruet. Res., 20, 91 (1967). MATSUI,C. and YAMAGUCHI,A., Virology 22, 40 (1964). McDoNALD, J. G. and HIEBERT,E., Virology 58, 200 (1974). MOOR, H. and M/3m.ET~ALER, K., J. Cell Biol. 17, 609 (1963). PURCIFULL,D. E. and EDWARDSON,J. R., Phytopathology 58, 532 (1968). PURC~FULL,D. E., Er~WARDSON,J. R. and CHRISTIE,S. R., Phytopathology 60, 779 (1970). 10. RUBIO-HUERTOS,M. and HIDALGO,F. G., Virology 24, 84 (1964). 11. SHEFFIELD,F. M. L., J. Roy. Mierose. Soe. 61, 30 (1941). 12. SHEVARD,J. F., Virology 36, 20 (1968). 1. 2. 3. 4. 5. 6. 7. 8. 9.
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Ultrastructure of the Crystolline Inclusion Induced by Tobacco Etch Virus Visuolized by Freeze-Etching J. G. McDONALD and ERNEST HIEBERT Plant Pathology Department, Plant Virus Laboratory, University of Florida, Gainesville, Florida 32611 Received November 19, 1973 Structural subunits of the crystalline inclusion induced by tobacco etch virus were clearly resolved when inclusions were examined both in situ and in isolated preparations by freeze-etch electron microscopy. Data supported the contention that the crystal substructure is organized into laminae and that these laminae assemble into crystals by stacking vertically. The substructure of the laminae, revealed by examining isolated inclusions, appeared to consist of a single layer of subunits packed in rectangular array. When inclusions were cross-fractured in situ, subunits were evident in rectangular and semihexagonal arrays. Based on these observations a model is proposed for the arrangement of subunits in the crystal. The formation of intracellular inclusions, whose typical three dimensional morphology has been described as that of a truncate four-sided pyramid (3, 5), has been associated by various workers with infection by tobacco etch virus (TEV) (5, 8, 10, 11). These inclusions may be found in both the nucleus and the cytoplasm (5), but have been observed more frequently in the nucleus, and there is presumptive evidence, based on studies of the early events in the infection process (5), that this is their site of origin. Some workers who have not observed these inclusions in the cytoplasm have simply designated them as intranuclear inclusions. However, as it is clear that these crystalline inclusions may also occur in the cytoplasm, and so as to avoid confusion with the cytoplasmic cylindrical inclusions also induced by TEV (1), we will refer to them as TEV-induced crystalline inclusions (TEV-CrI). The substructure of TEV-CrI has not been extensively investigated. Substructural data have been obtained from thin sectioning (5) and negative staining (2, 3) studies, but, as yet, no model has been proposed for the arrangement of structural subunits. These subunits are most likely proteinaceous. Exhaustive chemical analysis has not been completed but protein is clearly the major, if not sole, inclusion component (3, 11, 12). Freeze-etch electron microscopy was previously found to be useful for substructural studies of proteinaceous cylindrical inclusions induced by several viruses in the
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p o t a t o virus Y g r o u p (TEV, p o t a t o virus Y, a n d t u r n i p mosaic) (6). These inclusions were e x a m i n e d b o t h in situ a n d in isolated form. W e r e p o r t here a similar investigation of the substructure of T E V - C r I . MATERIALS AND METHODS
Source of TEV-infected tissue. TEV (ATCC No. PV-69) was maintained in Datura stramonium L. by mechanical transmission, Source of isolated TEV-CrL Inclusions were isolated by purification method II of Knuhtsen et al. (3). Freshly isolated inclusions in 5 raM, p H 7.0, phosphate with 5 m M 2-mercaptoethanol, 0.01 M sodium chloride and 0.04 M sodium sulfite were prepared immediately for freeze-etching. Freeze-etching. Techniques for the preparation of leaf tissue and isolated inclusions for freeze-etching were those described earlier (6). The lower epidermis was removed from selected leaf tissue, and the tissue was immersed, exposed surface face down, in a cryoprotectant (30% glycerol in 0.02 M, p H 7.5, potassium phosphate buffer). The immersed tissue was then cut into l-ram squares, and exposed briefly to a mild vacuum. After an imbibition period of 2-4 hours at room temperature, single pieces were mounted on 3-ram gold disks, and frozen in liquid Freon. Single drops of isolated inclusions (without cryoprotectant) were frozen in a similar manner on 3-ram scratched copper disks. Freeze-etching was performed on a Balzers BA 360 M instrument (7). A standard etching time of 30 seconds at -100°C was used in all experiments. Replication was performed by platinum-carbon shadowing followed by carbon reinforcement. Replicas of leaf tissue were cleaned by flotation for 1 hour on 50% commercial bleach (Clorox), followed by flotation for 1.5 hours on 70% sulfuric acid, and finally by flotation overnight on 10% commercial bleach. Replicas of isolated inclusions were cleaned by flotation for 1 hour on 10% commercial bleach. Cleaned replicas were picked up on 200- or 300-mesh grids from distilled water. Negative staining. TEV-CrI were extracted in 1% ammonium molybdate from TEVinfected D. stramonium by the leaf chopping method (9). Extracts were mounted on Formvar carbon-coated 70 × 300-mesh grids. Electron microscopy. Material was examined with a Philips 200 electron microscope. During the course of this work the magnification of the electron micrograph image was periodically calibrated. Below a film (35 ram) magnification of 9 000 ×, calibration was made using a diffraction grating (54, 864 lines/inch), while calibration of higher magnifications was made using negatively stained (phosphotungstate) bovine liver catalase crystals [assuming a periodicity of 88 _+3 ~ (4)]. RESULTS
Freeze-etched isolated TEV-CrI Replicas of isolated inclusions revealed a variety of fracture face images. Structural subunits were m o s t clearly seen on fracture planes t h a t a p p e a r e d to be either the b a s a l or apical surface of the inclusion, or else some internal layer parallel to these surfaces. F r a c t u r e faces were frequently p a r t i a l l y o b s c u r e d b y r e m n a n t s of ice.
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Figure 1 reveals two distinct fracture face images. In one image, designated as image I, the subunits are clearly visible, while in the second image, designated as image II, the subunits are less apparent. In Fig. 1 the major fracture face (face A) shows image I, while various minor faces (faces B and C being the largest areas) show image II. These faces are interpreted as being structural layers lying parallel to the base of the truncated pyramid crystal, with faces B and C being a structural layer or layers beneath face A, that were revealed when portions of face A were removed. Face A shows structural subunits in rectangular array (indicated by arrows labeled P and Q) with a spacing of about 130 fit in direction P and about 135 fit in direction {2. The subunits appear to have a depression in the center, giving them the appearanc e of being doughnut-shaped. The platinum shadow striking these subunits leaves two spots on the subunit with relatively little platinum accumulation (in the center, and at the edge farthest from the platinum source). On the fractured qrystal face, these white dots form rectangular arrays (indicated by arrows labelled R and S) at an angle of 45 ° to the axis of the structural subunits, and have a spacing of about 100 A in direction R and 95 fit in direction S. Fracture faces B and C (image II) show these white dots prominently, and the arrays of dots on faces B and C are continuous with the array of dots on face A. The structural subunits on faces B and C (image II) are less apparent than those seen on face A (image I), but may be seen by viewing Fig. 1 at a low angle (in directions P and Q). The alignment of subunits on faces B and C appears to be continuous with the alignment of subunits on the adjoining portions of face A. Figure 2 also shows the surface of several layers of an inclusion. Fracture face A shows image I while faces B and C show image II. Small portions of faces B and C have been removed, revealing further faces also showing image II. Face C appears to lie above face B, but the position of face A relative to the other two faces is not clear. Faces B and C show structural subunits that are aligned parallel to the subUnits on face A. However, the alignment of subunits on faces B and C does not appear to be mutually continuous, but is offset by half a subunit in both directions. The spacing of subunits is about 145 A in direction P and about 140 fit in direction Q. This discrepancy, and those in the spacing of subunits in various directions in Fig. 1,
FIO. 1. Laminae of an isolated inclusion showing fracture images I (face A) and II (faces B and C) Subunits are in rectangular array and are spaced at 130 A in direction P and at 135/~ in direction Q. Also visible is a rectangular array of white dots oriented at 45° to the array of subunits, and spaced at 100/~ in direction R and at 95 A in direction S. In this and subsequent figures, the encircled arrows show direction of platinum shadow deposition, x 107 000. FIa. 2. Laminae of an isolated inclusion showing fracture images I (face A) and II (faces B and C). Subunits are spaced at 145 A in direction P and at 140 A in direction Q. x 97 000. FIG. 3. Laminae of an isolated inclusion showing fracture images I (faces A and B) and II (face C) Subunits are spaced at 145 ~ in directions P and Q. x 151 000.
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were probably due to tilting of the specimen out of the plane normal to the beam path in the electron microscope. A higher magnification view of fracture images I (faces A and B) and II (face C) is shown in Fig. 3. The spacing in both directions between the subunits is about 145 N, while the spacing between the white dots is about 100 A. Not all inclusion fractures showed both fracture face images. In Fig. 4 only image I is revealed (face A). This fracture face appears to have been shadowed at a low angle, with the result that only the centers and edges of doughnut-shaped subunits show accumulation of platinum. The white dot pattern seen in Figs. 1-3 is not evident. Fracture face B, presumably a cross-sectional fracture through the inclusion, reveals little detail. Figure 5 shows a high magnification view of image I, and again the white dot pattern is not evident. Freeze-etched T E V - C r I in situ
Replicas of freeze-etch fractures through TEV-infected tissue revealed abundant crystalline inclusions. In the tissue selected, most of the inclusions were observed in the cytoplasm rather than the nucleus. Inclusions were cross-sectionally fractured, revealing a variety of fracture face images (Figs. 6 and 7). Where structural subunits were clearly resolved they appeared in rectangular array (Fig. 8) and in two distinct semihexagonal (imperfectly hexagonal) arrays (Figs. 9 and 10). Clear resolution of subunits presumably resulted from fractures that tended to follow structural planes within the crystal. Conversely, where little substructure was discernible it was assumed that the fracture crossed structural planes obliquely. In Fig. 8 the subunits in this rectangular array are spaced at about 185 A on the horizontal axis (arrow labeled J) and at about 145 A on the vertical axis (arrow labeled K). Figure 9 shows a semihexagonal array where subunits are spaced at about 190 A on the horizontal axis (arrow labled L), at about 150 A on one diagonal axis (arrow labeled M), and at about 140 A on the other diagonal axis (arrow labeled N). Figure 10 shows a different kind of semihexagonal array where the subunits are less clearly resolved. Here the subunits are spaced at about 130 A on the horizontal axis (arrow labled T), at about 135 A on one diagonal axis (arrow labeled W), and at about 130 A on the other diagonal axis (arrow labeled U).
FIG. 4. Lamina of an isolated inclusion showing fracture image I (face A). An apparent cross-fracture of the inclusion is shown in fracture face B. × 87 000. FIG. 5. Higher magnification view of a lamina showing fracture image I. × 310 000. Fro. 6. Cross-fractured inclusions in situ in the cytoplasm adjacent to a chloroplast (CH). Various inclusion fracture face images are shown. × 51 000. FIG. 7. Cross-fractured inclusions in situ in the cytoplasm. Various inclusion fracture face images are shown, x 37 000.
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Negatively stained T E V - C r I
Crystalline inclusions were easily identified in crude extracts by their characteristic rectangular (often square) morphology, and were similar in gross and fine structure to those described by Knuhtsen et al. (3). The surface substructure of an inclusion is shown in Fig. 11 and consists of a symmetrical array of black dots that have an interval of about 105 bk in directions Z and V (indicated by arrows) and an interval of about 150 A in directions X and Y (indicated by arrows). Connecting the dots are dark striations that run parallel to the rectangular sides of the inclusion. These striations, intersecting at right angles, appear to delineate structural subunits spaced at about 105 A. In some areas the striations in both directions appear to alternate in intensity. Here these darker striations (indicated by the small arrows) appear to delineate structural subunits that are spaced at about 150/k. This latter structural subunit has a black dot in its center and at its four corners.
DISCUSSION In this study freeze-etching was found to be useful for studying the substructure of TEV-CrI. Structural subunits were resolved on inclusions both in situ and in isolated preparations. The dimensions of the subunits varied when inclusions were fractured in a similar fashion. Since tilt affects most replicas to some extent, it was assumed that the m a x i m u m measurements were the most accurate. It has previously been shown that TEV-CrI typically have the shape of a truncate four-sided pyramid (3, 5). That the bonds between the subunits on the horizontal axis are stronger than those on the vertical axis, is suggested by thin-sectioned inclusions, where frequently the inclusions appear partially dissociated on the horizontal axis (3, 5, 10). This gives the crystal a laminated appearance and suggests that the crystal is formed by vertical stacking of rectangular laminae. The substructure of these laminae was revealed when isolated inclusions were freeze-etched. Doughnut-shaped subunits in rectangular array were spaced at about 145 ~ in both directions. The appearance of the subunits was more distinct in fracture image I than in fracture image II. Where both fracture images were observed on the same inclusion, laminae showing image II appeared to lie beneath laminae showing image I, suggesting that the two images resulted from different processes. Possibly FIG. 8. Cross-fractured inclusion in situ showing a rectangular array of subunits spaced at 185 /~ in direction J and at 145 A in direction K. x 182 000. Fie. 9. Cross-fractured inclusion in situ showing a semihexagonal array of subunits spaced at 190 in direction L, at 150 A in direction M and at 140 A_in direction N. x 182 000. FIG. 10. Cross-fractured inclusion in situ showing a semihexagonal array of subunits spaced at 130 in direction T, at 135 ,~ in direction W, and at 130 ~ in direction U. x 182 000,
!
150
MCDONALD AND HIEBERT
image I resulted from fracturing the specimen, while image II resulted from subsequent etching. Where several laminae of the same inclusion were observed, the subunits on the different layers sometimes appeared to lie directly on top of each other (Fig. 1), while at other times the subunits appeared to be offset by half a subunit (Fig. 2). It is assumed that this difference did not result from shearing at the time of fracture; its significance will be discussed below. Various cross-sectional views of TEV-CrI were revealed when they were freezeetched in situ. Where subunits were clearly resolved, both rectangular and semihexagonal arrays were evident. A crystal model that is compatible with these fracture images is one where the crysta t substructure is organized into rectangular laminae composed of single layers of sabunits packed in rectangular array. These laminae are then vertically stacked so that the subunits on adjacent laminae are mutually offset by half a subunit in both directions. Consistent with this model are the results from freeze-etching isolated inclusions where subunits on different layers sometimes appeared perpendicular to one another (Fig. 1) and at other times were offset by half a subunit (Fig. 2). Arrays of subunits, similar to the rectangular and semihexagonal arrays shown in Figs. 8 and 9, would be produced by making crosssectional fractures of this model at an angle of 45 ° to the rows of subunits on each lamina. A rectangular array would be produced by an oblique fracture (at 45 ° to the base of the crystal), whereas a semihexagonal array would be produced by a perpendicular fracture. In this model, if it is assumed that the spacing of subunits on a lamina is 145 A, then theoretically the spacing of subunits in direction J (185 A) in Fig. 8, and direction L (190 ~ ) in Fig. 9 should be 206 A. However, this apparent discrepancy is within the limits of error that may result from specimen tilt, and from the difficulty in measuring subunit spacing accurately (for Figs. 8 and 9). Subunits on the horizontal axis in Fig. 10 are spaced much closer together than the subunits on the same axis in Figs. 8 and 9. However, an image similar to Fig. 10 (with similar subunit spacing) would be produced by an oblique fracture (at 45 ° to the base) of the model running parallel to the rows of subunits on each lamina. Results from freeze-etching did not provide an indication of the orientation of the rectangular array of structural subunits on a lamina with respect to the sides of the truncated pyramid crystal. Presumably, they are oriented either parallel, or at 45 °, to the sides. Negative staining studies may possibly provide this answer; however, the results presented here seem indecisive. Interpretation of the image produced by negatively staining a three-dimensional object is made difficult by the fact that more than one surface of the crystal may be stained. The image produced may therefore be a composite of several stained layers. If, however, the subunits delineated by the small arrows in Fig. 11 correspond to the structural units seen by freeze-etching, then the subunits on a lamina are oriented paralled to the edges of the crystal. The
FREEZE-ETCHED CRYSTALLINE INCLUSIONS
151
FIG. 1I. Inclusion in a tissue extract, negatively stained with 1% a m m o n i u m molybdate. Black dots are spaced at 105/~ in directions Z and V, and at 150 ~ in directions X and Y. x 175 000.
152
M C D O N A L D A N D HIEBERT
black dots (indicating an accumulation of stain) seen at the centers of these subunits may correspond to the depressions seen on structural subunits when isolated inclusions were freeze-etched. Substructural information may also be obtained from thin-sectioning studies. Matsui and Yamaguchi (5) showed a cross-sectional view of a crystalline inclusion with striations running perpendicular to the base [see Fig. 8 of (5)]. The periodicity of these striations was not stated, but from the magnification estimate a periodicity of about 70 A may be calculated. The striated appearance of a protein crystal presumably results when rows of subunits are viewed in register. Sectioning of the proposed model at an angle of 45 ° to the rows of subunits on the laminae would result in the appearance of perpendicular rows of subunits, lying in register, and spaced at about 70 ~. Thus these data (5) are not inconsistent with proposed model. Further thin-sectioning studies may yield additional structural information, but, in general, this type of investigation is hampered by the necessity for subunits to be in register for them to be resolved. The authors are most grateful to Dr H. C. Aldrich for allowing us the use of his Balzers BA 360 M instrument, and for many helpful suggestions during the course of this work. We also thank Mrs R. C. Wase for technical assistance. This work was supported in part by Grant GB-32093 from the National Science Foundation. Submitted as Paper No. 5195 of the Florida Agricultural Experiment Station's Journal Series.
REFERENCES EDWARDSON, J. R., PURC~rULL, D. E. and CHRISTIE, R. G., Virology 34, 250 (1968). HITCHBORN,J. H. and HILLS, G. J., Virology 27, 528 (1965). KNUHXSEN,H., HIEB~RT,E. and PURCIFULL,D. E., Virology submitted (1974). LUFTIG, R., J. Ultrastruet. Res., 20, 91 (1967). MATSUI,C. and YAMAGUCHI,A., Virology 22, 40 (1964). McDoNALD, J. G. and HIEBERT,E., Virology 58, 200 (1974). MOOR, H. and M/3m.ET~ALER, K., J. Cell Biol. 17, 609 (1963). PURCIFULL,D. E. and EDWARDSON,J. R., Phytopathology 58, 532 (1968). PURC~FULL,D. E., Er~WARDSON,J. R. and CHRISTIE,S. R., Phytopathology 60, 779 (1970). 10. RUBIO-HUERTOS,M. and HIDALGO,F. G., Virology 24, 84 (1964). 11. SHEFFIELD,F. M. L., J. Roy. Mierose. Soe. 61, 30 (1941). 12. SHEVARD,J. F., Virology 36, 20 (1968). 1. 2. 3. 4. 5. 6. 7. 8. 9.