JOURNAL OF ULTRASTRUCTURE RESEARCH 5 4 , 176- 182 (1976)
The Hexagonal Lattice Spacing of Intracellular Crystalline Tobacco Mosaic Virus J. H. M. WILLISON Department of Microbiology and Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada Received April 10, 1975 Previous estimates of hexagonal lattice spacing of the highly unstable, lamellar-twinned, intracellular tobacco mosaic virus (TMV) crystals have varied between 15 and 23 nm. In this study, the crystals have been examined by freeze-etching. Short periods (1-5 min) of glycerol or sucrose infiltration were applied before freezing. Glycerol t r e a t m e n t caused plasmolysis and a concomitant reduction in crystal lattice spacing as a result of water withdrawal. Cells penetrated by glycerol commonly contained well-ordered TMV crystals with hexagonal lattice spacings in the range 21-25 nm. These spacings probably represent stages in a glycerol-induced expansion of plasmolytically contracted crystals. Crystals in unplasmolyzed sucrose-treated cells always contained ice. The relative proportions of virus and ice (55:45) were stereometrically determined and the interparticle spacing (17.6 nm) was measured using TMV rod length as a standard. On the assumption t h a t intracrystalline ice arises from intracrystalline water, the hexagonal lattice spacing must be 23.6 _+ 1.2 nm.
The intracellular tobacco mosaic virus (TMV) crystal is polymorphic, but its most usual form is a thin hexagonal plate (1) in which the virus particles are arranged in layers parallel to the crystal hexagonal face (13). The virus particles of alternate layers lie roughly parallel to each other, but adjacent layers are inclined so as to produce a zig-zag or herringbone appearance (9, 13). Electron microscopy of thin sections of the crystals shows that the particles are hexagonally packed (10, 11 ). The crystals are highly labile and will change their form, or break down, under the influence of dilute acids, salt solutions, or even slight pressure from outside the cell (1, 13). This feature has made them difficult to study and left some doubt as to the hexagonal packing distance. Bernal and Frankuchen (2), who made early classic Xray diffraction studies of TMV gels, were of the opinion that intracellular crystals would be spaced similarly to isoelectric gels with a hexagonal packing distance of 18.5 nm. Wilkins and his co-workers (13) compared the birefringence of intracellular crystals with that of TMV gel and concluded that the crystals contained either
about 30 or about 60% water, probably the latter. Since the interparticle spacing in dried TMV gels is 15.2 nm (2) this would make the hydrated intracellular spacing either about 18 or about 23 nm if one assumes that the amount of water lying between adjacent layers of rods is insignificant, and therefore, that hydration simply moves the hexagonally packed rods apart. In an early freeze-etching study of intracellular TMV crystals, Steere (9) did not measure interparticle spacings, but it is clear from his micrographs that the particles lay 17-18 nm apart after freezing. However, Warmke and Edwardson (11) proposed, on the basis of thin section evidence, that the spacing was 15-16 nm. In the present study, intracellular TMV crystals have been reexamined using the freze-etching technique to attempt to account for these disparate estimates of the hexagonal lattice spacing. METHODS
Nicotiana tabacum L. var H a v a n a plants were systemically infected with TMV (vulgare strain). Pieces of infected leaves were diced in either 25% glycerol-0.1 M phosphate buffer or 20% w/v sucrose0.1 M phosphate buffer and frozen for freeze-etching 176
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
INTRACELLULAR TMV LATTICE SPACING after 2-5 min (glycerol) or 1-2 min (sucrose). Samples were freeze-etched in a Balzers apparatus using the procedure of Moor and Mfihlethaler (7) and etched for 30 sec at - 105°C before shadowing. Replicas were treated with 2% Meicelase (Meija Seika Kaishi Ltd., Tokyo, Japan), pH 6.0, for 24 hr before cleaning, sequentially, with sodium hypochlorite and 40% chromic acid, 8-18 hr each. For stereometric analysis, a t r a n s p a r e n t "Ethulon" overlay, marked with a standard set of points arranged in an hexagonal lattice (12), was layed over 8 x 10 in. photographic prints. Points were counted in two groups: virus or ice. In each case, the overlay was placed, randomly, in three different positions over the micrograph and an average of the three sets of points was obtained. Points t h a t lay over capsid surfaces t h a t has been exposed by ice etching were counted as ice. TMV rod length, 300 nm (4), was used as an internal calibration standard where required.
177
plasmolysis had begun, but in other regions there was no evidence of the onset of plasmolysis nor of ice development between the cell wall and plasmalemma. TMV crystals within these unplasmolyzed cells contained large ice crystals that had apparently arisen as a result of the crystallization of water from within the TMV crystals themselves (Fig. 6). The stereometric ratio of total crystal volume (virus + ice) to virus was 1.83 _+ 4.6% (1 SD, seven values). Where ice etching revealed closepacked virus rods (Fig. 5), the lattice spacing was 17.6 +_ 0.2 nm (1 SD, 10 values). DISCUSSION
Ultrastructural studies of intracellular TMV crystals are complicated by the fact RESULTS that the crystals are disrupted by glutaralGlycerol treatment. Treatment of leaf dehyde fixation (14) and, at best, are only pieces with 25% glycerol usually resulted poorly preserved by osmium tetroxide or in gross plasmolysis. In highly plasmo- potassium permanganate (10). Freezing of lyzed cells, virions within the TMV crys- untreated leaves results in ice crystals tals were tightly packed such that individ- within virus crystals (14), but effective ual rods could rarely be made out (Fig. 1). penetration of glycerol as a cryoprotectant In moderately plasmolyzed cells, ice usu- is virtually impossible (14, this work). The ally was found within the virus crystals deleterious properties of glycerol in its use (Fig. 2). Cells also were found in which as a cryoprotectant have been recognized glycerol had penetrated into the cell to for some time (8). It seems probable that provide conventional cryoprotected im- the effect of glycerol in the present study ages. These cells sometimes appear to be has been sequentially: severe plasmolysis, unplasmolyzed, but tonoplast rupture, entry through the plasmalemma, deplasTMV crystal disruption, and other forms molysis, tonoplast rupture, and TMV crysof cell injury were commonplace. Neverthe- tal disruption. If this is the case, then the less, in such cells, TMV crystals that had ~'cryoprotected" TMV crystals (e.g., Fig. 3) retained both the ~'herringbone" structure have undergone shrinkage prior to the ob(Fig. 3) and the hexagonal lattice (Fig. 4) served glycerol-expanded state. Consewere sometimes found. Adjacent layers of quently the 21-25 nm spacings of herringvirus rods were not separated by an appre- bone crystals, described above in glycerolciable gap (Fig. 3). The hexagonal lattice treated material, probably represent spacing of these cryoprotected crystals was stages in the glycerol expansion and bear variable, with values lying between 21 and only a passing relevance to the in vivo 25 nm. The mean spacing obtained from spacing. Nevertheless, the striking regufive such crystals in which rod length larity of these crystals both in terms of the could be used reliably for calibration (e.g., constant angle between adjacent layers Fig. 3) was 23.5 nm. and the straightness of virus rods conSucrose treatment. In 20% w/v sucrose- trasts with the numerous disaggregated treated material gross plasmolysis was forms described by Esau (3) in glutaraldenever found. In some regions of the tissue, hyde-fixed material.
FIG. 1. Part of a mesophyll cell plasmolyzed by glycerol treatment. The TMV crystal (TMV) is completely ice-free as a result of the plasmolytic withdrawal of water. P, plasmalemma; W, cell wall. Bar, 1 t~m. FIG. 2. As a result of moderate glycerol plasmolysis, sufficient water has remained in this TMV crystal (TMV) for some of the water to segregate and appear as ice (arrows) after freezing. The interparticle spacing is 17.5 nm. W, cell wall. Bar, 1 t~m. 178
Fro. 3. Part of TMV crystal lying within a mesophyll cell that has been penetrated by glycerol. The crystal has retained its herringbone structure and the lattice spacing is about 24 nm, despite the fact that the tonoplast is ruptured. P, plasmalemma. Bar, 1 t~m. 179
FIG. 4. P a r t of TMV crystal, in a mesophyll cell penetrated by glycerol, fractured normally to the particle long axis. The particles lie in a n hexagonal lattice and a 120° angle is present in the crystal outline (arrow). Bar, 500 nm. Fro. 5. P a r t of a n ice-containing TMV crystal in a sucrose-treated cell. Ice-etching h a s revealed capsid surfaces (arrows) and enables the m e a s u r e m e n t of interparticle spacings. Several complete virus rods, suitable for calibration purposes, are clearly revealed. In the virus containing regions of the segregated crystal virions h a v e fractured internally. TMV rod length, 300 nm. 180
FIG. 6. P a r t of a TMV crystal in a n unplasmolyzed mesophyll cell from a leaf piece treated with 20% sucrose for about 1 min. The water present within the hydrated crystal before freezing has segregated to form ice (asterisks) upon freezing. The virus particles (V) in the segregated crystal are 17.6 n m apart and constitute 55% of the total crystal volume. In places (arrows) ice etching reveals capsid surfaces comparable to those in Fig. 5. Bar, 1 tLm. 181
182
J. H. M. WILLISON
The ice-containing virus crystals in su- tals are g e n e r a l l y spaced in the r a n g e 23crose-treated m a t e r i a l used for stereome- 24 n m suggests t h a t t h e y correspond with t r y were chosen carefully to ensure, as far "equilibrium gels" (2) of isolated virus, in as possible, t h a t cells had not lost apprecia- which the spacing is d e t e r m i n e d by salt ble quantities of w a t e r and that, as a conse- concentration and pH. This would imply quence, the a m o u n t of w a t e r s e p a r a t i n g t h a t the v a r i a t i o n in the m e a s u r e d v a l u e is the virions h a d not been reduced. Sucrose not due e n t i r e l y to the m e t h o d of estimatt r e a t m e n t was used to e l i m i n a t e the possi- ing w a t e r c o n t e n t using s t e r e o m e t r y , b u t bility of the loss of i n t r a c e l l u l a r w a t e r to r a t h e r t h a t t h e r e is some n a t u r a l v a r i a t i o n contribute to e x t r a c e l l u l a r ice (6). The rela- in the interparticle spacing arising from tively small s t a n d a r d deviation (5%) of the variations in the local p H and ionic compostereometrically d e t e r m i n e d ice-content of sition of the cells in which t h e y are conthe TMV crystals suggests t h a t the value tained. However, the i n vitro TMV equilibof 45% CrystalliZable w a t e r is valid, partic- r i u m gel is n e v e r in the same form as the u l a r l y in view of the probability t h a t t h e r e intracellular crystal, which is characterisis some n a t u r a l v a r i a t i o n in the interparti- tically l a m e l l a r - t w i n n e d (13). T h e r e m u s t cle spacing of i n vivo TMV crystals (see be a unique, b u t as yet u n d e t e r m i n e d , feadiscussion below). During freezing of bio- t u r e of the cellular e n v i r o n m e n t t h a t gives logical materials, ice segregates from the rise to lamellar-twinning. other cellular constituents giving rise, temI am grateful to W. C. Kimmins (Biology Departporarily, to high electrolyte concentrations ment, Dalhousie University) for the TMV used in (5)'. Thus, the a v e r a g e particle spacing in this study and to K. B. Easterbrook (Microbiology ice-containing crystals (17.6 nm) corre- Department, Dalhousie) for the use of equipment granted to him. The author is a postdoctoral fellow sponds well with the m i n i m u m interparti- supported by the Killam Trust. cle distance of h y d r a t e d TMV gels at high REFERENCES salt concentrations (17.5 nm) obtained by 1. BAWDEN, F. C., AND SHEFFIELD, F. M. L., Ann. Bernal and F r a n k u c h e n using X-ray difAppl. Biol. 26, 102 (1939). fraction (2). Since it can be hypothesized 2. BERNAL, J. D., AND FRANKUCHEN, I. J., Gen. reasonably t h a t i n t r a c r y s t a l l i n e ice arises Physiol. 25,111 (1941). 3. ESAU,K., Viruses in Plant Hosts. University of from i n t r a c r y s t a l l i n e water, the i n vivo Wisconsin Press, Madison, 1968. hexagonal lattice spacing of these TMV 4. MATTHEWS,R. E. L., Plant Virology. Academic crystals m u s t h a v e been 23.8 _+ 1.2 n m Press, New York/London, 1970. (i.e., (17.6) (1.83)°'5). This v a l u e contrasts 5. MAZUR,P., Science 168,939 (1970). 6. MOOR,H., Z. Zellforsch. 62, 546 (1964). with most previous estimates. However, 7. MOOR, H., AND MUHLETHALER,K., J. Cell. Biol. Steere, in e a r l y freeze-etching work (9), 17,609 (1963). used no cryoprotectant, and therefore, was 8. PLATTNER, H., FISCHER, W. M., SCHMITT, W. observing ice-containing crystals comparaW., AND BACHMANN, L., J. Cell. Biol. 53,116 ble with the sucrose-treated m a t e r i a l de(1972). 9. STEERE, R. L., J. Biophys. Biochem. Cytol. 3, 45 scribed here. W a r m k e and E d w a r d s o n (11) (1957). m e a s u r e d thin-sectioned crystals in which several layers of particles were p r e s e n t in 10. WARMKE, H. E., AND CHRISTIE, R. G., Virology 32,534 (1967). the thickness of the section m a k i n g relia- 11. WARMKE,H. E., ANDEDWARDSON, J. R., Virolble m e a s u r e m e n t v e r y difficult. However, ogy 30, 45 (1966). the above v a l u e does correspond well with 12. WEIBEL,E. R., KISTLER,G. S., AND SCHERLE, W. F., J. Cell. Biol. 30, 23 (1966). the e s t i m a t e impli'ed ~by Wilki~s and his co-workers (13), which was based upon reli- 13. WILmNS,M. F. H., STOKES,A. R., SEEDS,W. E., AND OSIER, G. Nature (London) 166, 127 able physical m e a s u r e m e n t s of crystals in (1950). living c e l l s . ' 14. WILHSON,J. H. M., AND COCKING, E. C . , J . Gen. T h a t virions in i n t r a c e l l u l a r T M V crys- : Virol. 4,229 (1969).
The Hexagonal Lattice Spacing of Intracellular Crystalline Tobacco Mosaic Virus J. H. M. WILLISON Department of Microbiology and Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada Received April 10, 1975 Previous estimates of hexagonal lattice spacing of the highly unstable, lamellar-twinned, intracellular tobacco mosaic virus (TMV) crystals have varied between 15 and 23 nm. In this study, the crystals have been examined by freeze-etching. Short periods (1-5 min) of glycerol or sucrose infiltration were applied before freezing. Glycerol t r e a t m e n t caused plasmolysis and a concomitant reduction in crystal lattice spacing as a result of water withdrawal. Cells penetrated by glycerol commonly contained well-ordered TMV crystals with hexagonal lattice spacings in the range 21-25 nm. These spacings probably represent stages in a glycerol-induced expansion of plasmolytically contracted crystals. Crystals in unplasmolyzed sucrose-treated cells always contained ice. The relative proportions of virus and ice (55:45) were stereometrically determined and the interparticle spacing (17.6 nm) was measured using TMV rod length as a standard. On the assumption t h a t intracrystalline ice arises from intracrystalline water, the hexagonal lattice spacing must be 23.6 _+ 1.2 nm.
The intracellular tobacco mosaic virus (TMV) crystal is polymorphic, but its most usual form is a thin hexagonal plate (1) in which the virus particles are arranged in layers parallel to the crystal hexagonal face (13). The virus particles of alternate layers lie roughly parallel to each other, but adjacent layers are inclined so as to produce a zig-zag or herringbone appearance (9, 13). Electron microscopy of thin sections of the crystals shows that the particles are hexagonally packed (10, 11 ). The crystals are highly labile and will change their form, or break down, under the influence of dilute acids, salt solutions, or even slight pressure from outside the cell (1, 13). This feature has made them difficult to study and left some doubt as to the hexagonal packing distance. Bernal and Frankuchen (2), who made early classic Xray diffraction studies of TMV gels, were of the opinion that intracellular crystals would be spaced similarly to isoelectric gels with a hexagonal packing distance of 18.5 nm. Wilkins and his co-workers (13) compared the birefringence of intracellular crystals with that of TMV gel and concluded that the crystals contained either
about 30 or about 60% water, probably the latter. Since the interparticle spacing in dried TMV gels is 15.2 nm (2) this would make the hydrated intracellular spacing either about 18 or about 23 nm if one assumes that the amount of water lying between adjacent layers of rods is insignificant, and therefore, that hydration simply moves the hexagonally packed rods apart. In an early freeze-etching study of intracellular TMV crystals, Steere (9) did not measure interparticle spacings, but it is clear from his micrographs that the particles lay 17-18 nm apart after freezing. However, Warmke and Edwardson (11) proposed, on the basis of thin section evidence, that the spacing was 15-16 nm. In the present study, intracellular TMV crystals have been reexamined using the freze-etching technique to attempt to account for these disparate estimates of the hexagonal lattice spacing. METHODS
Nicotiana tabacum L. var H a v a n a plants were systemically infected with TMV (vulgare strain). Pieces of infected leaves were diced in either 25% glycerol-0.1 M phosphate buffer or 20% w/v sucrose0.1 M phosphate buffer and frozen for freeze-etching 176
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
INTRACELLULAR TMV LATTICE SPACING after 2-5 min (glycerol) or 1-2 min (sucrose). Samples were freeze-etched in a Balzers apparatus using the procedure of Moor and Mfihlethaler (7) and etched for 30 sec at - 105°C before shadowing. Replicas were treated with 2% Meicelase (Meija Seika Kaishi Ltd., Tokyo, Japan), pH 6.0, for 24 hr before cleaning, sequentially, with sodium hypochlorite and 40% chromic acid, 8-18 hr each. For stereometric analysis, a t r a n s p a r e n t "Ethulon" overlay, marked with a standard set of points arranged in an hexagonal lattice (12), was layed over 8 x 10 in. photographic prints. Points were counted in two groups: virus or ice. In each case, the overlay was placed, randomly, in three different positions over the micrograph and an average of the three sets of points was obtained. Points t h a t lay over capsid surfaces t h a t has been exposed by ice etching were counted as ice. TMV rod length, 300 nm (4), was used as an internal calibration standard where required.
177
plasmolysis had begun, but in other regions there was no evidence of the onset of plasmolysis nor of ice development between the cell wall and plasmalemma. TMV crystals within these unplasmolyzed cells contained large ice crystals that had apparently arisen as a result of the crystallization of water from within the TMV crystals themselves (Fig. 6). The stereometric ratio of total crystal volume (virus + ice) to virus was 1.83 _+ 4.6% (1 SD, seven values). Where ice etching revealed closepacked virus rods (Fig. 5), the lattice spacing was 17.6 +_ 0.2 nm (1 SD, 10 values). DISCUSSION
Ultrastructural studies of intracellular TMV crystals are complicated by the fact RESULTS that the crystals are disrupted by glutaralGlycerol treatment. Treatment of leaf dehyde fixation (14) and, at best, are only pieces with 25% glycerol usually resulted poorly preserved by osmium tetroxide or in gross plasmolysis. In highly plasmo- potassium permanganate (10). Freezing of lyzed cells, virions within the TMV crys- untreated leaves results in ice crystals tals were tightly packed such that individ- within virus crystals (14), but effective ual rods could rarely be made out (Fig. 1). penetration of glycerol as a cryoprotectant In moderately plasmolyzed cells, ice usu- is virtually impossible (14, this work). The ally was found within the virus crystals deleterious properties of glycerol in its use (Fig. 2). Cells also were found in which as a cryoprotectant have been recognized glycerol had penetrated into the cell to for some time (8). It seems probable that provide conventional cryoprotected im- the effect of glycerol in the present study ages. These cells sometimes appear to be has been sequentially: severe plasmolysis, unplasmolyzed, but tonoplast rupture, entry through the plasmalemma, deplasTMV crystal disruption, and other forms molysis, tonoplast rupture, and TMV crysof cell injury were commonplace. Neverthe- tal disruption. If this is the case, then the less, in such cells, TMV crystals that had ~'cryoprotected" TMV crystals (e.g., Fig. 3) retained both the ~'herringbone" structure have undergone shrinkage prior to the ob(Fig. 3) and the hexagonal lattice (Fig. 4) served glycerol-expanded state. Consewere sometimes found. Adjacent layers of quently the 21-25 nm spacings of herringvirus rods were not separated by an appre- bone crystals, described above in glycerolciable gap (Fig. 3). The hexagonal lattice treated material, probably represent spacing of these cryoprotected crystals was stages in the glycerol expansion and bear variable, with values lying between 21 and only a passing relevance to the in vivo 25 nm. The mean spacing obtained from spacing. Nevertheless, the striking regufive such crystals in which rod length larity of these crystals both in terms of the could be used reliably for calibration (e.g., constant angle between adjacent layers Fig. 3) was 23.5 nm. and the straightness of virus rods conSucrose treatment. In 20% w/v sucrose- trasts with the numerous disaggregated treated material gross plasmolysis was forms described by Esau (3) in glutaraldenever found. In some regions of the tissue, hyde-fixed material.
FIG. 1. Part of a mesophyll cell plasmolyzed by glycerol treatment. The TMV crystal (TMV) is completely ice-free as a result of the plasmolytic withdrawal of water. P, plasmalemma; W, cell wall. Bar, 1 t~m. FIG. 2. As a result of moderate glycerol plasmolysis, sufficient water has remained in this TMV crystal (TMV) for some of the water to segregate and appear as ice (arrows) after freezing. The interparticle spacing is 17.5 nm. W, cell wall. Bar, 1 t~m. 178
Fro. 3. Part of TMV crystal lying within a mesophyll cell that has been penetrated by glycerol. The crystal has retained its herringbone structure and the lattice spacing is about 24 nm, despite the fact that the tonoplast is ruptured. P, plasmalemma. Bar, 1 t~m. 179
FIG. 4. P a r t of TMV crystal, in a mesophyll cell penetrated by glycerol, fractured normally to the particle long axis. The particles lie in a n hexagonal lattice and a 120° angle is present in the crystal outline (arrow). Bar, 500 nm. Fro. 5. P a r t of a n ice-containing TMV crystal in a sucrose-treated cell. Ice-etching h a s revealed capsid surfaces (arrows) and enables the m e a s u r e m e n t of interparticle spacings. Several complete virus rods, suitable for calibration purposes, are clearly revealed. In the virus containing regions of the segregated crystal virions h a v e fractured internally. TMV rod length, 300 nm. 180
FIG. 6. P a r t of a TMV crystal in a n unplasmolyzed mesophyll cell from a leaf piece treated with 20% sucrose for about 1 min. The water present within the hydrated crystal before freezing has segregated to form ice (asterisks) upon freezing. The virus particles (V) in the segregated crystal are 17.6 n m apart and constitute 55% of the total crystal volume. In places (arrows) ice etching reveals capsid surfaces comparable to those in Fig. 5. Bar, 1 tLm. 181
182
J. H. M. WILLISON
The ice-containing virus crystals in su- tals are g e n e r a l l y spaced in the r a n g e 23crose-treated m a t e r i a l used for stereome- 24 n m suggests t h a t t h e y correspond with t r y were chosen carefully to ensure, as far "equilibrium gels" (2) of isolated virus, in as possible, t h a t cells had not lost apprecia- which the spacing is d e t e r m i n e d by salt ble quantities of w a t e r and that, as a conse- concentration and pH. This would imply quence, the a m o u n t of w a t e r s e p a r a t i n g t h a t the v a r i a t i o n in the m e a s u r e d v a l u e is the virions h a d not been reduced. Sucrose not due e n t i r e l y to the m e t h o d of estimatt r e a t m e n t was used to e l i m i n a t e the possi- ing w a t e r c o n t e n t using s t e r e o m e t r y , b u t bility of the loss of i n t r a c e l l u l a r w a t e r to r a t h e r t h a t t h e r e is some n a t u r a l v a r i a t i o n contribute to e x t r a c e l l u l a r ice (6). The rela- in the interparticle spacing arising from tively small s t a n d a r d deviation (5%) of the variations in the local p H and ionic compostereometrically d e t e r m i n e d ice-content of sition of the cells in which t h e y are conthe TMV crystals suggests t h a t the value tained. However, the i n vitro TMV equilibof 45% CrystalliZable w a t e r is valid, partic- r i u m gel is n e v e r in the same form as the u l a r l y in view of the probability t h a t t h e r e intracellular crystal, which is characterisis some n a t u r a l v a r i a t i o n in the interparti- tically l a m e l l a r - t w i n n e d (13). T h e r e m u s t cle spacing of i n vivo TMV crystals (see be a unique, b u t as yet u n d e t e r m i n e d , feadiscussion below). During freezing of bio- t u r e of the cellular e n v i r o n m e n t t h a t gives logical materials, ice segregates from the rise to lamellar-twinning. other cellular constituents giving rise, temI am grateful to W. C. Kimmins (Biology Departporarily, to high electrolyte concentrations ment, Dalhousie University) for the TMV used in (5)'. Thus, the a v e r a g e particle spacing in this study and to K. B. Easterbrook (Microbiology ice-containing crystals (17.6 nm) corre- Department, Dalhousie) for the use of equipment granted to him. The author is a postdoctoral fellow sponds well with the m i n i m u m interparti- supported by the Killam Trust. cle distance of h y d r a t e d TMV gels at high REFERENCES salt concentrations (17.5 nm) obtained by 1. BAWDEN, F. C., AND SHEFFIELD, F. M. L., Ann. Bernal and F r a n k u c h e n using X-ray difAppl. Biol. 26, 102 (1939). fraction (2). Since it can be hypothesized 2. BERNAL, J. D., AND FRANKUCHEN, I. J., Gen. reasonably t h a t i n t r a c r y s t a l l i n e ice arises Physiol. 25,111 (1941). 3. ESAU,K., Viruses in Plant Hosts. University of from i n t r a c r y s t a l l i n e water, the i n vivo Wisconsin Press, Madison, 1968. hexagonal lattice spacing of these TMV 4. MATTHEWS,R. E. L., Plant Virology. Academic crystals m u s t h a v e been 23.8 _+ 1.2 n m Press, New York/London, 1970. (i.e., (17.6) (1.83)°'5). This v a l u e contrasts 5. MAZUR,P., Science 168,939 (1970). 6. MOOR,H., Z. Zellforsch. 62, 546 (1964). with most previous estimates. However, 7. MOOR, H., AND MUHLETHALER,K., J. Cell. Biol. Steere, in e a r l y freeze-etching work (9), 17,609 (1963). used no cryoprotectant, and therefore, was 8. PLATTNER, H., FISCHER, W. M., SCHMITT, W. observing ice-containing crystals comparaW., AND BACHMANN, L., J. Cell. Biol. 53,116 ble with the sucrose-treated m a t e r i a l de(1972). 9. STEERE, R. L., J. Biophys. Biochem. Cytol. 3, 45 scribed here. W a r m k e and E d w a r d s o n (11) (1957). m e a s u r e d thin-sectioned crystals in which several layers of particles were p r e s e n t in 10. WARMKE, H. E., AND CHRISTIE, R. G., Virology 32,534 (1967). the thickness of the section m a k i n g relia- 11. WARMKE,H. E., ANDEDWARDSON, J. R., Virolble m e a s u r e m e n t v e r y difficult. However, ogy 30, 45 (1966). the above v a l u e does correspond well with 12. WEIBEL,E. R., KISTLER,G. S., AND SCHERLE, W. F., J. Cell. Biol. 30, 23 (1966). the e s t i m a t e impli'ed ~by Wilki~s and his co-workers (13), which was based upon reli- 13. WILmNS,M. F. H., STOKES,A. R., SEEDS,W. E., AND OSIER, G. Nature (London) 166, 127 able physical m e a s u r e m e n t s of crystals in (1950). living c e l l s . ' 14. WILHSON,J. H. M., AND COCKING, E. C . , J . Gen. T h a t virions in i n t r a c e l l u l a r T M V crys- : Virol. 4,229 (1969).
