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Electron microscopic autoradiography of leaf cells infected with tobacco mosaic virus I. Uridine-H3 uptake
VIROLOGY
24, 102-106
Electron
(1964)
Microscopic
Autoradiography Tobacco
of Leaf
Mosaic
I. Uridine-H3 HIROYUKI Plant
Pathology
Laboratory,
Faculty
HIBINO
Infected
with
Virus
Uptake’ AND
of Agriculture, Accepted
Cells
May
CHIAKI Nagoya
MATSUI University,
Anjo,
Aichiken,
Japan
6, 1964
Autoradiographic techniques were applied to thin sections of tobacco leaves labeled with tritiated uridine for 24 hours at 3 days after infection with tobacco mosaic virus or rubbing with distilled water. In both cases, the developed silver grains were restricted to areas over the nucleus and cytoplasmic ground substance. Tritiated uridine uptake of the leaf cells was increased by virus infection, and the greater part of the actively incorporated uridine was in the masses of virus particles.
taken, and the present paper deals with uptake of tritiated uridine into intracellular virus particles.
INTRODUCTION
Light microscopic autoradiography has been widely used as a cytochemical method. According to limitations in resolution imposed by emulsion thickness as well as section thickness, however, it is difficult to obtain high resolution autoradiography by conventional microautoradiographic procedures. In researches on virus-infected tissue cells, especially, there arises a need for high resolution autoradiography, because the size of virus particles is far beyond the resolution of the light microscope and fine structural details of the cell are not clearly resolved under a light microscope. Recently, electron microscopic autoradiography has begun to provide real contributions to the relation between the fine structures of the cell organelles and their biosynthetic activities. Thus Dales (1963) first succeeded in the application of electron microscopic autoradiography for the investigation of intracellular vaccinia virus. A series of electron microscopic autovirus radiographies of tobacco mosaic (TMV) infected leaf cells has been under-
MATERIALS
AND
METHODS
Upper and lower surfaces of detached matured leaves of Nicotiuna tabacum L. var. Bright Yellow were inoculated mechanically with the common strain of TMV. The inoculated leaves were kept at 25”C, under continuous illumination of fluorescent lamps, with their petioles immersed in distilled water. Three days after inoculation, rectangular pieces (2 X 1 cm) cut from the inoculated leaves were floated on 0.5 ml of distilled water containing 50 PC of uridine-H3 (The Radiochemical Center, specific activity, 1.1 c/mmole), and were incubated at 25” under illumination. After 24 hours of incubation, small pieces of the floated leaf rectangles were fixed. Fixative, washing, dehydrating, and plastic embedding procedures were those described by Matsui and Yamaguchi (1964). Thin sections 50-100 rnp thick were obtained using a JUXl-4 ultramicrotome (Japan Electron Optics Laboratory Co., Ltd.) with glass knives, and were picked up on copper specimen grids coated with a slightly thicker Formvar film. The
1 This study was supported by a grant (EF60447-01) from United States Department of Health, Education, and Welfare, Public Health Service. 102
AUTORADIOGRAPHY
specimen grids with thin sections were affixed by one edge with double-coated Scotch tape to a glass microscope slide which had been coated previously with a mixture of 0.1% gelatin and 0.01% chromealum (Care, 1961). This coating facilitated the homogeneousadherence of the emulsion f&n to the grass slide (mentioned later). Under safelight illumination (red lamp), melted (43’) NR-Ml-G nuclear emulsion (Konishiroku Photo Ind. Co., Ltd. Japan) was diluted 1:3 with distilled water (43’). The glass slide with the specimen grids was coated with an emulsion film by the loop method described by Caro et al. (1962). After coating, the emulsion film was dried, and the emulsion-coated grids were placed in a light-tight metal box with desiccant, and stored at 4” for autoradio~aphic exposure. After 2 weeks, the emulsion-coated glass slide was developed in Konidol X (Konishiroku Photo Ind. Co., Ltd.) or Fuji Rend01 (Fuji Photo Film Co., Ltd. Japan) at 18” for 4 minutes and fixed in Konifix (Konishiroku Photo Ind. Co., Ltd.) for 10 minutes. The developed glass slide was washed for 10 minutes in distilled water and dried. The developed specimens were examined in a JEM-T6 electron microscope (JEOL. Co., Ltd.) without removal of gelatin of the emulsion. For comparison, healthy leaves rubbed with distilled water were examined in the same way. For light microscopic autoradio~aphy, thicker sections (about 1 FL)of the same materials were placed on a gelatin chromealum coated glass slide. The grassslide with the sections was coated with an emulsion film (diluted 1:0.5 with distilled water) by the loop method. The subsequent procedures were those described previously. RESULTS
AND DISCUSSION
Healthy Leaf Cells In the light microscopic autoradiographs of healthy leaf cells labeled with uridine-H3, the developed silver grains were seen over all cells, and no grains were found over the intercellular spaces. Accordingly, it is clear that almost all cells were labeled by the present procedures.
103
OF TMV
In the electron microscopic autoradiographs, on the other hand, the developed silver grains were so few that it was laborious to search for them. Pres~ably, the laborious search for the developed silver grains is due to thin spec~ne~ used for electron microscopic autoradiography. Furthermore, it has been commonly accepted that the synthesis of nucleic acid within the matured leaf cells is lower than in animal cells. Accordingly, it is natural that the developed silver grains detected in the present autoradiographs are few in number. In the electron microscopic autoradiographs of healthy leaf cells, the greater part of the developed silver grains was seen over the nuclei and cytoplasmic ground substance, whereas no grains were detected over the mitocho~ldria, vacuoles, and ~tercellular spaces (Figs. l-3). Silver grains over the chloroplasts were seldom encountered. Although Jacobson et al. (1963) demonstrated clearly the active occurrence of RNA within plastids of actively growing young leaves, since the silver grains over the chloroplasts were rarely detected in the present autoradiographs, it is appropriate to conclude that newly synthesized RNA of the chloroplasts of matured leaf cells is at a low level. TMV-Infected
Leaf Cells
In the autoradiographs of TMV-infected leaf cells labeled with uridine-H3, the nuclei and cytoplasmic ground substance associated with develops silver grains were somewhat more in number than in healthy leaf cells. There was no labeling in the mitochondria, vacuoles, and intercellular spaces (Figs. 4 and 5). Sometimes a vacuole-like profile was encountered within a nucleus as shown in Fig. 6, whereas most nucleoplasm uniformly consisted of a finely textured component. In the former, however, the developed silver grains were restricted to the area over the nucleoplasm. In addition to the developed silver grains over the nuclei and cytoplasmic ground substance, the most striking feature of the autor~io~aphs of T~~V-infected leaf cells labeled with uridine-H3 was a heavy deposit of the developed silver grains over the fibrous masses (Fig. 7). According to pre-
tr FIGS. 1-8. The key to the labeling is as follows: C, chloroplast; G, silver grain; ICS, intercellul: N, nucleus; 0, osmiophilic granule; 8, starch grain; TMV, mass of TM V sP lace; M, mitochondrion; V, vacuole; W, cell wall. Ps articles; FIG. 1. Healthy leaf cell nucleus. Note an irregularly coiled silver grain. FIGS. 2 and 3. Healthy leaf cell periphery. Note the silver grains over the cytoplasm. FIGS. 4 and 5. TMV-infected leaf cell periphery. 104
FIGS. G. Survey
view
of TMV-infected
leaf cells.
The
silver
grains
are seen over
the TMV
masses
and
a nucleus.
FIG. 7. The
silver
grains
over
the
mass
of TM\’
particles
cut
obliquely
to the
long
axis
of virus
particles.
FIG. 8. The silver
grain
over
the mass
of TMV
particles 105
cut parallel
to the long
axis
of the particles.
106
HIBINO
AND
vious observations (Matsui, 19X4), it is apparent that these fibrous masses correspond to the masses of intracellular TMV particles. Figure 6 represents a survey view of an autoradiograph of TMV-infected leaf cells. The developed silver grains associated with the virus masses varied in number (Fig. 8). Occasionally, masses of TMV particles without a developed silver grain were encountered, but grains were usually detected in other sections of these same masses of TMV particles. Accordingly it is apparent that most masses of TMV particles were labeled with uridine-H3. Moreover, the developed silver grains were seen over a peculiar region of the cytoplasm as shown in Fig. 5. This region appeared as a fine reticular profile, and different from the general profile of the cytoplasmic ground substance. Whether this region corresponds to a mass of irregularly disposed TMV particles or relates to some process of virus reproduction, will be discussed in a later paper. In the light of the present evidence, at any rate, it is concluded that uridine-H3 uptake of the leaf cells is increased by virus infection and the greater part of the actively incorporated uridine is distributed in masses
MATSUI
of newly produced virus particles ably, RNA of TMV particles).
(presum-
ACKNOWLEDGMENT Our sincere thanks Ind. Co., Ltd. for emulsion.
are due Konishiroku kindly supplying
Photo nuclear
REFERENCES CARO, L. G. (1961). Electron microscopic radioautography of thin sections: The Golgi zone as a site of protein concentration in pancreatic acinar cells. J. Biophys. Biochem. Cytol. 10, 3745. CARO, L. G., v.4~ TUBERGEN, R. P., and KOLB, J. A. (1962). High-resolution autoradiography. I. J. Cell Biol. 15, 173-188. DALES, S. (1963). The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid. J. Cell. Biol. 18, 51-72. JACOBSON, A. B., SWIFT, H., and BOGORAD, L. (1963). Cytochemical studies concerning the occurrence and distribution of RNA in plastids of Zeamays. J. Cell Biol. 17, 557-570. MATSUI, C. (1958). Pathological cytology of tobacco leaf infected with tobacco mosaic virus. III. J. Biophys. Biochem. Cytol. 4,831~832. MATSUI, C., and YAMAGUCHI, A. (1964). Electron microscopy of host cells infected with tobacco etch virus. I. Virology 22, 4047.
24, 102-106
Electron
(1964)
Microscopic
Autoradiography Tobacco
of Leaf
Mosaic
I. Uridine-H3 HIROYUKI Plant
Pathology
Laboratory,
Faculty
HIBINO
Infected
with
Virus
Uptake’ AND
of Agriculture, Accepted
Cells
May
CHIAKI Nagoya
MATSUI University,
Anjo,
Aichiken,
Japan
6, 1964
Autoradiographic techniques were applied to thin sections of tobacco leaves labeled with tritiated uridine for 24 hours at 3 days after infection with tobacco mosaic virus or rubbing with distilled water. In both cases, the developed silver grains were restricted to areas over the nucleus and cytoplasmic ground substance. Tritiated uridine uptake of the leaf cells was increased by virus infection, and the greater part of the actively incorporated uridine was in the masses of virus particles.
taken, and the present paper deals with uptake of tritiated uridine into intracellular virus particles.
INTRODUCTION
Light microscopic autoradiography has been widely used as a cytochemical method. According to limitations in resolution imposed by emulsion thickness as well as section thickness, however, it is difficult to obtain high resolution autoradiography by conventional microautoradiographic procedures. In researches on virus-infected tissue cells, especially, there arises a need for high resolution autoradiography, because the size of virus particles is far beyond the resolution of the light microscope and fine structural details of the cell are not clearly resolved under a light microscope. Recently, electron microscopic autoradiography has begun to provide real contributions to the relation between the fine structures of the cell organelles and their biosynthetic activities. Thus Dales (1963) first succeeded in the application of electron microscopic autoradiography for the investigation of intracellular vaccinia virus. A series of electron microscopic autovirus radiographies of tobacco mosaic (TMV) infected leaf cells has been under-
MATERIALS
AND
METHODS
Upper and lower surfaces of detached matured leaves of Nicotiuna tabacum L. var. Bright Yellow were inoculated mechanically with the common strain of TMV. The inoculated leaves were kept at 25”C, under continuous illumination of fluorescent lamps, with their petioles immersed in distilled water. Three days after inoculation, rectangular pieces (2 X 1 cm) cut from the inoculated leaves were floated on 0.5 ml of distilled water containing 50 PC of uridine-H3 (The Radiochemical Center, specific activity, 1.1 c/mmole), and were incubated at 25” under illumination. After 24 hours of incubation, small pieces of the floated leaf rectangles were fixed. Fixative, washing, dehydrating, and plastic embedding procedures were those described by Matsui and Yamaguchi (1964). Thin sections 50-100 rnp thick were obtained using a JUXl-4 ultramicrotome (Japan Electron Optics Laboratory Co., Ltd.) with glass knives, and were picked up on copper specimen grids coated with a slightly thicker Formvar film. The
1 This study was supported by a grant (EF60447-01) from United States Department of Health, Education, and Welfare, Public Health Service. 102
AUTORADIOGRAPHY
specimen grids with thin sections were affixed by one edge with double-coated Scotch tape to a glass microscope slide which had been coated previously with a mixture of 0.1% gelatin and 0.01% chromealum (Care, 1961). This coating facilitated the homogeneousadherence of the emulsion f&n to the grass slide (mentioned later). Under safelight illumination (red lamp), melted (43’) NR-Ml-G nuclear emulsion (Konishiroku Photo Ind. Co., Ltd. Japan) was diluted 1:3 with distilled water (43’). The glass slide with the specimen grids was coated with an emulsion film by the loop method described by Caro et al. (1962). After coating, the emulsion film was dried, and the emulsion-coated grids were placed in a light-tight metal box with desiccant, and stored at 4” for autoradio~aphic exposure. After 2 weeks, the emulsion-coated glass slide was developed in Konidol X (Konishiroku Photo Ind. Co., Ltd.) or Fuji Rend01 (Fuji Photo Film Co., Ltd. Japan) at 18” for 4 minutes and fixed in Konifix (Konishiroku Photo Ind. Co., Ltd.) for 10 minutes. The developed glass slide was washed for 10 minutes in distilled water and dried. The developed specimens were examined in a JEM-T6 electron microscope (JEOL. Co., Ltd.) without removal of gelatin of the emulsion. For comparison, healthy leaves rubbed with distilled water were examined in the same way. For light microscopic autoradio~aphy, thicker sections (about 1 FL)of the same materials were placed on a gelatin chromealum coated glass slide. The grassslide with the sections was coated with an emulsion film (diluted 1:0.5 with distilled water) by the loop method. The subsequent procedures were those described previously. RESULTS
AND DISCUSSION
Healthy Leaf Cells In the light microscopic autoradiographs of healthy leaf cells labeled with uridine-H3, the developed silver grains were seen over all cells, and no grains were found over the intercellular spaces. Accordingly, it is clear that almost all cells were labeled by the present procedures.
103
OF TMV
In the electron microscopic autoradiographs, on the other hand, the developed silver grains were so few that it was laborious to search for them. Pres~ably, the laborious search for the developed silver grains is due to thin spec~ne~ used for electron microscopic autoradiography. Furthermore, it has been commonly accepted that the synthesis of nucleic acid within the matured leaf cells is lower than in animal cells. Accordingly, it is natural that the developed silver grains detected in the present autoradiographs are few in number. In the electron microscopic autoradiographs of healthy leaf cells, the greater part of the developed silver grains was seen over the nuclei and cytoplasmic ground substance, whereas no grains were detected over the mitocho~ldria, vacuoles, and ~tercellular spaces (Figs. l-3). Silver grains over the chloroplasts were seldom encountered. Although Jacobson et al. (1963) demonstrated clearly the active occurrence of RNA within plastids of actively growing young leaves, since the silver grains over the chloroplasts were rarely detected in the present autoradiographs, it is appropriate to conclude that newly synthesized RNA of the chloroplasts of matured leaf cells is at a low level. TMV-Infected
Leaf Cells
In the autoradiographs of TMV-infected leaf cells labeled with uridine-H3, the nuclei and cytoplasmic ground substance associated with develops silver grains were somewhat more in number than in healthy leaf cells. There was no labeling in the mitochondria, vacuoles, and intercellular spaces (Figs. 4 and 5). Sometimes a vacuole-like profile was encountered within a nucleus as shown in Fig. 6, whereas most nucleoplasm uniformly consisted of a finely textured component. In the former, however, the developed silver grains were restricted to the area over the nucleoplasm. In addition to the developed silver grains over the nuclei and cytoplasmic ground substance, the most striking feature of the autor~io~aphs of T~~V-infected leaf cells labeled with uridine-H3 was a heavy deposit of the developed silver grains over the fibrous masses (Fig. 7). According to pre-
tr FIGS. 1-8. The key to the labeling is as follows: C, chloroplast; G, silver grain; ICS, intercellul: N, nucleus; 0, osmiophilic granule; 8, starch grain; TMV, mass of TM V sP lace; M, mitochondrion; V, vacuole; W, cell wall. Ps articles; FIG. 1. Healthy leaf cell nucleus. Note an irregularly coiled silver grain. FIGS. 2 and 3. Healthy leaf cell periphery. Note the silver grains over the cytoplasm. FIGS. 4 and 5. TMV-infected leaf cell periphery. 104
FIGS. G. Survey
view
of TMV-infected
leaf cells.
The
silver
grains
are seen over
the TMV
masses
and
a nucleus.
FIG. 7. The
silver
grains
over
the
mass
of TM\’
particles
cut
obliquely
to the
long
axis
of virus
particles.
FIG. 8. The silver
grain
over
the mass
of TMV
particles 105
cut parallel
to the long
axis
of the particles.
106
HIBINO
AND
vious observations (Matsui, 19X4), it is apparent that these fibrous masses correspond to the masses of intracellular TMV particles. Figure 6 represents a survey view of an autoradiograph of TMV-infected leaf cells. The developed silver grains associated with the virus masses varied in number (Fig. 8). Occasionally, masses of TMV particles without a developed silver grain were encountered, but grains were usually detected in other sections of these same masses of TMV particles. Accordingly it is apparent that most masses of TMV particles were labeled with uridine-H3. Moreover, the developed silver grains were seen over a peculiar region of the cytoplasm as shown in Fig. 5. This region appeared as a fine reticular profile, and different from the general profile of the cytoplasmic ground substance. Whether this region corresponds to a mass of irregularly disposed TMV particles or relates to some process of virus reproduction, will be discussed in a later paper. In the light of the present evidence, at any rate, it is concluded that uridine-H3 uptake of the leaf cells is increased by virus infection and the greater part of the actively incorporated uridine is distributed in masses
MATSUI
of newly produced virus particles ably, RNA of TMV particles).
(presum-
ACKNOWLEDGMENT Our sincere thanks Ind. Co., Ltd. for emulsion.
are due Konishiroku kindly supplying
Photo nuclear
REFERENCES CARO, L. G. (1961). Electron microscopic radioautography of thin sections: The Golgi zone as a site of protein concentration in pancreatic acinar cells. J. Biophys. Biochem. Cytol. 10, 3745. CARO, L. G., v.4~ TUBERGEN, R. P., and KOLB, J. A. (1962). High-resolution autoradiography. I. J. Cell Biol. 15, 173-188. DALES, S. (1963). The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid. J. Cell. Biol. 18, 51-72. JACOBSON, A. B., SWIFT, H., and BOGORAD, L. (1963). Cytochemical studies concerning the occurrence and distribution of RNA in plastids of Zeamays. J. Cell Biol. 17, 557-570. MATSUI, C. (1958). Pathological cytology of tobacco leaf infected with tobacco mosaic virus. III. J. Biophys. Biochem. Cytol. 4,831~832. MATSUI, C., and YAMAGUCHI, A. (1964). Electron microscopy of host cells infected with tobacco etch virus. I. Virology 22, 4047.