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Intranuclear and intracytoplasmic structures of Rhodotorula glutinis as revealed by electron micrographs of serial sections
Experimental
Cell Research 29, 235-241 (1963)
INTRANUCLEAR
235
AND INTRACYTOPLASMIC
STRUCTURES
OF RHODOTORULA GLUTINIS AS REVEALED BY ELECTRON MICROGRAPHS OF SERIAL SECTIONS S. F. CONTI, Department Division
T. R. THYAGARAJANI
and H. B. NAYLOR
of Microbiology, Dartmouth Medical School, Hanover, New Hampshire, and of Bacteriology, Department of Dairy and Food Science, Cornell University, Ithaca, N.Y.,
U.S.A.
Received April 9, 1962
ACCOKDISG to Mundkur [la] and Townsend [21], the yeast nucleus is a homogeneous vesicle with no observable internal differentiation whereas the presence of chromocenters and nucleolar equivalents, identified by cytochemical staining techniques, has been reported by others [ 14, 15, 171. Chromosome counts on stained preparations have been reported by a number of workers 11, 4, 8, 9, 10, 16, 241. On the other hand, early electron microscope studies of vegetative cells failed to reveal any intranuclear structure [2, 61. Internal differentiation of the nucleus in sporulating cells of Saccharomyces cereuisiae was reported in a study of Hashimoto et al. [7]. Totsuyanagi [25, 261 observed in the nucleus a dense nucleolus and some areas of low electron density which he interpreted to be chromosomal. The recent work of Vitals et al. [22] also reveals areas of contrasting electron density in the nucleoplasm of S. cerevisiae. The presence in the nucleus of a membrane bounded structure and areas of low electron density in Rhodotorula glutinis has been reported [ 181. The difficulties involved in observing intranuclear structures in ultrathin sections with the electron microscope have been pointed out 12, 3, 111. During a study of the nuclei in living cells of Rh. glutinis a dense central body surrounded by a shell of optically uniform material of lower density was observed, Cytochemical staining techniques revealed that the central body is Feulgen-negative but stainable with iron alum haematoxylin and on this basis was identified as the nucleolus. The area surrounding the nucleolus was found to contain Feulgen-positive structures. Although the small size of the chromatin granules did not permit as exact count, the staining reaction 1 Present address: Central
Drug Research
Institute,
Lucknow,
India. Experimental
Cell Research 29
236
S. F. Conti, T. R. Thyagarajan
and H. S. Naylor
indicated that deoxyribonucleic acid is contained in this region of the nucleus [20]. The presence of an intranulcear structure prompted LB to study this yeast bp means of electron microscopy of ultrathin sections.
MATERIAL
AND
METHODS
obtained from Dr. L. J. Wickerham was routinely grown at room Rh. glutinis temperature for 4X hr in a pH 4.6 barley malt wort medium of specific gravity 1.005. Cells were fixed in 1.5 per cent aqueous potassium permanganate for 10 min at room temperature, rinsed in distilled water, and resuspended in an aqueous solution of 2.0 per cent osmium tetroxide for 90 min. The specimens were dehydrated by passage through a graded alcohol series, embedded in butyl methacrylate, and polymerized at 55°C. Ultrathin sections were obtained with an LKB 4800 ultrotome using a glass knife. Sections were mounted on carbon coated grids and examined with an Akashi Tronscope TRS-50 electron microscope equipped with a 50 p aperture.
RESULTS
The appearance of the nucleus in living cells under the phase microscope is shown in Fig. 1. The dense central nucleolus is surrounded by a shell of uniform material of lower density in which no structural details are observable. In the course of preparing thin sections of this organism for examination with the electron microscope consecutive sections of a single resting cell were obtained. Electron micrographs of seven of these sections are presented in Figs. 3-Y. The nucleus contains a prominent inclusion (Figs. :1-S) which may be regarded as the same structure identified as the nucleolus in previous studies [Xl]. This structure is surrounded bv a membrane and in cross section appears to have a maximum diameter of approximately 0.5 p (Figs. 5-7). In addition to the nucleolus irregular areas of low electron density can
Fig. l.-Cells of Rh. glufinis tral body which is identified Fig. 2.-An
under the phase microscope. The nucleus (Iv) contains as the nucleolus (Nu). x 5500.
iron alum haematoxylin
stain of the yeast cell.
a dense ccn-
Y 5500.
Figs. 3-g.-Micrographs of 7 consecutive sections of a resting yeast cell. Note the areas of low electron density in the nucleus, and the membrane bounded nucleolus. An area of low electron density between the nucleolus and nucleus is seen in b’ig. 7, and the connection of a mitochondrion (M) to the nuclear membrane (NfiI) in Fig. 5, and plasma membrane (CM) in Fig. 9 is apparent. The circular or spherical inclusions (I,) of low electron density located in the cytoplasm are lipoidal in nature and stainable with Sudan Black B. Further discussion of these figures appears in the text. x 22,000. Experimental
Cell Research 29
Structures of Rhodotorula glutinis
Experimental
237
Cell Research 29
238
Experimental
S. F. Conti, T. R. Thyagarajan and H. B. Naylor
Cell Reseureh 29
Structures
of Rhodotorula
glutinis
be observed in the nucleoplasm. The diffuse nature of these areas makes it difficult to follow them from one section to another, consequently, only a rough approximation of their three dimensional configuration can be made. The presence of an extensive intracytoplasmic membrane system is evident from the electron micrographs. Certain membrane segments such as the one in close proximity to the cytoplasmic membrane at the upper left portion of the sectioned cell and the one lying close to the nucleus can be seen in each of the serial sections (Figs. S-9). The close association of mitochondria with the membrane system is also clearly shown. In some instances, the membrane seems to form a bridge between a mitochondrion and the plasma membrane (Fig. 9). In other instances, the mitochondrion is closely associated with a membrane which lies very near to, and perhaps connects with, the nuclear membrane. This can be observed most clearly in Figs. 3, 4, and 5 where three successive sections of the mitochondrion lying just to the right of the nucleus all show the connection with the internal membrane. Although this mitochondrion has disappeared from subsequent sections, the membrane can still be seen (Figs. 6, 7, and 8). The well defined areas of low electron density in the cytoplasm are lipoidal inclusions which increase in size and number during the stationary phase of growth of this yeast. DISCUSSION
As has been pointed out by Williams and Kallman [23], it is difficult to reconstruct detailed three-dimensional models from electron micrographs of serial sections of biological materials. It is possible, however, to obtain valuable information about those structures that can be followed through succeeding sections with a high degree of certainty. The electron micrographs presented here indicate the presence of a membrane bounded intranuclear structure in Rh. glufinis. The complete series of sections through this structure indicate that it is intranuclear, and not formed by an infolding of the nuclear membrane. While investigators have observed nucleoli in yeasts with the light microscope, electron microscopy has yielded very little information about this structure. Edwards et al. [5] observed nucleoli in thin sections of yeast-like cells of Hisfoplasma, and Yotsuyanagi [25] observed similar bodies within the nuclei in thin sections of S. cereuisiae. In both cases, the nucleoli were considerably more electron dense than the surrounding nucleoplasm, and they did not appear to be enclosed by a membrane. The membrane bounded Experimental
Cell Research 29
240
S. F. Confi, T. R. Thyagarajan
and H. R. Nay/or
intranuclear component of Rh. glutinis, which is tentatively considered to be the nucleolus, has about the same electron density as the nucleoplasm except for the membrane, which is of greater electron density. The structure of the nucleolus may vary with the physiological state of the cells since we have not been able to observe it at all in thin sections of dividing cells. Studies of Lipomyces lipofer with the light microscope [ 131 illustrate that the nucleolus disappears during mitosis and then reappears in the nuclei of the resulting daughter cells. Areas of low electron density within the nucleus of Rh. glutinis can also be seen in the accompanying electron micrographs. However, due to the diffuse nature of the areas, they cannot be followed readily from one section to the next. It is probable that the areas referred to here are comparable to those observed in the nucleus of S. cerevisiae by others [i, 22, 25, 261. Totsuyanagi [a,?] has presented some evidence that the material is chromosomal in nature. Careful study of the serial sections reveals some interesting information about the intracytoplasmic membrane system of this yeast. Although thin in cross section, the membrane(s) must have considerable width since in certain areas it can be followed through the entire series of electron micrographs. The close association of the membrane with the nucleus is also evident. In some instances, the mitochondria appear to be connected to the plasma membrane by means of the internal membrane system. The development, nature and function of the membrane(s) in yeast cells is not known, but a number of workers have reported similar findings for several different yeasts [2, 5, 221. Further details on the ultrastructure of Rh. glutinis have been Lxesented elsewhere [ 18, 191. SUMMARY
The fine structure of the yeast Rhodotorula glutinis was studied in electron micrographs of serial sections of a resting cell. The nucleus contains a prominent membrane bounded structure which is tentatively identified as the nucleolus. Poorly defined areas of low electron density were also observed within the membrane bounded nucleus. The cytoplasm contained lipoidal inclusions, mitochondria and intracptoplasmic membranes. Apparent connections between the mitochondria, plasma membrane, and nuclear membrane were observed. The significance and nature of the intracytoplasmic structures is discussed.
Experimental
Cell Research 29
Sfrucfures of Rhodoforula glufinis
241
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25. 26.
BADIAN, J., Bull. Intern. Acad. I’olon. Ser. 13. 1, 61 (1937). H. B., .I. Bacterial. 78, 868 (1959). COSTI, S. F. and NAYLOK, COSSLETT, V. E., Brif. .I. Appl. Phys. 9, 253 (195X). DEL.knmTm, E. D., J. Hacferiol. 60, 321 (1950). EDWARDS, M. R., HAGEN, E. L. and EL)WAKDS, G. A., J. (ien. Microbial. 20, 496 (1959). HASHIMOTO, T., COSTI, S. F. and NAYLOK, H. B., J. Bacterial. 77, 344 (1959). HASHIMOTO, T., GEMIARDT, P., CONTI, S. F. and NAYLOH, H. B., J. Riophgs. Biochem. Cytol. 7, 305 (1960). LEVAN, A., Hereditas 33, 457 (1947). LIETZ, Ii.,‘Arch. Microbio/. 18, 275’(1951). MCCLAIIY. D. 0.. \VILLI.knfs. M. A.. LINDEGHES. C. C. and OGUR, AI.. J. Racferiol. 73. 360 (19j7). MOSES, 31. .J., .I. Riophys. Riochem. Cytol. (Suppl.) 2, 397 (1956). i%NDKUN, B. I)., .I. Racteriol. 68, 514 (1954). RORISOW, C. F., .J. Biophys. Biochem. Cytol. 9, 879 (1961). ROYAS, Y., Curr. Sci. 27, 400 (1958). ROYAT, S. and SUBRAMANIAM, hf. I<., I’roc. Indian Acad. Sci. 51 R, 205 (1961). SUBRAMAXIAM, M. I<., Proc. Natl. Inst. Sci. (India) 12, 143 (1946). SU~RAMANIAM, >I. I<., ROYAN, S., THYAGARAJAS; T. R., ASWATIIANARAYAN.~, N. V. and SUBR.AYANYAM, S.. Indian Inst. Sci.. Golden JubiZee Research Volume. D. 96. 1959. TIXYAGARAJAN, T. RI, C&NTI, S. I’. and NAGLOX, H. B., J. Bacterial. 83, 381 (i962).’ __ Exptl. Cell Research 25, 216 (1961). THYAGARAJAN, T. R. and NAYLOR, H. B., .I. Hacteriol. 83, 127 (1962). TOWNSEND, G. F., Stain Technol. 36, 40 (1961). VITOLS, E:., NORTI~, R. .J. and LJSNAN.F., 4. W., J. Riophys. Biochem. Cytol. 9, 689 (1961). WILLIAMS, R. C. and I
16 - 621806
Experimental
Cell Research 29
Cell Research 29, 235-241 (1963)
INTRANUCLEAR
235
AND INTRACYTOPLASMIC
STRUCTURES
OF RHODOTORULA GLUTINIS AS REVEALED BY ELECTRON MICROGRAPHS OF SERIAL SECTIONS S. F. CONTI, Department Division
T. R. THYAGARAJANI
and H. B. NAYLOR
of Microbiology, Dartmouth Medical School, Hanover, New Hampshire, and of Bacteriology, Department of Dairy and Food Science, Cornell University, Ithaca, N.Y.,
U.S.A.
Received April 9, 1962
ACCOKDISG to Mundkur [la] and Townsend [21], the yeast nucleus is a homogeneous vesicle with no observable internal differentiation whereas the presence of chromocenters and nucleolar equivalents, identified by cytochemical staining techniques, has been reported by others [ 14, 15, 171. Chromosome counts on stained preparations have been reported by a number of workers 11, 4, 8, 9, 10, 16, 241. On the other hand, early electron microscope studies of vegetative cells failed to reveal any intranuclear structure [2, 61. Internal differentiation of the nucleus in sporulating cells of Saccharomyces cereuisiae was reported in a study of Hashimoto et al. [7]. Totsuyanagi [25, 261 observed in the nucleus a dense nucleolus and some areas of low electron density which he interpreted to be chromosomal. The recent work of Vitals et al. [22] also reveals areas of contrasting electron density in the nucleoplasm of S. cerevisiae. The presence in the nucleus of a membrane bounded structure and areas of low electron density in Rhodotorula glutinis has been reported [ 181. The difficulties involved in observing intranuclear structures in ultrathin sections with the electron microscope have been pointed out 12, 3, 111. During a study of the nuclei in living cells of Rh. glutinis a dense central body surrounded by a shell of optically uniform material of lower density was observed, Cytochemical staining techniques revealed that the central body is Feulgen-negative but stainable with iron alum haematoxylin and on this basis was identified as the nucleolus. The area surrounding the nucleolus was found to contain Feulgen-positive structures. Although the small size of the chromatin granules did not permit as exact count, the staining reaction 1 Present address: Central
Drug Research
Institute,
Lucknow,
India. Experimental
Cell Research 29
236
S. F. Conti, T. R. Thyagarajan
and H. S. Naylor
indicated that deoxyribonucleic acid is contained in this region of the nucleus [20]. The presence of an intranulcear structure prompted LB to study this yeast bp means of electron microscopy of ultrathin sections.
MATERIAL
AND
METHODS
obtained from Dr. L. J. Wickerham was routinely grown at room Rh. glutinis temperature for 4X hr in a pH 4.6 barley malt wort medium of specific gravity 1.005. Cells were fixed in 1.5 per cent aqueous potassium permanganate for 10 min at room temperature, rinsed in distilled water, and resuspended in an aqueous solution of 2.0 per cent osmium tetroxide for 90 min. The specimens were dehydrated by passage through a graded alcohol series, embedded in butyl methacrylate, and polymerized at 55°C. Ultrathin sections were obtained with an LKB 4800 ultrotome using a glass knife. Sections were mounted on carbon coated grids and examined with an Akashi Tronscope TRS-50 electron microscope equipped with a 50 p aperture.
RESULTS
The appearance of the nucleus in living cells under the phase microscope is shown in Fig. 1. The dense central nucleolus is surrounded by a shell of uniform material of lower density in which no structural details are observable. In the course of preparing thin sections of this organism for examination with the electron microscope consecutive sections of a single resting cell were obtained. Electron micrographs of seven of these sections are presented in Figs. 3-Y. The nucleus contains a prominent inclusion (Figs. :1-S) which may be regarded as the same structure identified as the nucleolus in previous studies [Xl]. This structure is surrounded bv a membrane and in cross section appears to have a maximum diameter of approximately 0.5 p (Figs. 5-7). In addition to the nucleolus irregular areas of low electron density can
Fig. l.-Cells of Rh. glufinis tral body which is identified Fig. 2.-An
under the phase microscope. The nucleus (Iv) contains as the nucleolus (Nu). x 5500.
iron alum haematoxylin
stain of the yeast cell.
a dense ccn-
Y 5500.
Figs. 3-g.-Micrographs of 7 consecutive sections of a resting yeast cell. Note the areas of low electron density in the nucleus, and the membrane bounded nucleolus. An area of low electron density between the nucleolus and nucleus is seen in b’ig. 7, and the connection of a mitochondrion (M) to the nuclear membrane (NfiI) in Fig. 5, and plasma membrane (CM) in Fig. 9 is apparent. The circular or spherical inclusions (I,) of low electron density located in the cytoplasm are lipoidal in nature and stainable with Sudan Black B. Further discussion of these figures appears in the text. x 22,000. Experimental
Cell Research 29
Structures of Rhodotorula glutinis
Experimental
237
Cell Research 29
238
Experimental
S. F. Conti, T. R. Thyagarajan and H. B. Naylor
Cell Reseureh 29
Structures
of Rhodotorula
glutinis
be observed in the nucleoplasm. The diffuse nature of these areas makes it difficult to follow them from one section to another, consequently, only a rough approximation of their three dimensional configuration can be made. The presence of an extensive intracytoplasmic membrane system is evident from the electron micrographs. Certain membrane segments such as the one in close proximity to the cytoplasmic membrane at the upper left portion of the sectioned cell and the one lying close to the nucleus can be seen in each of the serial sections (Figs. S-9). The close association of mitochondria with the membrane system is also clearly shown. In some instances, the membrane seems to form a bridge between a mitochondrion and the plasma membrane (Fig. 9). In other instances, the mitochondrion is closely associated with a membrane which lies very near to, and perhaps connects with, the nuclear membrane. This can be observed most clearly in Figs. 3, 4, and 5 where three successive sections of the mitochondrion lying just to the right of the nucleus all show the connection with the internal membrane. Although this mitochondrion has disappeared from subsequent sections, the membrane can still be seen (Figs. 6, 7, and 8). The well defined areas of low electron density in the cytoplasm are lipoidal inclusions which increase in size and number during the stationary phase of growth of this yeast. DISCUSSION
As has been pointed out by Williams and Kallman [23], it is difficult to reconstruct detailed three-dimensional models from electron micrographs of serial sections of biological materials. It is possible, however, to obtain valuable information about those structures that can be followed through succeeding sections with a high degree of certainty. The electron micrographs presented here indicate the presence of a membrane bounded intranuclear structure in Rh. glufinis. The complete series of sections through this structure indicate that it is intranuclear, and not formed by an infolding of the nuclear membrane. While investigators have observed nucleoli in yeasts with the light microscope, electron microscopy has yielded very little information about this structure. Edwards et al. [5] observed nucleoli in thin sections of yeast-like cells of Hisfoplasma, and Yotsuyanagi [25] observed similar bodies within the nuclei in thin sections of S. cereuisiae. In both cases, the nucleoli were considerably more electron dense than the surrounding nucleoplasm, and they did not appear to be enclosed by a membrane. The membrane bounded Experimental
Cell Research 29
240
S. F. Confi, T. R. Thyagarajan
and H. R. Nay/or
intranuclear component of Rh. glutinis, which is tentatively considered to be the nucleolus, has about the same electron density as the nucleoplasm except for the membrane, which is of greater electron density. The structure of the nucleolus may vary with the physiological state of the cells since we have not been able to observe it at all in thin sections of dividing cells. Studies of Lipomyces lipofer with the light microscope [ 131 illustrate that the nucleolus disappears during mitosis and then reappears in the nuclei of the resulting daughter cells. Areas of low electron density within the nucleus of Rh. glutinis can also be seen in the accompanying electron micrographs. However, due to the diffuse nature of the areas, they cannot be followed readily from one section to the next. It is probable that the areas referred to here are comparable to those observed in the nucleus of S. cerevisiae by others [i, 22, 25, 261. Totsuyanagi [a,?] has presented some evidence that the material is chromosomal in nature. Careful study of the serial sections reveals some interesting information about the intracytoplasmic membrane system of this yeast. Although thin in cross section, the membrane(s) must have considerable width since in certain areas it can be followed through the entire series of electron micrographs. The close association of the membrane with the nucleus is also evident. In some instances, the mitochondria appear to be connected to the plasma membrane by means of the internal membrane system. The development, nature and function of the membrane(s) in yeast cells is not known, but a number of workers have reported similar findings for several different yeasts [2, 5, 221. Further details on the ultrastructure of Rh. glutinis have been Lxesented elsewhere [ 18, 191. SUMMARY
The fine structure of the yeast Rhodotorula glutinis was studied in electron micrographs of serial sections of a resting cell. The nucleus contains a prominent membrane bounded structure which is tentatively identified as the nucleolus. Poorly defined areas of low electron density were also observed within the membrane bounded nucleus. The cytoplasm contained lipoidal inclusions, mitochondria and intracptoplasmic membranes. Apparent connections between the mitochondria, plasma membrane, and nuclear membrane were observed. The significance and nature of the intracytoplasmic structures is discussed.
Experimental
Cell Research 29
Sfrucfures of Rhodoforula glufinis
241
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25. 26.
BADIAN, J., Bull. Intern. Acad. I’olon. Ser. 13. 1, 61 (1937). H. B., .I. Bacterial. 78, 868 (1959). COSTI, S. F. and NAYLOK, COSSLETT, V. E., Brif. .I. Appl. Phys. 9, 253 (195X). DEL.knmTm, E. D., J. Hacferiol. 60, 321 (1950). EDWARDS, M. R., HAGEN, E. L. and EL)WAKDS, G. A., J. (ien. Microbial. 20, 496 (1959). HASHIMOTO, T., COSTI, S. F. and NAYLOK, H. B., J. Bacterial. 77, 344 (1959). HASHIMOTO, T., GEMIARDT, P., CONTI, S. F. and NAYLOH, H. B., J. Riophgs. Biochem. Cytol. 7, 305 (1960). LEVAN, A., Hereditas 33, 457 (1947). LIETZ, Ii.,‘Arch. Microbio/. 18, 275’(1951). MCCLAIIY. D. 0.. \VILLI.knfs. M. A.. LINDEGHES. C. C. and OGUR, AI.. J. Racferiol. 73. 360 (19j7). MOSES, 31. .J., .I. Riophys. Riochem. Cytol. (Suppl.) 2, 397 (1956). i%NDKUN, B. I)., .I. Racteriol. 68, 514 (1954). RORISOW, C. F., .J. Biophys. Biochem. Cytol. 9, 879 (1961). ROYAS, Y., Curr. Sci. 27, 400 (1958). ROYAT, S. and SUBRAMANIAM, hf. I<., I’roc. Indian Acad. Sci. 51 R, 205 (1961). SUBRAMAXIAM, M. I<., Proc. Natl. Inst. Sci. (India) 12, 143 (1946). SU~RAMANIAM, >I. I<., ROYAN, S., THYAGARAJAS; T. R., ASWATIIANARAYAN.~, N. V. and SUBR.AYANYAM, S.. Indian Inst. Sci.. Golden JubiZee Research Volume. D. 96. 1959. TIXYAGARAJAN, T. RI, C&NTI, S. I’. and NAGLOX, H. B., J. Bacterial. 83, 381 (i962).’ __ Exptl. Cell Research 25, 216 (1961). THYAGARAJAN, T. R. and NAYLOR, H. B., .I. Hacteriol. 83, 127 (1962). TOWNSEND, G. F., Stain Technol. 36, 40 (1961). VITOLS, E:., NORTI~, R. .J. and LJSNAN.F., 4. W., J. Riophys. Biochem. Cytol. 9, 689 (1961). WILLIAMS, R. C. and I
16 - 621806
Experimental
Cell Research 29