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Ultrastructure of intracellular and extracellular vesicles, membranes, and myelin figures produced by Ochromonas danica
© 1971 by Academic Press, Inc.
418
J. U L T R A S T R U C T U R E R E S E A R C H
35, 418-430 (1971)
Ultrastructure of Intracellular and Extracellular Vesicles, Membranes, and Myelin Figures Produced by O c h r o m o n a s d a n i c a 1 S. AARONSON, URSULA BEHRENS, RENA ORNER2 AND T. H. HAINES
Biology Department, Queens College, City University of New York Flushing, New York 11367 Chemistry Department, City College, City University of New York, New York, New York 10031 Received April 20, 1970, and in revised form October 29, 1970 Ochromonas danica synthesized a variety of large and small intra- and extracellular membrane-bounded structures including vesicles and myelin-like figures. Vesicles were derived from membranes associated with the flagella, mitochondria, chloroplasts, plasma membrane, and leucosin vacuole. Myelin-like figures were derived from the chloroplast membrane and lamellae. Most membranous structures were bounded by unit membranes ca. 75-80 It wide. There are an increasing number of reports on the occurrence of unusual membranous structures intra- and extracellularly as well as within the organelles of a large variety of organisms. Membranous structures including extra membranes, vesicles, myelin-like figures, membranous whorls have been reported in several organelles: mitochondria (3, 4, 12, 13, 16, 25, 30), nucleus (1I), chloroplast (20), in the cytoplasm (2, 4, 11), in metazoan cells (5, 6, 17, 21), in phagocytic cells (8, 9, 18, 22, 27, 29), in a protozoan (2), in anaerobic yeast (15); in permanently altered but viable cells: in chloroplasts (19), or in mitochondria (24), as a result of cell stress (6, 7, 10, 27), cell age (23), or change in protein nutrition (17). Membranous structures are also secreted extracellularly by mammalian cells (26) and phagocytic cells (18, 29). The role of these membranes in the several cells and cell organelles remains uncertain. In this paper we present ultrastructural evidence for the production of a wide variety of intra- and extracellular membranous structures in Ochromonas danica, a phytoflagellate, grown photoheterotrophically in a chemically defined medium. This work z Aided by a National Science Foundation grant GB 7833 to S. A. and a Department of the Interior Water Pollution Control Administration grant 18050DCW to T. H. H. 2 Present address: Department of Chemistry, Fairleigh Dickinson University, Teaneck, New Jersey 07666. Mrs. Orner was supported during part of this work by a National Science Foundation Faculty Fellowship.
ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
419
was done independently in our two laboratories, and our results were found to be complementary. MATERIALS AND METHODS O. danica Pringsheim was grown axenically in a chemically defined medium (1) in 125-150 foot-candles of white fluorescent light at 25°C in a refrigerated incubator. All chemicals were reagent grade and were obtained commercially. Organisms were collected for electron microscopy after varying periods of growth. One week is mid to late log stage, and 2 weeks is the beginning of the stationary stage under these growth conditions. Organisms were centrifuged at 860 g in a Sorvall angle centrifuge for 5 minutes at room temperature, and the pellets were fixed for 1 hour in 2.5 % glutaraldehyde (or 1% osmic acid) containing 10/~g/100 ml CaC12 and buffered with Na cacodylate (0.1 M) at pH 7.4. After 2 rinses in cacodylate buffer, the organisms fixed with glutaraldehyde were postfixed for 1 hour in 1% osmic acid buffered with Na cacodylate (0.1 M) at pH 7.4. During fixation the pellets were placed on crushed ice. After the osmic acid was removed, the pellets were stained in 1% aqueous uranyl acetate for 20 minutes at room temperature and then dehydrated in a graded alcohol series. The organisms were cleared in propylene oxide, embedded in Epon 812, and dried in a 60°C incubator for 48 hours. Sections were prepared with a diamond knife on the MT-2 Porter-Blum ultramicrotome and stained with uranyl acetate and lead citrate. Negative staining was done with 2% phosphotungstic acid. Micrographs were taken with a Phillips 300 electron microscope.
OBSERVATIONS O. danica produced a large variety of intra- and extracellular small and large membrane-bounded vesicles and myelin-like figures (Fig. 1) bounded by unit membranes ca. 75-80 A wide. Some parts of the cytoplasm seem to be particularly rich in vesicles (Figs. 1 and 2), and these are surrounded by dense areas of ribosomes which are involved in the synthesis of membrane proteins and perhaps the contents of the vesicles as well; the vesicles contained ribosome-rich material with their membranes. This region is probably equivalent to a region called the "tongue" (23). Vesicles and other membranous structures arose from several cell organelles. A longitudinal section of a flagellum (Fig. 3) showed a side view of membranes arising from the flagellar surface. Cross sections of flagella (Figs. 4, 18, and 19) showed similar evaginations from the membrane surrounding the flagellum. Fig. 5 is a negative stain of the flagellar surface showing extrusions (vesicles) at several sites. The mitochondria may also give rise to vesicles, although these were rarely seen (Figs. 6 and 7). The chloroplast gave rise to a variety of vesicles, membranes, and myelin-like figures. Figures 8 and 17 illustrate an early stage in the formation of myelin-like figures; the chloroplast membrane and a number of lamellae loosen up and extend, "ripple", from the chloroplast surface. These extend farther from the chloroplast surface (Figs. 9 and 10) until they break away into the cytoplasm, and then into the 28-- 711838 J . Ultrastructure Research
420
AARONSON ET AL.
large leucosln vacuole (Figs. 1 a n d 9) o r to the exterior (Figs. 1 a n d 11). N o t e t h a t t h e internal m e m b r a n e s of the myelin-like figures became increasingly d i s a r r a n g e d as their distance increased f r o m the p a r e n t c h l o r o p l a s t (Figs. 9-1 I). The leucosin vacuole (Fig. 12) a n d the cell m e m b r a n e (Fig. 13) also gave rise to vesicles of assorted shapes a n d sizes. Negative staining revealed the a p p e a r a n c e of the surface of a whole O. d a n i c a cell (Fig. 14) showing the l o n g flagellum with its m a s t i g o n e m e s a n d the s h o r t flagellum which lacked m a s t i g o n e m e s a n d the p o i n t e d p o s t e r i o r end. The surface of the cell has p r o t r u s i o n s (Fig. 14) a n d a c c u m u l a t i o n s of vesicular a n d m e m b r a n o u s m a t e r i a l (Figs. 14 a n d 15) similar to those seen in sections. N o t e mass of vesicles a n d m e m b r a n e s seemingly escaping f r o m the surface of the cell a n d the myelin-like structures which w o u l d a p p e a r if this m a t e r i a l were sectioned (Fig. 15). F i g u r e 16 shows an e n l a r g e m e n t of some of the surface p r o t r u s i o n s seen on negatively stained cells. M a n y of the vesicles c o n t a i n e d smaller vesicles a n d electron-lucent m a t e r i a l (Figs. 1 a n d 2), a n d these were secreted into the external m e d i u m a l o n g with a diverse g r o u p Key to abbreviations
C cl cm
F fm
LV M mt my
chloroplast chloroplast lamellae cell membrane flagellum flagellar membrane leucosin vacuole mitochondrion mastigonemes mitochondrial vesicle
N nucleus nucleolus R ribosomes V vesicle vl large vesicle vm myelin-like vesicle vrr vesicle-rich region vs small vesicle nl
FIG. 1. Nine-day-old Ochromonas daniea cell showing a variety of intracellular and extracellular membranous structures, x 16 000. FIG. 2. Four-day-old cell showing vesicle-rich region of cytoplasm. × 20 230. FIG. 3. Longitudinal section of the flagellum of a 9-day-old cell showing vesicles arising from flagellar membrane. × 25 600. FIG. 4. Cross sections of flagella with vesicles containing small vesicles extending from surface of 9-day-old cells. × 25 600. FIG. 5. Negative stain of flagellum from 9-day-old cell showing protrusions (vesicles) at surface and entangled in the mastigonemes, x 30 800. FIGS. 6 and 7. Serial sections of 4-day-old cell with evagination of cell surface trapping part of a mitochondrion in vesicle. Fig. 6, × 16 700; Fig. 7, × 26 300. FIG. 8. Section through chloroplast of a 9-day-old cell showing "rippling" of chloroplast surface and lamellae giving rise to myelin-like vesicles (arrows). x 28 300. FIG. 9. Section through chloroplast of 9-day-old cell showing later development of myelin-like vesicles. Note continuation of lamellae and chloroplast membrane into vesicle, x 20 200. FIG. 10. Section through chloroplast of 9-day-old cell showing loosening and disarrangement of lamellar membranes as they enter myelin-like vesicle, x 51 300. FIG. 11. Cross section of chloroplast and cell surface of 9 day-old cell showing the disarrangement of myelin-like vesicles as they move away from the chloroplast. × 51 300. FIG. 12. Numerous vesicles in the interior and arising from the membrane of the leucosin vacuole of 4-day-old cell. × 20 500.
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ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
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FIG. 13. Vesicles emerging from the surface of the cell membrane of a 9-day-old cell; see arrows. x 17 100. of micromolecules (amino acids, vitamins, UV-absorbing material, etc.) and macromolecules (proteins, including at least one enzyme, acid phosphatase; R N A ; carbohydrate; and lipids). The secretion of molecules will be described elsewhere (Aaronson, in press). DISCUSSION The n u m b e r of reports on the occurrence of unusual membranes and membraneb o u n d organelles seems to be increasing with the use of glutaraldehyde as a fixative. This has raised the possibility that these m e m b r a n o u s structures, particularly the myelin-like figures, are artifacts of glutaraldehyde fixation (5). That the m e m b r a n o u s structures synthesized by O. danica are not artifacts m a y be adduced f r o m the following: 1. Electron microscopy of O. danica fixed with osmic acid as well as glutaraldehydeosmic acid revealed vesicles arising f r o m the chlorophlast and flagellar surface. FIG. 14. Negative stain of a 9-day-old cell showing protrusions (vesicles) and smaller vesicular material at cell surface and trapped in the mastigonemes; see arrows, x 11 200.
ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
427
428
AARONSON ET AL.
FIG. 15. Negative stain of 9-day-old cell showing vesicular material escaping from cell surface at the flageUar end. Some of this material would appear myelin-like in cross section, x 37 680. FIG. 16. Enlargement of the surface of negatively-stained 9-day-old cell showing protrusions (vesicles) at or near cell surface, x 47 900.
ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
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FIG. 17. Osmic acid-fixed cross section showing early myelin-like vesicle arising from chloroplast. × 39 900. FIGS. 18 and 19. Osmic acid-fixed cross section of flagellum showing vesicle arising from surface. 2. Negative staining of O. danica cells showed protrusions, vesicles, and myelinlike figures at or near the cell surface in the absence of glutaraldehyde fixation. 3. Electron micrographs of the centrifugates of the cell-free O. danica supernatant at 39 000 and 105 000 g revealed the presence of a varied group of vesicles in the pellet. Few myelin figures were seen in these preparations, but these may have been broken during the high speed centrifugation. The details of these observations are to be published elsewhere (Orner, Haines, Aaronson, and Behrens, in preparation). 4. Multimembrane systems (14) and myelin-like membranes are seen in mitochonddria (12, 21), in Euglena chloroplasts (19), and cytoplasm (7) fixed with KMnO4 or osmic acid in place of glutaraldehyde and also in alveolar spaces of embryonic and postpartum rat lung fixed with osmic acid (26). Myelin-like configurations have been seen in micrographs of O. malhamensis (2) and in another chrysophyte Hymenornonas sp. (20) as well as here, suggesting that they may be common in the Chrysophyceae. The function of these internal and external membrane-bound structures remains uncertain despite the assumption of some
430
AARONSON ET AL.
that they represent autophagic vesicles (14), areas of cellular disintegration (7, 10, 12, 26, 27), or normal patterns in the morphogenesis of organelles (3, 21). It appears then that the function of these unusual membranous structures varies from cell to cell and organism to organism, and their function in O. danica remains to be discovered. We are grateful to John Bodnaruk of the Department of Chemical Engineering, City College, City University of New York, for his cooperation in the use of the electron microscope facility. 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. 27. 28. 29. 30.
AARONSON, S. and BAKER, H., J. Protozool. 6, 282 (1959). ANDERSON,O. R. and ROELS, D. A., J. Ultrastract. Res. 20, 127 (1967). BEAULATON,J. A., J. Cell Biol. 39, 501 (1968). BECK, D. P. and GREENAWALT,J. W., at. CellBiol. 39, l l a (1968). CUR6Y, J. J., J. Mieroscop. (Paris) 7, 63 (1968). DIETERT,S. E. and SCALLEN,T. J., J. Cell Biol. 40, 44 (1969). FAWCETT,D. W. and Iro, S., J. Biophys. Biochem. Cytol. 4, 135 (1958). GEZELIUS,K., Exp. Cell Res. 18, 425 (1959). - - - - ibid. 23, 300 (1961). HOLTZMAN,E. and NOVIKOFF,A. B., J. Cell BioL 27, 651 (1965). HUMEAU,C., C. R. Soc. Biol. 162, 183 (1968). JOURNEY,L. J. and GOLDSTEIN,M. N., Can. Res. 23, 551 (1963). LEBECX,Y., HETENYI,G., JR. and PHILLIPS,M. J., Z. Zellforsch. Mikrosk. Anat. 99, 491 (1969). LEVY, M. R. and ELLIOTT,A. M., J. ProtozooL 15, 208 (1968). LINNANE,A. W., VITOLS,E. and NOWLAND, P. G., J. Cell Biol. 13, 345 (1962). MALHOTRA,S. K., Nature (London) 219, 1267 (1968). MENErEE, M. G. and EVANS, V. J., J. Nat. Can. Inst. 25, 1303 (1960). MERCER,E. H. and SHAFFER, N. M., J. Biophys. Bioehem. Cytol. 7, 353 (1960). MORIBER, L. G., HERSHENOV, B., AARONSON, S. and BENSKY, B., J. Protozool. 10, 80 (1963). OLSON, R. A., JENNINGS,W. H. and ALLEN, M. B., J. CellPhysiol. 70, 133 (1967). PANNESE,E., J. Ultrastruc. Res. 15, 57 (1966). SCHUSTER,F., J. ProtozooL 11, 207 (1964). SCHUSTER,F. L., HERSHENOV,B. and AARONSON, S., J. ProtozooL 15, 335 (1968). SIMON,J., BHATNAGAR,P. L. and MELBURN, N. S., J. Insect Physiol. 15, 135 (1969). SMIT~, D. G., MARCHANT,R., MAROUDAS,N. G. and WILKIE, D., Y. Gen. Microbiol. 56, 47 (1969). SUN, C. N., J. Ultrastruet. Res. 15, 380 (1966). SWIFT, H. and HRUBAN, Z., Fed. Proc., Fed. Amer. Soe. Exp. Biol. 23, 1026 (1964). TANAKA,Y., Exp. Cell Res. 52, 338 (1968). VICKERMAN,K., Exp. Cell Res. 26, 497 (1962). YOTSUYANAGLY., C. R. Acad. Sei. Ser. D 262, 1348 (1966).
418
J. U L T R A S T R U C T U R E R E S E A R C H
35, 418-430 (1971)
Ultrastructure of Intracellular and Extracellular Vesicles, Membranes, and Myelin Figures Produced by O c h r o m o n a s d a n i c a 1 S. AARONSON, URSULA BEHRENS, RENA ORNER2 AND T. H. HAINES
Biology Department, Queens College, City University of New York Flushing, New York 11367 Chemistry Department, City College, City University of New York, New York, New York 10031 Received April 20, 1970, and in revised form October 29, 1970 Ochromonas danica synthesized a variety of large and small intra- and extracellular membrane-bounded structures including vesicles and myelin-like figures. Vesicles were derived from membranes associated with the flagella, mitochondria, chloroplasts, plasma membrane, and leucosin vacuole. Myelin-like figures were derived from the chloroplast membrane and lamellae. Most membranous structures were bounded by unit membranes ca. 75-80 It wide. There are an increasing number of reports on the occurrence of unusual membranous structures intra- and extracellularly as well as within the organelles of a large variety of organisms. Membranous structures including extra membranes, vesicles, myelin-like figures, membranous whorls have been reported in several organelles: mitochondria (3, 4, 12, 13, 16, 25, 30), nucleus (1I), chloroplast (20), in the cytoplasm (2, 4, 11), in metazoan cells (5, 6, 17, 21), in phagocytic cells (8, 9, 18, 22, 27, 29), in a protozoan (2), in anaerobic yeast (15); in permanently altered but viable cells: in chloroplasts (19), or in mitochondria (24), as a result of cell stress (6, 7, 10, 27), cell age (23), or change in protein nutrition (17). Membranous structures are also secreted extracellularly by mammalian cells (26) and phagocytic cells (18, 29). The role of these membranes in the several cells and cell organelles remains uncertain. In this paper we present ultrastructural evidence for the production of a wide variety of intra- and extracellular membranous structures in Ochromonas danica, a phytoflagellate, grown photoheterotrophically in a chemically defined medium. This work z Aided by a National Science Foundation grant GB 7833 to S. A. and a Department of the Interior Water Pollution Control Administration grant 18050DCW to T. H. H. 2 Present address: Department of Chemistry, Fairleigh Dickinson University, Teaneck, New Jersey 07666. Mrs. Orner was supported during part of this work by a National Science Foundation Faculty Fellowship.
ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
419
was done independently in our two laboratories, and our results were found to be complementary. MATERIALS AND METHODS O. danica Pringsheim was grown axenically in a chemically defined medium (1) in 125-150 foot-candles of white fluorescent light at 25°C in a refrigerated incubator. All chemicals were reagent grade and were obtained commercially. Organisms were collected for electron microscopy after varying periods of growth. One week is mid to late log stage, and 2 weeks is the beginning of the stationary stage under these growth conditions. Organisms were centrifuged at 860 g in a Sorvall angle centrifuge for 5 minutes at room temperature, and the pellets were fixed for 1 hour in 2.5 % glutaraldehyde (or 1% osmic acid) containing 10/~g/100 ml CaC12 and buffered with Na cacodylate (0.1 M) at pH 7.4. After 2 rinses in cacodylate buffer, the organisms fixed with glutaraldehyde were postfixed for 1 hour in 1% osmic acid buffered with Na cacodylate (0.1 M) at pH 7.4. During fixation the pellets were placed on crushed ice. After the osmic acid was removed, the pellets were stained in 1% aqueous uranyl acetate for 20 minutes at room temperature and then dehydrated in a graded alcohol series. The organisms were cleared in propylene oxide, embedded in Epon 812, and dried in a 60°C incubator for 48 hours. Sections were prepared with a diamond knife on the MT-2 Porter-Blum ultramicrotome and stained with uranyl acetate and lead citrate. Negative staining was done with 2% phosphotungstic acid. Micrographs were taken with a Phillips 300 electron microscope.
OBSERVATIONS O. danica produced a large variety of intra- and extracellular small and large membrane-bounded vesicles and myelin-like figures (Fig. 1) bounded by unit membranes ca. 75-80 A wide. Some parts of the cytoplasm seem to be particularly rich in vesicles (Figs. 1 and 2), and these are surrounded by dense areas of ribosomes which are involved in the synthesis of membrane proteins and perhaps the contents of the vesicles as well; the vesicles contained ribosome-rich material with their membranes. This region is probably equivalent to a region called the "tongue" (23). Vesicles and other membranous structures arose from several cell organelles. A longitudinal section of a flagellum (Fig. 3) showed a side view of membranes arising from the flagellar surface. Cross sections of flagella (Figs. 4, 18, and 19) showed similar evaginations from the membrane surrounding the flagellum. Fig. 5 is a negative stain of the flagellar surface showing extrusions (vesicles) at several sites. The mitochondria may also give rise to vesicles, although these were rarely seen (Figs. 6 and 7). The chloroplast gave rise to a variety of vesicles, membranes, and myelin-like figures. Figures 8 and 17 illustrate an early stage in the formation of myelin-like figures; the chloroplast membrane and a number of lamellae loosen up and extend, "ripple", from the chloroplast surface. These extend farther from the chloroplast surface (Figs. 9 and 10) until they break away into the cytoplasm, and then into the 28-- 711838 J . Ultrastructure Research
420
AARONSON ET AL.
large leucosln vacuole (Figs. 1 a n d 9) o r to the exterior (Figs. 1 a n d 11). N o t e t h a t t h e internal m e m b r a n e s of the myelin-like figures became increasingly d i s a r r a n g e d as their distance increased f r o m the p a r e n t c h l o r o p l a s t (Figs. 9-1 I). The leucosin vacuole (Fig. 12) a n d the cell m e m b r a n e (Fig. 13) also gave rise to vesicles of assorted shapes a n d sizes. Negative staining revealed the a p p e a r a n c e of the surface of a whole O. d a n i c a cell (Fig. 14) showing the l o n g flagellum with its m a s t i g o n e m e s a n d the s h o r t flagellum which lacked m a s t i g o n e m e s a n d the p o i n t e d p o s t e r i o r end. The surface of the cell has p r o t r u s i o n s (Fig. 14) a n d a c c u m u l a t i o n s of vesicular a n d m e m b r a n o u s m a t e r i a l (Figs. 14 a n d 15) similar to those seen in sections. N o t e mass of vesicles a n d m e m b r a n e s seemingly escaping f r o m the surface of the cell a n d the myelin-like structures which w o u l d a p p e a r if this m a t e r i a l were sectioned (Fig. 15). F i g u r e 16 shows an e n l a r g e m e n t of some of the surface p r o t r u s i o n s seen on negatively stained cells. M a n y of the vesicles c o n t a i n e d smaller vesicles a n d electron-lucent m a t e r i a l (Figs. 1 a n d 2), a n d these were secreted into the external m e d i u m a l o n g with a diverse g r o u p Key to abbreviations
C cl cm
F fm
LV M mt my
chloroplast chloroplast lamellae cell membrane flagellum flagellar membrane leucosin vacuole mitochondrion mastigonemes mitochondrial vesicle
N nucleus nucleolus R ribosomes V vesicle vl large vesicle vm myelin-like vesicle vrr vesicle-rich region vs small vesicle nl
FIG. 1. Nine-day-old Ochromonas daniea cell showing a variety of intracellular and extracellular membranous structures, x 16 000. FIG. 2. Four-day-old cell showing vesicle-rich region of cytoplasm. × 20 230. FIG. 3. Longitudinal section of the flagellum of a 9-day-old cell showing vesicles arising from flagellar membrane. × 25 600. FIG. 4. Cross sections of flagella with vesicles containing small vesicles extending from surface of 9-day-old cells. × 25 600. FIG. 5. Negative stain of flagellum from 9-day-old cell showing protrusions (vesicles) at surface and entangled in the mastigonemes, x 30 800. FIGS. 6 and 7. Serial sections of 4-day-old cell with evagination of cell surface trapping part of a mitochondrion in vesicle. Fig. 6, × 16 700; Fig. 7, × 26 300. FIG. 8. Section through chloroplast of a 9-day-old cell showing "rippling" of chloroplast surface and lamellae giving rise to myelin-like vesicles (arrows). x 28 300. FIG. 9. Section through chloroplast of 9-day-old cell showing later development of myelin-like vesicles. Note continuation of lamellae and chloroplast membrane into vesicle, x 20 200. FIG. 10. Section through chloroplast of 9-day-old cell showing loosening and disarrangement of lamellar membranes as they enter myelin-like vesicle, x 51 300. FIG. 11. Cross section of chloroplast and cell surface of 9 day-old cell showing the disarrangement of myelin-like vesicles as they move away from the chloroplast. × 51 300. FIG. 12. Numerous vesicles in the interior and arising from the membrane of the leucosin vacuole of 4-day-old cell. × 20 500.
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AARONSON ET AL.
ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
423
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ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
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FIG. 13. Vesicles emerging from the surface of the cell membrane of a 9-day-old cell; see arrows. x 17 100. of micromolecules (amino acids, vitamins, UV-absorbing material, etc.) and macromolecules (proteins, including at least one enzyme, acid phosphatase; R N A ; carbohydrate; and lipids). The secretion of molecules will be described elsewhere (Aaronson, in press). DISCUSSION The n u m b e r of reports on the occurrence of unusual membranes and membraneb o u n d organelles seems to be increasing with the use of glutaraldehyde as a fixative. This has raised the possibility that these m e m b r a n o u s structures, particularly the myelin-like figures, are artifacts of glutaraldehyde fixation (5). That the m e m b r a n o u s structures synthesized by O. danica are not artifacts m a y be adduced f r o m the following: 1. Electron microscopy of O. danica fixed with osmic acid as well as glutaraldehydeosmic acid revealed vesicles arising f r o m the chlorophlast and flagellar surface. FIG. 14. Negative stain of a 9-day-old cell showing protrusions (vesicles) and smaller vesicular material at cell surface and trapped in the mastigonemes; see arrows, x 11 200.
ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
427
428
AARONSON ET AL.
FIG. 15. Negative stain of 9-day-old cell showing vesicular material escaping from cell surface at the flageUar end. Some of this material would appear myelin-like in cross section, x 37 680. FIG. 16. Enlargement of the surface of negatively-stained 9-day-old cell showing protrusions (vesicles) at or near cell surface, x 47 900.
ULTRASTRUCTURE OF OCHROMONAS MEMBRANOUS ORGANELLES
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429
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FIG. 17. Osmic acid-fixed cross section showing early myelin-like vesicle arising from chloroplast. × 39 900. FIGS. 18 and 19. Osmic acid-fixed cross section of flagellum showing vesicle arising from surface. 2. Negative staining of O. danica cells showed protrusions, vesicles, and myelinlike figures at or near the cell surface in the absence of glutaraldehyde fixation. 3. Electron micrographs of the centrifugates of the cell-free O. danica supernatant at 39 000 and 105 000 g revealed the presence of a varied group of vesicles in the pellet. Few myelin figures were seen in these preparations, but these may have been broken during the high speed centrifugation. The details of these observations are to be published elsewhere (Orner, Haines, Aaronson, and Behrens, in preparation). 4. Multimembrane systems (14) and myelin-like membranes are seen in mitochonddria (12, 21), in Euglena chloroplasts (19), and cytoplasm (7) fixed with KMnO4 or osmic acid in place of glutaraldehyde and also in alveolar spaces of embryonic and postpartum rat lung fixed with osmic acid (26). Myelin-like configurations have been seen in micrographs of O. malhamensis (2) and in another chrysophyte Hymenornonas sp. (20) as well as here, suggesting that they may be common in the Chrysophyceae. The function of these internal and external membrane-bound structures remains uncertain despite the assumption of some
430
AARONSON ET AL.
that they represent autophagic vesicles (14), areas of cellular disintegration (7, 10, 12, 26, 27), or normal patterns in the morphogenesis of organelles (3, 21). It appears then that the function of these unusual membranous structures varies from cell to cell and organism to organism, and their function in O. danica remains to be discovered. We are grateful to John Bodnaruk of the Department of Chemical Engineering, City College, City University of New York, for his cooperation in the use of the electron microscope facility. 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. 27. 28. 29. 30.
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