- Email: [email protected]
New findings related to pinocytosis
PRELIMINARY
NEW FINDINGS PINOCYTOSIS
NOTES
RELATED
TO
E. MONROY,l
Institute de Investigaciones Biomtdicas, Universidad National Autdnoma de Mkxico, Apt Postal 70228, Ciudad Universitaria 20, D.F., Mexico
Summary Various culture media for maintenance of the newt Amhystoma mexicanum macrophages were tested. Once the most suitable medium was found, the cultivated cells were photographed and filmed. These cells show special features in their cytoplasm, that were studied under both light and electron microscopes. These features included an apparent fluidification of certain portions of the cytoplasm, as seen with timelapse cinematography and a marked orientation of cell inclusions and mitochondria. These observations are discussed in relation to pinocytosis.
The present paper describes some special features of newt macrophages maintained in culture, which seem important in relation to pinocytosis. Although pinocytosis is a general function of all cells and is present in all animal and certain plant cells [30], the extensive studies made in regard to its physiological aspects have been done mainly in amebae [5-91. The effect of physical factors such as temperature and high pressure, and also aerobic respiration correlated with pinocytosis has been studied also in amebae by Rustad et al. [12, 30, 31, 341.
method of Luft f191: sections were obtained with an LKB microtome,-either with glass or diamond knives, stained with uranyl acetate and lead hydroxide [28] and observed under an Akashi Tronscope TRS-80.
Results Fig. 1 show that the average count for all cultures after 15 days of incubation was close to 45 cells per explant. Migration of cells began after 10 days; on the 20th day of incubation the number of cells decreased onehalf and after 30 days the mean number of cells in the culture was 12 per explant. Fig. 2 shows that a part of the cytoplasm near the undulating membrane has a definite granular appearance different from the rest. Time lapse cinematography shows that this region changes constantly: the cytoplasm is even at first, but suddenly it begins to show the formation of “lagoons”, as if it became 92 48 44 40 36 32 28 24
Material and Methods
20
Spleen macrophages obtained from newts classified as Ambystoma mexicanum (Shaw) from the lake Xochimilco were used. For the maintenance of macrophages in culture, a combination of the coverslip and tube techniques [26] was employed, using 12 x 50 mm coverslips introduced in 16 mm tubes [24]. The tubes were laid still and incubated at 27°C. Basal Eagle medium plus 20 % calf serum, was the most suitable according to the results of trials in different media. To study the cells under the electron microscope, the technique of Robbins & Gonatas [29] was followed. The cells were embedded in Epon 812 by the
16
1 Present address: Department Hall, University of Virginia, 22903, USA.
of Biology, Gilmer Charlottesville, Va
12 8 4 4
8
12
15 20 24 28 32
123456
Abscissa: days; ordinate: mean number of cells per explant. Fig. I. Maintenance in culture of macrophagic cells from spleen explants of A. mexicanum in Eagle medium (basal plus 20 % calf serum) at 27°C. Abscissa: periods; ordinate: min. Fig. 3. Periods of apparent cytoplasmic fluidification (see text). Exptl
Cell Res 59
154 E. Monroy
2. In the upper left of this cell a zone of apparent cytoplasmic fluidification (CF) is seen. N ,nucleus; NC, nucleolus; FD, fat droplets; CZ, cytoplasmic inclusions; SP, small pinosomes; F, filopodia; A4, mitochondria, UM, undulating membrane. Magnification: x 40 (in the negative). Fig.
more fluid in certain zones; then, the “lagoons” disappear gradually and the grey cytoplasm regains the initial appearance, beginning, after a while, a new cycle. The entire process takes about 8 min. The undulating membrane is a completely transparent hyaline structure extending towards the outside ExptI
CeN Res 59
of the cell. It is a most characteristic morphological feature of macrophages in culture [18]. During this process it maintains its usual appearance. As seen in fig. 3, the above-mentioned cycle tends to be variable after 9 h of observation. The endoplasm of these macrophages
New findings in pinocytosis
155
Fig. 4. Part of a macrophage cytoplasm. Both kinds of pinosomes are seen, small and large. Mitochondria are oriented in a radial way. PNZ, perinuclear zone; M, mitochondria; LP, large pinosomes; SP, small pinosomes. Magnification: x 100 (in the negative).
Fig. 5. Electron micrograph of a cultured macrophage. The intracytoplasmic filaments (F) are seen. DB, dense bodies; A4, mitochondria; PV, pinocytic vesicle; GRE, granular endoplasmic reticulum, FD, fat droplets. Magnification: approx. x 22,000. Exptl
Cell Res 59
156 E. Monroy shows another feature: all cell inclusions and mitochondria are always oriented in a radial way (fig. 4). Pinocytosis is easily seen in these cells. However, as shown in fig. 4, there are two kinds of pinosomes: large and irregular, and small and spherical; the first travel very rapidly towards the perinuclear region; the others travel slowly through the cytoplasm. Both kinds of pinosomes can be seen in one cell at the same time. If the cell happens to be forming “lagoons” inside its cytoplasm, it is interesting that the pinosomes maintain their individuality all the time, i.e., the plasma membrane that forms them, persists; but they move about five times faster than the pinosomes of a cell without “lagoons”. In electron micrographics the cytoplasm of these cells show filaments of approx. 50 A in diameter, between the endoplasmic reticulum, the mitochondria and the cell inclusions (fig. 5). The filaments were constantly present in all the cells studied.
Discussion Pertinent literature contains data about tissue culture of different amphibian cells, but not on macrophages [3, 4, 14, 16, 271. Spleen macrophages of A. mexicanum never exhibit mitosis (fig. l), so they are maintained in culture and not grown in it. The increase in the number of cells is due to the migration of macrophages out of the explant; the decrease is due to the death of the cells and in some cases to explant detachment from the coverslip with subsequent injury of some cells. There is a latent period of 10 days before the migration begins, as it often occurs when adult tissues are cultured. Pinocytosis: Very little is known about how pinosomes move. Gey et al. [15] have shown that the force that moves the pinosomes is so Exptl
Cell
Res 59
great that it can break a filamentous mitochondria if it is on its way. However, this force is independent of the cytoplasm flux 1251. In A. mexicanum macrophages the radially oriented granules and mitochondria suggest that a cytoplasmic stream going in a plasma membrane-nucleus direction could very well carry the pinosomes to their final site. Allen [l, 2, 331, using the centrifuge microscope on three different species of amebae has found that the cytoplasmic inclusions are displaced discontinuously (at a variable velocity) in apparently all parts of the cell suggesting non-Newtonian behaviour and/or heterogenous consistency. Information is lacking in the case of macrophages. In the cells under study, in the sites where “lagoons” are formed, the cytoplasm has a fluid appearance. This is probably the case, since Allen himself states that very likely, changes in cytoplasmic consistency are taking place in amebae. The difficulty to use the centrifuge method in macrophages is that the apparent fluidification is transitory. Something similar to this has been observed by Gross & Riedel [17] in pulsating chicken embryo muscle cells, although this change in consistency of the cytoplasm is constant and not transitory as in the newt macrophages. At the electron microscopic level, fine intracytoplasmic filaments are present. Similar filaments have been reported by several authors in a variety of plant, animal and cancer cells [IO, 11, 13, 20, 21, 23, 321. It is interesting to note [22] the presence of these filaments in NiteZla [22] and their relation to the cytoplasmic stream, suggesting that they may be the initiators of such stream. It remains to be demonstrated whether the pinosomes are transported by a cytoplasmic stream flowing in a plasma membrane-nuclear direction, that becomes noticeable when the cytoplasm apparently fluidifies.
Sulfate in newt egg jelly This investigation was conducted at the Instituto de Investigaciones Biomedicas, UNAM, with a fehowship given to the author by the Instituto National de la Investigacibn Cientifica, INIC, Mexico.
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. E: 30. 31. 32. 33. 34.
Allen, R D, J biophys biochem cytol8 (1960) 379. - Proc natl acad sci US 54 (1965) 1153. Auclair, W, Nature 192 (1961) 467. Balls, M & Ruben, L N, Exptl cell res 43 (1966) 694. Chapman-Andresen, C & Halter, H, Exptl cell res. Suppl. 3 (1955) 52. Chapman-Andresen, C, Exptl cell res 12 (1957) 397. - Comut rend trav Carlsbere: 310958) 77. Chapman-Andresen, C & H&zer, H( J biophys biochem cvtol 8 (1960) 288. Chapman-Andre&n, d, Proc 2nd intern conf protozool. London (1965). Cloney, R A, J ultrastruct res 14 (1966) 300. De Petris, S, Karlsbad, G & Pernis, B, J ultrastruct res 7 (1962) 39. De Terra, N & Rustad, C, Exptl cell res 17 (1959) 191. Epstein, M A, J biophys biochem cytol 3 (1957) 567. Freed, J J, Exptl cell res 26 (1962) 327. Gey, G 0, Shapras, P & Borysko, E, Ann NY acad sci 58 (1954) 1089. Granoff, A & Came, P E, Ann N Y acad sci 126 (1965) 237. Gross, W 0 & Riedel, B, Exptl cell res 54 (1969) 237. Jacoby, F, Cells and tissues in culture (ed E N Willmer) p. 1. Academic Press, New York (1965). Luft, J H, J biouhvs biochem cvtol 9 (1961) 409. M&anus, S M A & Roth, L-E, J cell biol 25 (1965) 305. Nachmias, V T, J cell biol 23 (1964) 183. Nagi, R & Rebhun, L I, J ultrastruct res 14 (1966) 571. Overtone, J, J cell biol 29 (1966) 293. Pomerat, C M. Personal communication (1957). -Fed proc 17 (1958) 975. Parker, R C, Methods of tissue culture. Hoeber medical division, Harper & Row (1964). Rafferty, K A, Jr, Ann N Y acad sci 126 (1965) 3. Reynolds, E C, J cell biol 17 (1963) 208. Robbins, E & Gonatas, N K, J cell bio120 (1964) 356. Rustad, R C, Nature (1959) 1058. - Recent progress in surface science (ed J F Danielli) p. 353. Academic Press, New York (1964). Tanaka, Y, J natl cancer inst 33 (1964) 467. Wohlman, A & Allen, R D, J cell sci 3 (1968) 105. Zimmerman, A & Rustad, R C, J cell biol 25 (1965) 397.
Received July 17, 1969 Revised version received October 30, 1969
157
INCORPORATION OF “5S-SULFATE INTO THE OVIDUCTS AND EGG JELLY OF THE NEWT, NOTOPHTHALMUS VIRIDESCENS A. A. HUMPHRIES, Emory
University,
Atlanta,
JR, Department Ga 30322,
of
Biology,
USA
Summary %-sulfate,
when injected into female newts (Notophwas incorporated into region A of the oviducts and secreted into layer Jl of the egg jelly. No label was noted in other regions of the oviduct. The evidence suggests that the label is incorporated into acid mucopolysaccharide, possibly into a compound with characteristics similar to those of chondroitin sulfate C or heparin. There was some indication that labeled material may move from layer Jl into layer 52, but there was no clear evidence of such movement into the oocyte or into jelly layers J3, 54, or J5. Suggestions as to the role of secretions of region A include possible involvement in sperm penetration and in the cell movements of gastrulation. thalmus
viridescens),
Although sulfate is known to be a prominent constituent of the egg jelly of many species, especially echinoderms, its presence in the jelly surrounding amphibian eggs has been in doubt, and indeed, “lack of sulfate” has been said to be one of the main differences between the jelly coats of amphibians and echinoderms ([ 193, p. 15). Chemical analysis of Bufo vulgaris jelly by Minganti [17] revealed no sulfate, and the use of 35S-sulfate by Lee [14] seemed to show that sulfate is not present in egg jelly of Rana pipiens. However, Minganti & D’Anna [18] reported that about 1 % of the dry weight of the jelly of Discoglossus pictus in sulfate, and Bolognani et al. [l] found traces of sulfate in jelly of Rana latastei and Bufo vulgaris. Furthermore, histochemical studies, using a variety of techniques, suggest the presence of sulfated polysaccharides in oviductal secretions of several species of amphibians [3, 4, 6, 7, 10-131. The oviductal secretions that make up amphibian egg jelly are clearly important in fertilization and possibly in other ways (see [6] for references). We have reported previously the results of a series of investigations Exptl
Cell Res 59
NEW FINDINGS PINOCYTOSIS
NOTES
RELATED
TO
E. MONROY,l
Institute de Investigaciones Biomtdicas, Universidad National Autdnoma de Mkxico, Apt Postal 70228, Ciudad Universitaria 20, D.F., Mexico
Summary Various culture media for maintenance of the newt Amhystoma mexicanum macrophages were tested. Once the most suitable medium was found, the cultivated cells were photographed and filmed. These cells show special features in their cytoplasm, that were studied under both light and electron microscopes. These features included an apparent fluidification of certain portions of the cytoplasm, as seen with timelapse cinematography and a marked orientation of cell inclusions and mitochondria. These observations are discussed in relation to pinocytosis.
The present paper describes some special features of newt macrophages maintained in culture, which seem important in relation to pinocytosis. Although pinocytosis is a general function of all cells and is present in all animal and certain plant cells [30], the extensive studies made in regard to its physiological aspects have been done mainly in amebae [5-91. The effect of physical factors such as temperature and high pressure, and also aerobic respiration correlated with pinocytosis has been studied also in amebae by Rustad et al. [12, 30, 31, 341.
method of Luft f191: sections were obtained with an LKB microtome,-either with glass or diamond knives, stained with uranyl acetate and lead hydroxide [28] and observed under an Akashi Tronscope TRS-80.
Results Fig. 1 show that the average count for all cultures after 15 days of incubation was close to 45 cells per explant. Migration of cells began after 10 days; on the 20th day of incubation the number of cells decreased onehalf and after 30 days the mean number of cells in the culture was 12 per explant. Fig. 2 shows that a part of the cytoplasm near the undulating membrane has a definite granular appearance different from the rest. Time lapse cinematography shows that this region changes constantly: the cytoplasm is even at first, but suddenly it begins to show the formation of “lagoons”, as if it became 92 48 44 40 36 32 28 24
Material and Methods
20
Spleen macrophages obtained from newts classified as Ambystoma mexicanum (Shaw) from the lake Xochimilco were used. For the maintenance of macrophages in culture, a combination of the coverslip and tube techniques [26] was employed, using 12 x 50 mm coverslips introduced in 16 mm tubes [24]. The tubes were laid still and incubated at 27°C. Basal Eagle medium plus 20 % calf serum, was the most suitable according to the results of trials in different media. To study the cells under the electron microscope, the technique of Robbins & Gonatas [29] was followed. The cells were embedded in Epon 812 by the
16
1 Present address: Department Hall, University of Virginia, 22903, USA.
of Biology, Gilmer Charlottesville, Va
12 8 4 4
8
12
15 20 24 28 32
123456
Abscissa: days; ordinate: mean number of cells per explant. Fig. I. Maintenance in culture of macrophagic cells from spleen explants of A. mexicanum in Eagle medium (basal plus 20 % calf serum) at 27°C. Abscissa: periods; ordinate: min. Fig. 3. Periods of apparent cytoplasmic fluidification (see text). Exptl
Cell Res 59
154 E. Monroy
2. In the upper left of this cell a zone of apparent cytoplasmic fluidification (CF) is seen. N ,nucleus; NC, nucleolus; FD, fat droplets; CZ, cytoplasmic inclusions; SP, small pinosomes; F, filopodia; A4, mitochondria, UM, undulating membrane. Magnification: x 40 (in the negative). Fig.
more fluid in certain zones; then, the “lagoons” disappear gradually and the grey cytoplasm regains the initial appearance, beginning, after a while, a new cycle. The entire process takes about 8 min. The undulating membrane is a completely transparent hyaline structure extending towards the outside ExptI
CeN Res 59
of the cell. It is a most characteristic morphological feature of macrophages in culture [18]. During this process it maintains its usual appearance. As seen in fig. 3, the above-mentioned cycle tends to be variable after 9 h of observation. The endoplasm of these macrophages
New findings in pinocytosis
155
Fig. 4. Part of a macrophage cytoplasm. Both kinds of pinosomes are seen, small and large. Mitochondria are oriented in a radial way. PNZ, perinuclear zone; M, mitochondria; LP, large pinosomes; SP, small pinosomes. Magnification: x 100 (in the negative).
Fig. 5. Electron micrograph of a cultured macrophage. The intracytoplasmic filaments (F) are seen. DB, dense bodies; A4, mitochondria; PV, pinocytic vesicle; GRE, granular endoplasmic reticulum, FD, fat droplets. Magnification: approx. x 22,000. Exptl
Cell Res 59
156 E. Monroy shows another feature: all cell inclusions and mitochondria are always oriented in a radial way (fig. 4). Pinocytosis is easily seen in these cells. However, as shown in fig. 4, there are two kinds of pinosomes: large and irregular, and small and spherical; the first travel very rapidly towards the perinuclear region; the others travel slowly through the cytoplasm. Both kinds of pinosomes can be seen in one cell at the same time. If the cell happens to be forming “lagoons” inside its cytoplasm, it is interesting that the pinosomes maintain their individuality all the time, i.e., the plasma membrane that forms them, persists; but they move about five times faster than the pinosomes of a cell without “lagoons”. In electron micrographics the cytoplasm of these cells show filaments of approx. 50 A in diameter, between the endoplasmic reticulum, the mitochondria and the cell inclusions (fig. 5). The filaments were constantly present in all the cells studied.
Discussion Pertinent literature contains data about tissue culture of different amphibian cells, but not on macrophages [3, 4, 14, 16, 271. Spleen macrophages of A. mexicanum never exhibit mitosis (fig. l), so they are maintained in culture and not grown in it. The increase in the number of cells is due to the migration of macrophages out of the explant; the decrease is due to the death of the cells and in some cases to explant detachment from the coverslip with subsequent injury of some cells. There is a latent period of 10 days before the migration begins, as it often occurs when adult tissues are cultured. Pinocytosis: Very little is known about how pinosomes move. Gey et al. [15] have shown that the force that moves the pinosomes is so Exptl
Cell
Res 59
great that it can break a filamentous mitochondria if it is on its way. However, this force is independent of the cytoplasm flux 1251. In A. mexicanum macrophages the radially oriented granules and mitochondria suggest that a cytoplasmic stream going in a plasma membrane-nucleus direction could very well carry the pinosomes to their final site. Allen [l, 2, 331, using the centrifuge microscope on three different species of amebae has found that the cytoplasmic inclusions are displaced discontinuously (at a variable velocity) in apparently all parts of the cell suggesting non-Newtonian behaviour and/or heterogenous consistency. Information is lacking in the case of macrophages. In the cells under study, in the sites where “lagoons” are formed, the cytoplasm has a fluid appearance. This is probably the case, since Allen himself states that very likely, changes in cytoplasmic consistency are taking place in amebae. The difficulty to use the centrifuge method in macrophages is that the apparent fluidification is transitory. Something similar to this has been observed by Gross & Riedel [17] in pulsating chicken embryo muscle cells, although this change in consistency of the cytoplasm is constant and not transitory as in the newt macrophages. At the electron microscopic level, fine intracytoplasmic filaments are present. Similar filaments have been reported by several authors in a variety of plant, animal and cancer cells [IO, 11, 13, 20, 21, 23, 321. It is interesting to note [22] the presence of these filaments in NiteZla [22] and their relation to the cytoplasmic stream, suggesting that they may be the initiators of such stream. It remains to be demonstrated whether the pinosomes are transported by a cytoplasmic stream flowing in a plasma membrane-nuclear direction, that becomes noticeable when the cytoplasm apparently fluidifies.
Sulfate in newt egg jelly This investigation was conducted at the Instituto de Investigaciones Biomedicas, UNAM, with a fehowship given to the author by the Instituto National de la Investigacibn Cientifica, INIC, Mexico.
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. E: 30. 31. 32. 33. 34.
Allen, R D, J biophys biochem cytol8 (1960) 379. - Proc natl acad sci US 54 (1965) 1153. Auclair, W, Nature 192 (1961) 467. Balls, M & Ruben, L N, Exptl cell res 43 (1966) 694. Chapman-Andresen, C & Halter, H, Exptl cell res. Suppl. 3 (1955) 52. Chapman-Andresen, C, Exptl cell res 12 (1957) 397. - Comut rend trav Carlsbere: 310958) 77. Chapman-Andresen, C & H&zer, H( J biophys biochem cvtol 8 (1960) 288. Chapman-Andre&n, d, Proc 2nd intern conf protozool. London (1965). Cloney, R A, J ultrastruct res 14 (1966) 300. De Petris, S, Karlsbad, G & Pernis, B, J ultrastruct res 7 (1962) 39. De Terra, N & Rustad, C, Exptl cell res 17 (1959) 191. Epstein, M A, J biophys biochem cytol 3 (1957) 567. Freed, J J, Exptl cell res 26 (1962) 327. Gey, G 0, Shapras, P & Borysko, E, Ann NY acad sci 58 (1954) 1089. Granoff, A & Came, P E, Ann N Y acad sci 126 (1965) 237. Gross, W 0 & Riedel, B, Exptl cell res 54 (1969) 237. Jacoby, F, Cells and tissues in culture (ed E N Willmer) p. 1. Academic Press, New York (1965). Luft, J H, J biouhvs biochem cvtol 9 (1961) 409. M&anus, S M A & Roth, L-E, J cell biol 25 (1965) 305. Nachmias, V T, J cell biol 23 (1964) 183. Nagi, R & Rebhun, L I, J ultrastruct res 14 (1966) 571. Overtone, J, J cell biol 29 (1966) 293. Pomerat, C M. Personal communication (1957). -Fed proc 17 (1958) 975. Parker, R C, Methods of tissue culture. Hoeber medical division, Harper & Row (1964). Rafferty, K A, Jr, Ann N Y acad sci 126 (1965) 3. Reynolds, E C, J cell biol 17 (1963) 208. Robbins, E & Gonatas, N K, J cell bio120 (1964) 356. Rustad, R C, Nature (1959) 1058. - Recent progress in surface science (ed J F Danielli) p. 353. Academic Press, New York (1964). Tanaka, Y, J natl cancer inst 33 (1964) 467. Wohlman, A & Allen, R D, J cell sci 3 (1968) 105. Zimmerman, A & Rustad, R C, J cell biol 25 (1965) 397.
Received July 17, 1969 Revised version received October 30, 1969
157
INCORPORATION OF “5S-SULFATE INTO THE OVIDUCTS AND EGG JELLY OF THE NEWT, NOTOPHTHALMUS VIRIDESCENS A. A. HUMPHRIES, Emory
University,
Atlanta,
JR, Department Ga 30322,
of
Biology,
USA
Summary %-sulfate,
when injected into female newts (Notophwas incorporated into region A of the oviducts and secreted into layer Jl of the egg jelly. No label was noted in other regions of the oviduct. The evidence suggests that the label is incorporated into acid mucopolysaccharide, possibly into a compound with characteristics similar to those of chondroitin sulfate C or heparin. There was some indication that labeled material may move from layer Jl into layer 52, but there was no clear evidence of such movement into the oocyte or into jelly layers J3, 54, or J5. Suggestions as to the role of secretions of region A include possible involvement in sperm penetration and in the cell movements of gastrulation. thalmus
viridescens),
Although sulfate is known to be a prominent constituent of the egg jelly of many species, especially echinoderms, its presence in the jelly surrounding amphibian eggs has been in doubt, and indeed, “lack of sulfate” has been said to be one of the main differences between the jelly coats of amphibians and echinoderms ([ 193, p. 15). Chemical analysis of Bufo vulgaris jelly by Minganti [17] revealed no sulfate, and the use of 35S-sulfate by Lee [14] seemed to show that sulfate is not present in egg jelly of Rana pipiens. However, Minganti & D’Anna [18] reported that about 1 % of the dry weight of the jelly of Discoglossus pictus in sulfate, and Bolognani et al. [l] found traces of sulfate in jelly of Rana latastei and Bufo vulgaris. Furthermore, histochemical studies, using a variety of techniques, suggest the presence of sulfated polysaccharides in oviductal secretions of several species of amphibians [3, 4, 6, 7, 10-131. The oviductal secretions that make up amphibian egg jelly are clearly important in fertilization and possibly in other ways (see [6] for references). We have reported previously the results of a series of investigations Exptl
Cell Res 59