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Concerning morphological evidence for pinocytosis in higher plants
Concerning
evidence for pinocytosis
5. KING, E. J., Biochem. J. 26, 292 (1932). 6. LOWRY, D. H., ROSEBROUGH, N. J., FARR, (1951). 7. PERINI, F., in Photosynthesis Mechanisms
A. L. and RAND.ILL,
207 R. J., J. Biol.
Chem.
193, 265
in Green Plants, Publication 1145. National Academy of S&n&-National Research Council, pp. 291-300, 1963. 8. PERINI, F., SCHIFF, J. A. and KAMEN, M. D., Biochim. Biophys. Beta (1964). In press.
9. PINCHOT, G. B., J. Biol. Chem. 10. ST.~I~L,W.L.,SMITH,J.C.,NAPOLITAXO,L.
CONCERNING
229,
11 (1957). M. and BASFORD,
MORPHOLOGICAL IN HIGHER
0. E. BRADFUTE,l Department
CICILY of Soils
EVIDENCE
R.E.,
J.CellBiol.
Nutrition, Berkeley,
(1963).
FOR PINOCYTOSIS
PLANTS
CHAPMAN-ANDRESENZ
and Plant California,
19,293
and W. A. JENSEN
Department of Botany, Calif., U.S.A.
University
of
Received July 10, 1964
I?INOCYTOSIS was first described as a bulk fluid ingestion of the external medium from observations of living macrophages in light microscopy [16]. In subsequent work [reviewed by IO, 121 indirect evidence for pinocytosis has been presented in the form of: (a) evidence suggesting the entry of macromolecules or colloidal particles into cells; (b) characteristic morphological features arising in light and electron micrographs of fixed tissues. These morphological features in fixed cells include bays or invaginations of the plasma membrane and cytoplasmic vesicles or vacuoles which contain evidence of having been formed from the plasma membrane. Studies with phase contrast microscopy showing the morphology and the sequence of events in living cells has substantiated the process of pinocytosis in amoebae and several types of animal cells from tissue cultures [lo, 121. In higher plants the evidence for pinocytosis is somewhat less definitive. Protein uptake by roots has been indicated by a variety of methods [3, 4, 14, 15, 181. Evidence suggesting active transport of basic proteins into plant roots [3] is now thought to have been misinterpreted [7], and the bulk of foreign basic protein is located in the cell walls of intact root epidermis and cap cells [5] and in the protoplasm of injured cells [6] not unlike that found in a variety of animal tissues [13]. Alcian blue is also concentrated in root cell walls and small amounts of this dye are found in small vacuoles of intact root epidermal cells [ll]. (In amoebae binding of both basic proteins and Alcian blue to the mucous coat is essential to subsequent pinocytic ingestion of the loaded membrane [lo].) Bays and invaginations in the plasma membrane 1 Present address: The Cell Research Institute, The University of Texas, U.S.h. 2 Permanent address: Carlsberg Laboratory, Copenhagen, Denmark. Experimental
Austin,
Texas
Cell
78712,
Research
36
208
0. E. Rradfute,
Cicily
Chapman-Andresen
and
W. A. .Jensen
of plant cells have been found in electron micrographs of the foliar tips of Elodea canadensis, the vegetative tips of Chrysanthemum segetum, and the roots of Triticum, sativum, Allium cepa [S, 91 and Zea mays [2O]. A somewhat more complex morphology thought to represent pinocytosis in microspore mother cells has been found in electron micrographs of Lycopersicum and Cucurbita 1191. Investigators presenting this graphic evidence for pinocytosis have suggested caution in the deduction of the sequence of events in living cells from micrographs of fixed tissue. We have studied living plant cells exposed to fluorescent-labeled, basic proteins in phase and fluorescence microscopy. Our premise was that the observation and description of the morphology of pinocytosis in a living plant cell would establish this process for higher plants and confirm the previous suggestions presented in the evidence for foreign protein entry and in the cellular morphology seen in electron micrographs. For this purpose, liquid endosperm of Pisum sativum was employed according to techniques developed for observation of intracellular detail of living plant endosperm [l, 21 and modifications thereof. Fluorescent labelled basic protein [l’i] was lyophilized and added directly to the native endosperm liquid. A Leitz phase microscope with Heine condenser and 35 mm camera with Eastman Kodak Tri-S film were employed. Pinocytosis was not observed and remains to be established in higher plants with the certainty that direct observation of the process in living cells would provide. Entry of the foreign basic protein concurrent with injury was observed and will be described fully elsewhere [6]. The purpose of this report is to demonstrate the importance of the direct observation of living cells in establishing morphological evidence for pinocytosis. Figs. 1-5 are phase contrast photomicrographs of living pea endosperm which might be presented as morphological evidence for pinocytosis if they had been derived from fixed tissue. In each case, however, the sequence of events before and after the photographic exposure clearly demonstrated that pinocytosis was not involved. The appearance of the endosperm differed according to the type of microchamber in which it was viewed. When the construction of the microchamber conformed to that of Bajer [l], i.e., the agar coat on the coverslip and agar coat on the slide were separated by a thin air space, the endosperm spread quite thinly at the upper agar: air interface (Figs. l-3). This multinucleate cell or syncitium had not changed in appearance during 1 hr of observation before the sequence of exposures was started. Degeneration of the cell resulted from the drying out of the microchamber, the edges of which had not been sealed. This in turn caused instability in the liquid film over the surface of the cells and strong and erratic currents in the external medium. The interval between each exposure of the sequence (Figs. 1-3) was less than 30 sec. Immediately after the last exposure (Fig. 3) the cell fragmented completely, so that instead of pinocytosis successive stages of degeneration have been recorded. When the microchamber was modified so as to include no air space, the endosperm spread and flowed between the two agar surfaces which were in contact. En masse flow of the endosperm occurred initially (Fig. 4) and was the result of pressure differences caused by unevenness in the opposing agar surfaces. Thus, instead of engulfing particles and part of the medium, the cell has been forced to flow around previously fixed particles. This en masse passive flow around the particles occurred Experimental
Cell Research 36
Concerning
evidence
for pinocy
fosis
l:igs. l-S.--Phase contrast photomicrographs of living pea endosperm cytosis. Figs. l-3 are a sequence of exposures resulting from degeneration. sive en masse flow of the cell around fixed particles. Fig. 5 resulted outward movement of a centrally located vacuole.
exhibiting pseudo-pinoFig. 4 resulted from pasfrom fragmentation and
Erperimerlfd
Cell Reseurch
36
210
0. E. Bradfute,
Cicily
Chapman-Andresen
and W. A. Jensen
within a few seconds after the preparation of the microchamber, but no other changes in shape or in the channels was observed in the following hr. Endosperm cells, viewed in an excess of their native liquid and without agar on either slide or coverslip, did not spread but remained generally spherical (Fig. 5). The channels or vesicles in this photomicrograph are not pinocytic vesicles transporting external medium in the direction of the central region of the cell, but are parts of a fragmented vacuole which had moved from the center to the periphery of the cell. Evidence presented here is limited to pea endosperm under a narrow range of conditions, which may not be optimal for physiological activity in these cells, hence this evidence cannot be assumed to exclude the possibility of pinocytosis in such cells or in other cells of higher plants; we wish, however, to emphasize that morphological evidence suggestive of vacuole formation by the cell membrane may be found as a result of phenomena other than pinocytosis. Additional uncertainty may be introduced by the fixation, dehydration, and embedding of cells used for electron microscopy. These considerations may be of general relevance in the deduction of the morphology of living processes from fixed cells. One of us (0. E. B.) gratefully acknowledges the support of grants USPH G-4236 and AEC AT (ll-l)-34, 50 made to Professor A. D. McLaren.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
BAJER, A., Experientia 11, 221 (1955). BAJER, A. and MOLT-BAJER, J., Acta. Sot. Uofan. Polon. 23, 69 (1954). BHIDE, S. V. and BRACHET, J., Ezptl Cell Res. 21, 303 (1960). BRACHET, J., Biochim. Biophys. Acfa 19, 583 (1956). BRADFUTE, 0. E., JENSES, W. A. and MCLAREX, A. D. In preparation. BRADFUTE, 0. E., JENSEN, W. A., SEEAR, J. and MCLAREN, A. D. In preparation. BRADFUTE, 0. E. and MCLARES, A. D., Physiof. Pfanfarum. 17, 677 (1964). BUVAT, R., Ann. Sci. Nafl lie s&e, Rot. 19, 121 (1955). BUVAT, R. and LANCE, A., Compf. Rend. Acad. Sci. Paris 245 (23), 2083 (1957). CHAPMAN-ANDRESEN, C., Compf. Rend. Trav. Lab. Carlsberg 33, 73 (1962). CHAPMAN-ANDRESEN, C., ASHTOS, MARY E. and JENSEN, W. A. In preparation. HOLTER, H., Intern. Rev. Cyfof. 8, 481 (1959). HOLTZER, H. and HOLTZER, S., Compf. Rend. Trav. Lab. Carlsberg, S&. Chim. 31,373 JENSEN, W. A. and MCLARES, A. D., Expfl Cell Res. 19, 414 (1960). KAUFMANN, B. P. and Das, N. K., Chromosoma 7, 19 (1955). LEWIS, W. H., Rull. Johns Hopkins Hosp. 49, 17 (1931). MELLORS, R. C., Analytical Cytology. 2nd ed., p. 1. McGraw-Hill, Xew York, 1959. MCLAREN, A. D., JENSEX, W. A. and JACOBSON, L., Pfanf Physiol. 35, 549 (1960). WEILING, F., Protoplasma 55/2, 372 (1962). WHALEY, W. G., MOLLEXHAUER, H. H. and LEECH, .J. H., Am. J. Rot. 47, 401 (1960).
Experimental
Cell Research136
(1960).
evidence for pinocytosis
5. KING, E. J., Biochem. J. 26, 292 (1932). 6. LOWRY, D. H., ROSEBROUGH, N. J., FARR, (1951). 7. PERINI, F., in Photosynthesis Mechanisms
A. L. and RAND.ILL,
207 R. J., J. Biol.
Chem.
193, 265
in Green Plants, Publication 1145. National Academy of S&n&-National Research Council, pp. 291-300, 1963. 8. PERINI, F., SCHIFF, J. A. and KAMEN, M. D., Biochim. Biophys. Beta (1964). In press.
9. PINCHOT, G. B., J. Biol. Chem. 10. ST.~I~L,W.L.,SMITH,J.C.,NAPOLITAXO,L.
CONCERNING
229,
11 (1957). M. and BASFORD,
MORPHOLOGICAL IN HIGHER
0. E. BRADFUTE,l Department
CICILY of Soils
EVIDENCE
R.E.,
J.CellBiol.
Nutrition, Berkeley,
(1963).
FOR PINOCYTOSIS
PLANTS
CHAPMAN-ANDRESENZ
and Plant California,
19,293
and W. A. JENSEN
Department of Botany, Calif., U.S.A.
University
of
Received July 10, 1964
I?INOCYTOSIS was first described as a bulk fluid ingestion of the external medium from observations of living macrophages in light microscopy [16]. In subsequent work [reviewed by IO, 121 indirect evidence for pinocytosis has been presented in the form of: (a) evidence suggesting the entry of macromolecules or colloidal particles into cells; (b) characteristic morphological features arising in light and electron micrographs of fixed tissues. These morphological features in fixed cells include bays or invaginations of the plasma membrane and cytoplasmic vesicles or vacuoles which contain evidence of having been formed from the plasma membrane. Studies with phase contrast microscopy showing the morphology and the sequence of events in living cells has substantiated the process of pinocytosis in amoebae and several types of animal cells from tissue cultures [lo, 121. In higher plants the evidence for pinocytosis is somewhat less definitive. Protein uptake by roots has been indicated by a variety of methods [3, 4, 14, 15, 181. Evidence suggesting active transport of basic proteins into plant roots [3] is now thought to have been misinterpreted [7], and the bulk of foreign basic protein is located in the cell walls of intact root epidermis and cap cells [5] and in the protoplasm of injured cells [6] not unlike that found in a variety of animal tissues [13]. Alcian blue is also concentrated in root cell walls and small amounts of this dye are found in small vacuoles of intact root epidermal cells [ll]. (In amoebae binding of both basic proteins and Alcian blue to the mucous coat is essential to subsequent pinocytic ingestion of the loaded membrane [lo].) Bays and invaginations in the plasma membrane 1 Present address: The Cell Research Institute, The University of Texas, U.S.h. 2 Permanent address: Carlsberg Laboratory, Copenhagen, Denmark. Experimental
Austin,
Texas
Cell
78712,
Research
36
208
0. E. Rradfute,
Cicily
Chapman-Andresen
and
W. A. .Jensen
of plant cells have been found in electron micrographs of the foliar tips of Elodea canadensis, the vegetative tips of Chrysanthemum segetum, and the roots of Triticum, sativum, Allium cepa [S, 91 and Zea mays [2O]. A somewhat more complex morphology thought to represent pinocytosis in microspore mother cells has been found in electron micrographs of Lycopersicum and Cucurbita 1191. Investigators presenting this graphic evidence for pinocytosis have suggested caution in the deduction of the sequence of events in living cells from micrographs of fixed tissue. We have studied living plant cells exposed to fluorescent-labeled, basic proteins in phase and fluorescence microscopy. Our premise was that the observation and description of the morphology of pinocytosis in a living plant cell would establish this process for higher plants and confirm the previous suggestions presented in the evidence for foreign protein entry and in the cellular morphology seen in electron micrographs. For this purpose, liquid endosperm of Pisum sativum was employed according to techniques developed for observation of intracellular detail of living plant endosperm [l, 21 and modifications thereof. Fluorescent labelled basic protein [l’i] was lyophilized and added directly to the native endosperm liquid. A Leitz phase microscope with Heine condenser and 35 mm camera with Eastman Kodak Tri-S film were employed. Pinocytosis was not observed and remains to be established in higher plants with the certainty that direct observation of the process in living cells would provide. Entry of the foreign basic protein concurrent with injury was observed and will be described fully elsewhere [6]. The purpose of this report is to demonstrate the importance of the direct observation of living cells in establishing morphological evidence for pinocytosis. Figs. 1-5 are phase contrast photomicrographs of living pea endosperm which might be presented as morphological evidence for pinocytosis if they had been derived from fixed tissue. In each case, however, the sequence of events before and after the photographic exposure clearly demonstrated that pinocytosis was not involved. The appearance of the endosperm differed according to the type of microchamber in which it was viewed. When the construction of the microchamber conformed to that of Bajer [l], i.e., the agar coat on the coverslip and agar coat on the slide were separated by a thin air space, the endosperm spread quite thinly at the upper agar: air interface (Figs. l-3). This multinucleate cell or syncitium had not changed in appearance during 1 hr of observation before the sequence of exposures was started. Degeneration of the cell resulted from the drying out of the microchamber, the edges of which had not been sealed. This in turn caused instability in the liquid film over the surface of the cells and strong and erratic currents in the external medium. The interval between each exposure of the sequence (Figs. 1-3) was less than 30 sec. Immediately after the last exposure (Fig. 3) the cell fragmented completely, so that instead of pinocytosis successive stages of degeneration have been recorded. When the microchamber was modified so as to include no air space, the endosperm spread and flowed between the two agar surfaces which were in contact. En masse flow of the endosperm occurred initially (Fig. 4) and was the result of pressure differences caused by unevenness in the opposing agar surfaces. Thus, instead of engulfing particles and part of the medium, the cell has been forced to flow around previously fixed particles. This en masse passive flow around the particles occurred Experimental
Cell Research 36
Concerning
evidence
for pinocy
fosis
l:igs. l-S.--Phase contrast photomicrographs of living pea endosperm cytosis. Figs. l-3 are a sequence of exposures resulting from degeneration. sive en masse flow of the cell around fixed particles. Fig. 5 resulted outward movement of a centrally located vacuole.
exhibiting pseudo-pinoFig. 4 resulted from pasfrom fragmentation and
Erperimerlfd
Cell Reseurch
36
210
0. E. Bradfute,
Cicily
Chapman-Andresen
and W. A. Jensen
within a few seconds after the preparation of the microchamber, but no other changes in shape or in the channels was observed in the following hr. Endosperm cells, viewed in an excess of their native liquid and without agar on either slide or coverslip, did not spread but remained generally spherical (Fig. 5). The channels or vesicles in this photomicrograph are not pinocytic vesicles transporting external medium in the direction of the central region of the cell, but are parts of a fragmented vacuole which had moved from the center to the periphery of the cell. Evidence presented here is limited to pea endosperm under a narrow range of conditions, which may not be optimal for physiological activity in these cells, hence this evidence cannot be assumed to exclude the possibility of pinocytosis in such cells or in other cells of higher plants; we wish, however, to emphasize that morphological evidence suggestive of vacuole formation by the cell membrane may be found as a result of phenomena other than pinocytosis. Additional uncertainty may be introduced by the fixation, dehydration, and embedding of cells used for electron microscopy. These considerations may be of general relevance in the deduction of the morphology of living processes from fixed cells. One of us (0. E. B.) gratefully acknowledges the support of grants USPH G-4236 and AEC AT (ll-l)-34, 50 made to Professor A. D. McLaren.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
BAJER, A., Experientia 11, 221 (1955). BAJER, A. and MOLT-BAJER, J., Acta. Sot. Uofan. Polon. 23, 69 (1954). BHIDE, S. V. and BRACHET, J., Ezptl Cell Res. 21, 303 (1960). BRACHET, J., Biochim. Biophys. Acfa 19, 583 (1956). BRADFUTE, 0. E., JENSES, W. A. and MCLAREX, A. D. In preparation. BRADFUTE, 0. E., JENSEN, W. A., SEEAR, J. and MCLAREN, A. D. In preparation. BRADFUTE, 0. E. and MCLARES, A. D., Physiof. Pfanfarum. 17, 677 (1964). BUVAT, R., Ann. Sci. Nafl lie s&e, Rot. 19, 121 (1955). BUVAT, R. and LANCE, A., Compf. Rend. Acad. Sci. Paris 245 (23), 2083 (1957). CHAPMAN-ANDRESEN, C., Compf. Rend. Trav. Lab. Carlsberg 33, 73 (1962). CHAPMAN-ANDRESEN, C., ASHTOS, MARY E. and JENSEN, W. A. In preparation. HOLTER, H., Intern. Rev. Cyfof. 8, 481 (1959). HOLTZER, H. and HOLTZER, S., Compf. Rend. Trav. Lab. Carlsberg, S&. Chim. 31,373 JENSEN, W. A. and MCLARES, A. D., Expfl Cell Res. 19, 414 (1960). KAUFMANN, B. P. and Das, N. K., Chromosoma 7, 19 (1955). LEWIS, W. H., Rull. Johns Hopkins Hosp. 49, 17 (1931). MELLORS, R. C., Analytical Cytology. 2nd ed., p. 1. McGraw-Hill, Xew York, 1959. MCLAREN, A. D., JENSEX, W. A. and JACOBSON, L., Pfanf Physiol. 35, 549 (1960). WEILING, F., Protoplasma 55/2, 372 (1962). WHALEY, W. G., MOLLEXHAUER, H. H. and LEECH, .J. H., Am. J. Rot. 47, 401 (1960).
Experimental
Cell Research136
(1960).