- Email: [email protected]
The isolation of motile cytoplasm from Amoeba proteus
156
C. M.
Thompson
and L. Wolpert
to be delivered. As the whole of the vitelline cell with its secretion products is removed after maturation, the activity of the vitelline glands can be classified as a type of holocrine secretion. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
MERCER, E. H., Nature 189, 558 (1961). N. and THORSELL, W., Exptl Cell Res. 27, 342 (1962). ~ ibid. In press. GOVAERT, J., ExpfZ Parasitol. 9, 141 (1960). HORSTMANN, H. J., Z. Parasitenk. 21, 437 (1962). KOURI, P. and NAUSS, R. W., J. Parasitol. 24, 291 (1938). RAO, H. I<., J. Parasitol. 45, 347 (1959). SMYTH, J. D., Nature 168, 322 (1951). SMYTH, J. D. and CLEGG, J. A., Exptl Parasitol. 8, 286 (1959). STEPHENSON, W., Parasitology 38, 128 (1947). GURAYA, S. S., Experentia 17, 561 (1961). Added in proof. BIRBECK, BJ~RKMAN,
M. S. C. and
THE
ISOLATION FROM
OF MOTILE
Zoology Department,
King’s Received
A
PROTEUS
AMOEBA
C. M. THOMPSON
CYTOPLASM
and L. WOLPERT
College,
University
June
17, 1963
of London, England
MAJOR obstacle to study of the molecular basis of amoeboid movement is absence of methods for isolating, in bulk, cellular components that are involved. The failure of light and electron microscopical studies to reveal structures, such as fibres, that can with confidence be assigneda motile function precludes the use of morphological criteria for recognising the motile components in isolated fractions. Although isolation of systems similar to actomyosin by extraction in relatively high salt concentrations has met with some success[3], the conclusion that such systems play a role in cell movement rests solely on the analogy that can be drawn between cell movement and muscular contraction. While suggestive, this analogy remains to be proved. There are, however, elegant demonstrations that the naked cytoplasm of single cells retains its motility [11, which support a physical rather than a chemical isolation procedure. We have therefore attempted a new approach by isolating cytoplasm which retains, in bulk, motile properties seenin the intact cell. Preparation of isolated cytoplasm.-Amoeba proteus (Dawson’s Strain) are mass cultured in Chalkley’s medium containing added MgCl, by feeding with washed Tetrahymena pyriformis following a schedule similar to that of Griffin [2]. Cells are well washed with medium and put in a cold-room for 24 hr. They remain rounded up at 4”C, but if healthy, begin to stream assoonasthey are brought to room temperature. Healthy cells are transferred as a slurry to transparent tubes. The slurry is
Experimental
Cell Research
32
Isolation
of motile
cytoplasm
from Amoeba
proteus
157
then spun at 4°C for 10 min at 35,000 g with gentle acceleration and deceleration. This results in the formation of five layers in the tube. The top layer is optically clear. The second layer, occupying most of the tube is opaque, white and viscous like a paste. It consists of amoebae with smooth surfaces, tightly packed together like a stack of inflated balloons. Fig. 1 shows the typical appearance of amoebae from layer 2 under phase contrast, The contents are stratified. At the light poles are fat droplets. The central areas appear empty except for a vacuole and at the heavy poles are heterogeneous masses in which vesicles and small granules can be seen. In the great majority, no nucleus, crystals or food vacuoles can be found. The formation of layer 2 is due to the heavy inclusions collecting at the heavy poles of the amoebae, becoming pinched off and being forced to the bottom of the tube. Layers 3 and 4 contain food vacuoles and nuclei surrounded by bags of membrane and the bottom layer-the fifth, consists of densely packed crystals. Only layer 2 is used and is gently homogenised so as to break open the membranes and destroy the paste-like consistency. The membrane fragments are spun out in the cold at 1000 to 2000 g for 5 min. The isolated cytoplasm remaining after removal of the cell membranes is kept in ice. Induction of motility in isolated cytoplasmic extracts.-The isolated cytoplasm is heterogeneous and contains fat droplets, granules and vesicles visible under phase contrast. There is usually only slight contamination by membranes (IO-100 per ml of extract). Motility is judged by observing the behaviour of granules in a drop of extract in a chamber 50 or 100 ,Udeep. Spacers support the coverslip which can be sealedwith liquid paraffin. At 4°C or at room temperature, only Brownian movement of granules can be seen. However, the addition of neutral ATP under appropriate conditions induces a striking motility. The first signs of movement, appearing after about 30 set, are saltatory movements of individual granules; that is, sudden jerky displacements of granules in any direction. The distance covered may be only a few microns, but the displacements are quite distinct from Brownian motion. Displacements continue in any direction but gradually become more widespread, whole areas moving as single blocks. The entire field begins to twitch. The moving blocks of cytoplasm appear to become connected to each other, so that the motion of one area is transmitted to others through a constantly changing network of oppositely directed streams of granules. The movement then appears to become more definitely organised into one of two alternative patterns. In the first case, streams of granules become arranged in parallel lines moving in opposite directions through the bulk of the cytoplasm. The streams persist for up to 15 min and die out slowly, leaving a uniform distribution of granular material over the whole area of the chamber. There’ is no net transfer of material in any direction although streams of particles may be moving in opposite directions to each other at 80 p/set for distancesof 600 ,u. In general, this ‘type of movement is similar to that observed by Allen et al. [ll, in single ruptured cells. In the second case, granules ceaseto move in oppositely directed streams. Instead<,large areas of the field appear to gel into sheets which contract rapidly to a small fraction of the original size. This type of movement does not persist for so long as streaming. As gelled areasform and contract, many larger granules are trapped and drawn together, including any membrane fragments. In this pattern of movement there is always a net transfer of material as denselocalised massesare formed (Fig. 2). When two sheets of gel contract in different directions Experimental
Cell Research 32
158
Experimental
C. M.
Cell
Research
32
Thompson
and L. Wolpert
Isolation
of motile
cytoplasm
from
Amoeba
159
proteus
and tear apart from each other, the connections between them become drawn out and sometimes clearly visible strands are formed which can extend for 500 p [6]. These thin strands have been observed to shorten and pull in smaller clumps of contracted material towards the main mass over a distance of several hundred microns. The formation and contraction of the gel is seen particularly well when a mixture of cytoplasm and ATP is introduced into a capillary, of about 500 ,u diameter (Fig. 3). Electron micrographs [5, 61 of the contracted gel from isolated cytoplasm confirms its heterogeneity. Most striking, however, are regions of partially orientated fibres. These fibres (about 120 A thick and 0.5 ,U long) occur in such forms as to suggest a relation with the contractile mechanism [6]. Conditions for motility and dependence on A TP.-The types of motility described have been induced, so far, only by the addition of ATP (i-5 mM) or ADP, ADP showing less activity. AMP and all other compounds tested are without effect (pyrophosphate, orthophosphate, SH compoundsand salts). Salts, particularly potassium chloride or traces of calcium, abolish the effect of ATP. i-5 m&f calcium chloride causesirreversible clumping of the granules and vesicles. Motility is observed only if cytoplasm, previously mixed with ATP in the cold, is allowed to come to room temperature. ATP doesnot have any effect on cytoplasm kept at a steady temperature of 4°C or 22°C. The capacity of pure cytoplasm stored in ice to respond to ATP and rising temperature gradually diminishes and disappears after i-2 hr, although its stability is prolonged by dilution with cold distilled water. Fresh extracts can be diluted I : 5 without loss of ATP responseand routine addition of an equal volume of water to layer 2 before homogenisation aids the removal of membranes. Discussion.-All the features of motility in our preparations of isolated cytoplasm show striking similarities to the behaviour of the cytoplasm in intact cells and to the behaviour of the naked cytoplasm of single cells ruptured in capillaries described by Allen et al. [l]. In intact cells, gelation, saltatory movements of particles and two directional streaming are all observed. Syneresis is particularly clearly seenin whole amoebaereleasedfrom high hydrostatic pressure[4]. ATP has beenrepeatedly claimed to play a central role in amoeboid movement soits specific effect on isolated cytoplasm is very suggestive. The action of ADP may be due to the presenceof myokinase in the system. Pressure-temperature studies of whole cells [4] suggestthat the structure forming capacity of the cytoplasm is reversibly disrupted by cooling. This is in line with the effects of temperature on the isolated cytoplasm. These similarities taken together with the minimal interference with the cytoplasm during its isolation, suggestthat the motility in the extracts has a similar basis to that in intact cells (61. Although the possibility of artefact cannot be completely excluded, the system seems Fig. l.-Amoeba centrifuged at 35,000 9 for IO min. The heavy inclusions have from the heavy pole (bottom). The remaining contents show stratification. V, vacuoles; H, hyaline region; G, vesicles and small granules. Fig. 2.-Edge G, gel region; porated into
of contracted gel formed X, larger inclusions trapped gel.
been pinched off F, fat droplets;
from isolated cytoplasm in presence of 2.5 mM ATP. during contraction; S, cytoplasmic material not incor-
Fig. 3.-The formation in capillary tubes of a thread-like gel from isolated cytoplasm in presence of 2.5 mM ATP. The tube on the left (A) showsthe thread and a fragment of surface membrane is seen at M. The tube B is the control containing 2.5 mM AMP. No contracted thread forms. ExDerimenfcd
Cell Research
32
I. L. Cameron to offer a very favourable amoeboid movement.
starting
This work has been supported
material for the study of the molecular
by the Nuffield
basis of
Foundation.
REFERENCES 1. ALLEN, R. D., COOLEDGE, J. W. and HALL, P. J., Nature 187, 896 (1960) 2. GRIFFIN, J. L., Exptl Cell Res. 21, 170 (1960). 3. HOFFMANN-BERLING, H., in Comparative Biochemistry, Vol. 2, p. 341. M. FLORKIN and H. S. MASON (eds). Academic Press, New York, 1960. 4. LANDAU, J. V., Ann. N. Y. Acad. Sci. 78, 487 (1959). 5. THOMPSON, C. M. and MERGER, E. H., In preparation, 1963. 6. WOLPERT, L., THOMPSON, C. M. and O’NEILL, C. H., in Primitive Motile Systems. R. D. ALLEN and N. KAMIYA (eds). Academic Press, New York, 1963. In press.
ORGANISMAL
REGULATION
ACTIVITY
OF MITOTIC
IN MICE
I. L. CAMERON Biology
Division, Oak Oak Ridge,
Ridge National Term.,
Laboratory,’
U.S.A.
Received June 25, 1963
A LTHOUGH
each tissue has its own mean mitotic
rate, there
is also the assumption
that all tissues of the body may be under a common mitotic control [l]. Since the blood stream is common to all tissues, factors influencing the mitotic activity of one tissue of the body may also influence others. Diurnal variation of mitotic rates occurs in various body tissues and is the most commonly cited example of systemic control of mitosis. Slowly dividing tissues(i.e., esophagusand skin) show considerable diurnal variation, whereas the rapidly proliferating cell systems (i.e., duodenum and hair bulbs) fail to show diurnal variation to any significant extent [2]. The present investigation examines the rapid and the relatively slowly proliferating tissue types to determine if systemic regulation affects only the slowly dividing tissues or if it is also acting on the rapidly dividing tissues. To assurethat a general organismal effect was being tested, three widely separated and different mouse epithelial cell populations were chosen: the plantar epidermis of the hind paw, the lower segment of the esophagus, and the upper portion of duodenum. Eleven male mice of the highly inbred SwissAlbino strain, 90 days of age, were injected at 11.00 a.m. with colchicine in 0.2 ml of saline (1 mg/kg body weight) to arrest and collect metaphase figures. Four hours later the animals were killed by a blow on the head. As the tissues were 1 Operated by Union Carbide Corporation for the United States Atomic Energy Commission. Experimental
Cell Research
32
C. M.
Thompson
and L. Wolpert
to be delivered. As the whole of the vitelline cell with its secretion products is removed after maturation, the activity of the vitelline glands can be classified as a type of holocrine secretion. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
MERCER, E. H., Nature 189, 558 (1961). N. and THORSELL, W., Exptl Cell Res. 27, 342 (1962). ~ ibid. In press. GOVAERT, J., ExpfZ Parasitol. 9, 141 (1960). HORSTMANN, H. J., Z. Parasitenk. 21, 437 (1962). KOURI, P. and NAUSS, R. W., J. Parasitol. 24, 291 (1938). RAO, H. I<., J. Parasitol. 45, 347 (1959). SMYTH, J. D., Nature 168, 322 (1951). SMYTH, J. D. and CLEGG, J. A., Exptl Parasitol. 8, 286 (1959). STEPHENSON, W., Parasitology 38, 128 (1947). GURAYA, S. S., Experentia 17, 561 (1961). Added in proof. BIRBECK, BJ~RKMAN,
M. S. C. and
THE
ISOLATION FROM
OF MOTILE
Zoology Department,
King’s Received
A
PROTEUS
AMOEBA
C. M. THOMPSON
CYTOPLASM
and L. WOLPERT
College,
University
June
17, 1963
of London, England
MAJOR obstacle to study of the molecular basis of amoeboid movement is absence of methods for isolating, in bulk, cellular components that are involved. The failure of light and electron microscopical studies to reveal structures, such as fibres, that can with confidence be assigneda motile function precludes the use of morphological criteria for recognising the motile components in isolated fractions. Although isolation of systems similar to actomyosin by extraction in relatively high salt concentrations has met with some success[3], the conclusion that such systems play a role in cell movement rests solely on the analogy that can be drawn between cell movement and muscular contraction. While suggestive, this analogy remains to be proved. There are, however, elegant demonstrations that the naked cytoplasm of single cells retains its motility [11, which support a physical rather than a chemical isolation procedure. We have therefore attempted a new approach by isolating cytoplasm which retains, in bulk, motile properties seenin the intact cell. Preparation of isolated cytoplasm.-Amoeba proteus (Dawson’s Strain) are mass cultured in Chalkley’s medium containing added MgCl, by feeding with washed Tetrahymena pyriformis following a schedule similar to that of Griffin [2]. Cells are well washed with medium and put in a cold-room for 24 hr. They remain rounded up at 4”C, but if healthy, begin to stream assoonasthey are brought to room temperature. Healthy cells are transferred as a slurry to transparent tubes. The slurry is
Experimental
Cell Research
32
Isolation
of motile
cytoplasm
from Amoeba
proteus
157
then spun at 4°C for 10 min at 35,000 g with gentle acceleration and deceleration. This results in the formation of five layers in the tube. The top layer is optically clear. The second layer, occupying most of the tube is opaque, white and viscous like a paste. It consists of amoebae with smooth surfaces, tightly packed together like a stack of inflated balloons. Fig. 1 shows the typical appearance of amoebae from layer 2 under phase contrast, The contents are stratified. At the light poles are fat droplets. The central areas appear empty except for a vacuole and at the heavy poles are heterogeneous masses in which vesicles and small granules can be seen. In the great majority, no nucleus, crystals or food vacuoles can be found. The formation of layer 2 is due to the heavy inclusions collecting at the heavy poles of the amoebae, becoming pinched off and being forced to the bottom of the tube. Layers 3 and 4 contain food vacuoles and nuclei surrounded by bags of membrane and the bottom layer-the fifth, consists of densely packed crystals. Only layer 2 is used and is gently homogenised so as to break open the membranes and destroy the paste-like consistency. The membrane fragments are spun out in the cold at 1000 to 2000 g for 5 min. The isolated cytoplasm remaining after removal of the cell membranes is kept in ice. Induction of motility in isolated cytoplasmic extracts.-The isolated cytoplasm is heterogeneous and contains fat droplets, granules and vesicles visible under phase contrast. There is usually only slight contamination by membranes (IO-100 per ml of extract). Motility is judged by observing the behaviour of granules in a drop of extract in a chamber 50 or 100 ,Udeep. Spacers support the coverslip which can be sealedwith liquid paraffin. At 4°C or at room temperature, only Brownian movement of granules can be seen. However, the addition of neutral ATP under appropriate conditions induces a striking motility. The first signs of movement, appearing after about 30 set, are saltatory movements of individual granules; that is, sudden jerky displacements of granules in any direction. The distance covered may be only a few microns, but the displacements are quite distinct from Brownian motion. Displacements continue in any direction but gradually become more widespread, whole areas moving as single blocks. The entire field begins to twitch. The moving blocks of cytoplasm appear to become connected to each other, so that the motion of one area is transmitted to others through a constantly changing network of oppositely directed streams of granules. The movement then appears to become more definitely organised into one of two alternative patterns. In the first case, streams of granules become arranged in parallel lines moving in opposite directions through the bulk of the cytoplasm. The streams persist for up to 15 min and die out slowly, leaving a uniform distribution of granular material over the whole area of the chamber. There’ is no net transfer of material in any direction although streams of particles may be moving in opposite directions to each other at 80 p/set for distancesof 600 ,u. In general, this ‘type of movement is similar to that observed by Allen et al. [ll, in single ruptured cells. In the second case, granules ceaseto move in oppositely directed streams. Instead<,large areas of the field appear to gel into sheets which contract rapidly to a small fraction of the original size. This type of movement does not persist for so long as streaming. As gelled areasform and contract, many larger granules are trapped and drawn together, including any membrane fragments. In this pattern of movement there is always a net transfer of material as denselocalised massesare formed (Fig. 2). When two sheets of gel contract in different directions Experimental
Cell Research 32
158
Experimental
C. M.
Cell
Research
32
Thompson
and L. Wolpert
Isolation
of motile
cytoplasm
from
Amoeba
159
proteus
and tear apart from each other, the connections between them become drawn out and sometimes clearly visible strands are formed which can extend for 500 p [6]. These thin strands have been observed to shorten and pull in smaller clumps of contracted material towards the main mass over a distance of several hundred microns. The formation and contraction of the gel is seen particularly well when a mixture of cytoplasm and ATP is introduced into a capillary, of about 500 ,u diameter (Fig. 3). Electron micrographs [5, 61 of the contracted gel from isolated cytoplasm confirms its heterogeneity. Most striking, however, are regions of partially orientated fibres. These fibres (about 120 A thick and 0.5 ,U long) occur in such forms as to suggest a relation with the contractile mechanism [6]. Conditions for motility and dependence on A TP.-The types of motility described have been induced, so far, only by the addition of ATP (i-5 mM) or ADP, ADP showing less activity. AMP and all other compounds tested are without effect (pyrophosphate, orthophosphate, SH compoundsand salts). Salts, particularly potassium chloride or traces of calcium, abolish the effect of ATP. i-5 m&f calcium chloride causesirreversible clumping of the granules and vesicles. Motility is observed only if cytoplasm, previously mixed with ATP in the cold, is allowed to come to room temperature. ATP doesnot have any effect on cytoplasm kept at a steady temperature of 4°C or 22°C. The capacity of pure cytoplasm stored in ice to respond to ATP and rising temperature gradually diminishes and disappears after i-2 hr, although its stability is prolonged by dilution with cold distilled water. Fresh extracts can be diluted I : 5 without loss of ATP responseand routine addition of an equal volume of water to layer 2 before homogenisation aids the removal of membranes. Discussion.-All the features of motility in our preparations of isolated cytoplasm show striking similarities to the behaviour of the cytoplasm in intact cells and to the behaviour of the naked cytoplasm of single cells ruptured in capillaries described by Allen et al. [l]. In intact cells, gelation, saltatory movements of particles and two directional streaming are all observed. Syneresis is particularly clearly seenin whole amoebaereleasedfrom high hydrostatic pressure[4]. ATP has beenrepeatedly claimed to play a central role in amoeboid movement soits specific effect on isolated cytoplasm is very suggestive. The action of ADP may be due to the presenceof myokinase in the system. Pressure-temperature studies of whole cells [4] suggestthat the structure forming capacity of the cytoplasm is reversibly disrupted by cooling. This is in line with the effects of temperature on the isolated cytoplasm. These similarities taken together with the minimal interference with the cytoplasm during its isolation, suggestthat the motility in the extracts has a similar basis to that in intact cells (61. Although the possibility of artefact cannot be completely excluded, the system seems Fig. l.-Amoeba centrifuged at 35,000 9 for IO min. The heavy inclusions have from the heavy pole (bottom). The remaining contents show stratification. V, vacuoles; H, hyaline region; G, vesicles and small granules. Fig. 2.-Edge G, gel region; porated into
of contracted gel formed X, larger inclusions trapped gel.
been pinched off F, fat droplets;
from isolated cytoplasm in presence of 2.5 mM ATP. during contraction; S, cytoplasmic material not incor-
Fig. 3.-The formation in capillary tubes of a thread-like gel from isolated cytoplasm in presence of 2.5 mM ATP. The tube on the left (A) showsthe thread and a fragment of surface membrane is seen at M. The tube B is the control containing 2.5 mM AMP. No contracted thread forms. ExDerimenfcd
Cell Research
32
I. L. Cameron to offer a very favourable amoeboid movement.
starting
This work has been supported
material for the study of the molecular
by the Nuffield
basis of
Foundation.
REFERENCES 1. ALLEN, R. D., COOLEDGE, J. W. and HALL, P. J., Nature 187, 896 (1960) 2. GRIFFIN, J. L., Exptl Cell Res. 21, 170 (1960). 3. HOFFMANN-BERLING, H., in Comparative Biochemistry, Vol. 2, p. 341. M. FLORKIN and H. S. MASON (eds). Academic Press, New York, 1960. 4. LANDAU, J. V., Ann. N. Y. Acad. Sci. 78, 487 (1959). 5. THOMPSON, C. M. and MERGER, E. H., In preparation, 1963. 6. WOLPERT, L., THOMPSON, C. M. and O’NEILL, C. H., in Primitive Motile Systems. R. D. ALLEN and N. KAMIYA (eds). Academic Press, New York, 1963. In press.
ORGANISMAL
REGULATION
ACTIVITY
OF MITOTIC
IN MICE
I. L. CAMERON Biology
Division, Oak Oak Ridge,
Ridge National Term.,
Laboratory,’
U.S.A.
Received June 25, 1963
A LTHOUGH
each tissue has its own mean mitotic
rate, there
is also the assumption
that all tissues of the body may be under a common mitotic control [l]. Since the blood stream is common to all tissues, factors influencing the mitotic activity of one tissue of the body may also influence others. Diurnal variation of mitotic rates occurs in various body tissues and is the most commonly cited example of systemic control of mitosis. Slowly dividing tissues(i.e., esophagusand skin) show considerable diurnal variation, whereas the rapidly proliferating cell systems (i.e., duodenum and hair bulbs) fail to show diurnal variation to any significant extent [2]. The present investigation examines the rapid and the relatively slowly proliferating tissue types to determine if systemic regulation affects only the slowly dividing tissues or if it is also acting on the rapidly dividing tissues. To assurethat a general organismal effect was being tested, three widely separated and different mouse epithelial cell populations were chosen: the plantar epidermis of the hind paw, the lower segment of the esophagus, and the upper portion of duodenum. Eleven male mice of the highly inbred SwissAlbino strain, 90 days of age, were injected at 11.00 a.m. with colchicine in 0.2 ml of saline (1 mg/kg body weight) to arrest and collect metaphase figures. Four hours later the animals were killed by a blow on the head. As the tissues were 1 Operated by Union Carbide Corporation for the United States Atomic Energy Commission. Experimental
Cell Research
32