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Enucleation of CHO cells by means of cytochalasin B and centrifugation: The topography of enucleation
Experimental Cell Research 94 (1975) 47-55
ENUCLEATION OF CYTOCHALASIN THE
OF CHO B AND
TOPOGRAPHY
CELLS
BY MEANS
CENTRIFUGATION:
OF ENUCLEATION
J. W. SHAY,’ M. R. GERSHENBAUM2and
K. R. PORTER2
‘Department of Cell Biology, University of Texas Health Science Center, Dallas, TX 75235 and 2Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80302, USA
SUMMARY Scanning electron microscopy has been used to observe the changes in morphology associated with the enucleation of Chinese hamster ovary cells using cytochalasin B and centrifugal force. Cells grown in monolayer culture on glass coverslips were inserted into centrifuge tubes with growth medium or growth medium containing cytochalasin B and subsequently centrifuged for various periods up to 60 min at 3 000 g in a prewarmed RC-2B centrifuge containing an SS-34 rotor. The cells spun without cytochalasin B were not enucleated and, when fixed and examined, were found not to differ in their morphology from cells of control cultures not subjected to centrifugal force. In contrast, cells centrifuged in growth medium containing cytochalasin B immediately show gross morphological changes. Initially the nucleus of each cell protrudes as an outpacketing on the cell’s free surface. As the outpacketing extends from the cell body, a stalk of cytoplasm is formed, which gradually decreases in diameter. Finally, the stalk breaks and the nucleus and small amounts of associated cytoplasm (karyoplast) sediments to the bottom of the centrifuge tube. When placed in fresh growth medium without cytochalasin B the greatly distorted enucleated cells (cytoplasts) which remain attached to the glass coverslip, rapidly recover a morphology similar to that of the normal nucleated cells.
Cytochalasin B (CB), a drug isolated from the fungus Helminthosporium dematiodeum, has been used to achieve the enucleation of populations of mammalian cells growing in monolayer culture [l-32]. A few cells in populations exposed to CB for extended periods of time release their nuclei spontaneously [l, 11, 15, 16, 181.However, the percentage of cells enucleated can be increased enormously by using centrifugal force concurrently with CB treatment [19, 311. Thus, up to 99% of the cells of a given population can be separated into nuclear and cytoplasmic parts [23, 24, 28, 29, 301. The products of the procedure include a 4-751811
pellet of nuclei (karyoplasts) and a population of greatly distorted enucleated cells (cytoplasts) which are adherent to a coverslip [23, 241. Transmission electron microscopy reveals that the karyoplasts consist of a nucleus surrounded by a thin sheath of cytoplasm limited by an intact plasma membrane. Ribosomes, fragments of the endoplasmic reticulum and a few mitochondria can be identitied in the cytoplasm. The cytoplasts, which appear similar to whole cells without nuclei [23, 24, 29, 301 contain centrioles, microtubules, microfilaments, Golgi, endoplasmic reticulum, mitochondria and ribosomes. Cytoplasts apparExptl Cell Res 94 (1975)
48
Shay, Gershrnbaum and Porter
Fig. I. Phase contrast light micrographs of centrifuged
CHO cells and recovering cytoplasts. (a) 20 min centrifugation in CB-one cell still contains a centrally located nucleus (urron-). x500; (b) after 40 min centrifugation cells begin to lose their nuclei. x500; (c)
ently contain the mechanisms necessary for attachment to a substrate, spreading, anisometric shape formation [23, 241, pinocytosis, contact inhibition, and cell locomotion [8,9, lo]. The karyoplasts, on the other hand, remain essentially isometric and lack the capacity to move [23, 24, 281. Previous reports have described the ultrastructure, the viability and some of the uses of the cytoplasts and karyoplasts [l-32], and recently the reconstruction of viable whole cells from the two parts has been reported
[6,231. The effects of cytochalasin in this process are puzzling. It has been reported that CB induces the disassembly of the finer filamentous (50-70 A microfilaments) components of the cytoplasm [33, 34, 351 especially in the cell cortex and one may assume that this is accompanied by a drop in viscosity. Possibly it is this latter change that permits the nucleus to be centrifuged out of the cell. As a first step in a further study of this interesting phenomenon we decided to observe the response of Chinese hamster fiptl
Cell Res 94 (1975)
by 60 min of centrifugation most cells have lost their nucleus. These distorted cytoplasts sometimes reach over 100 pm long. x500; (d) after 20 min incubation in fresh medium, the cytoplasts begin to recover a somewhat normal morphology. x500.
ovary (CHO) cells to cytochalasin B and centrifugal force and to compare this to the response of cells subjected only to centrifugal force. Using the scanning electron microscope we will illustrate the actual processes of enucleation and describe the form and behavior of karyoplasts and cytoplasts following separation.
MATERIALS
AND METHODS
Chinese hamster ovarv cells were mown on round glass (18 mm) coverslibs at 37°C (5% C0.J in Ham’s F-12 medium containing 10% fetal calf serum and 1% antibiotics (50 III/ml) penicillin and 50 pglml streutomvcin) GIBCo.. Grand Island. NY.). A stock cytochalasin ’ B (Aldrich Chemical Co., Inc., Milwaukee, Wise.) solution was made up in absolute ethanol (I mglml) and added to the growth medium to give a final concentration of 10 pg/ml. Cells were
2. Scanning electron micrograph of nonsynchronous CHO cells in logarithmic growth. These cells were spun at 3 000 g for 40 min in growth medium. x800. Fig. 3. One minute centrifugation in 10 pg/ml CB. Note the filooodia knnws~ which extend out to the original celh& attachment points X950. Fia. 4. After 5 min centrifuaation in CB. the nucleus is segregated into a cytoplaimic outpocketing on the cell’s free surface. X900. Fig.
Topography
of cell enucleation
49
Exptl Cell Res 94 (1975)
50
Shay, Gershenbaum
and Porter
grown to semiconfluency in normal growth medium on coverglasses which were then inserted cell side down into centrifuge tubes (30 ml corex no. 8445) containing cytochalasin-B as previously described [8, 9, IO, 13, 19; 23-301. These tubes were then spun for various periods up to 60 min at 3000 g in a prewarmed (37°C) RC-2B centrifuge containing an SS-34 rotor. Examination of various-&ages of the enucleation processes on living material was accomplished by removing the glass inserts at various intervals and placing them cell side down on depression slides containing growth medium. Observations were‘ made with a Zeiss light microscope equipped with phase contrast optics. The cells or parts of cells were fixed in glutaraldehyde for scanning electron microscopy and subsequently prepared as described earlier [36]. They were examined in a Kent Cambridge S-4 scanning electron microscope operated at 20 kV.
OBSERVATIONS Phase contrast light microscopy
Within 20 min after beginning centrifugation in medium containing CB the nuclei of most CHO cells appear segregated into small outpacketings of cytoplasm that bulge out from the main body of the cell (fig. la). From one of these cells (fig. 1a, arrow) there extends a thin strand of cytoplasm but the nucleus is still centrally located. After 40 min centrifugation in the presence of CB (fig. lb) most cells have lost their nuclei. Occasionally cells are observed with a thin strand or stalk of cytoplasm connected to its nucleus. By 60 min centrifugation in CB (fig. 1c) usually 95-99 % of the cells still attached to the substrate are enucleated resulting in greatly distorted cytoplasts which frequently reach over 100 pm long and 0.5 pm in diameter. After the nucleus is lost many of these distorted cytoplasts also show evidence of blebbing along their slender stalk (fig. 1b, c). If the coverslips containing these distorted cytoplasts are removed from the centrifuge tubes and placed into fresh medium, the cytoplasts quickly (20 min) recover from this distortion and regain a morphology similar to the original cell (fig. 1d). Exptl Cell Res 94 (1975)
Scanning electron microscopy
Scanning electron microscopy can be a valuable tool in the study of ultrastructural changes in the surfaces of cells. Fig. 2 is a scanning electron micrograph of a culture of nonsynchronous CHO cells spun at 3 000 g for 40 min in growth medium. The variations of cell shape in this micrograph correspond to variations in the cell cycle [37] and do not reflect differences due to the centrifugation. Fig. 3 illustrates a similar culture that was spun at 3 000 g for 1 min in the presence of 10 pg/ml CB and then prepared for scanning electron microscopy. Centrifugation and CB causes the cytoplasm to rapidly draw up into a relatively compact mass leaving thin strands of cytoplasm (filopodia) extending out to the original cellular attachment points on the glass coverslip. After 5 min centrifugation in CB (fig. 4) the nucleus of each cell appears to protrude from an outpocketing at the cell’s free surface and at higher magnification (fig. 5a) these effects are observed more clearly. (CB treatment alone will also produce these changes.) With increasing periods of centrifugation in the presence of CB (fig. .5b, 10 min; fig. 5c, 20 min; fig. 5d, 40 min) the nuclei appear to extend from a thick stalk of cytoplasm (fig. 5 6) which with time become increasingly longer and thinner (fig. 5c, 5d) until the nuclei are removed by a breaking of this cytoplasmic stalk. After the nucleus is lost the cytoplasmic stalk of each distorted cytoplast frequently shows evidence of blebbing along its length especially at the end where the nucleus was severed (fig. 50 While still attached to the cytoplasmic stalk the surface membranes around the nucleus usually appear smooth (fig. 6), but as soon as detached, they develop surface microvilli or blebs but rarely both (fig. 7). Groups of karyoplasts are frequently observed in
Topography
of cell enucleation
51
Fig. 5. With increasing lengths of centrifugation in the presence of CB, the nuclei appear to extend out from the cell body on a stalk of cytoplasm. This stalk becomes increasingly longer and thinner with time. (a) 5
min centrifugation. X2 000; (b) 10 min centrifugation. X2 Ooo; (c) 20 min centrifugation. X 1 100; (d) 40 min centrifugation. X 1 100.
clumps (fig. 7) and do not show the effects of contact. Most karyoplasts have the ability to attach to a substrate but do not spread or move about. The cytoplasts when placed in fresh
medium begin rapidly to recover from the CB treatment (fig. 8a), occasionally leaving some cytoplasmic material behind (fig. 8a, arrows). Some of this cytoplasmic material left on the coverslip results from cells Exptl Cell Res 94 (1975)
52
Shay, Gershenbaum
and Porter
Fig. 6. The surface membrane around the nucleus usually appears smooth when still attached to the cell by a cytoplasmic stalk (arrow). ~6 500.
Fig. 7. Clump of karyoplasts after being replated in fresh medium. Note that blebs and microvilh rarely appear together. x2 650.
that detached during centrifugation. After 20 min recovery, the cytoplasts are usually slightly elongate (fig. 8b), but by 40 min, they appear similar to the overall morphology of whole cells (fig. 8~). The cytoplasts at this time usually show few surface details but by 2 h recovery in fresh medium most develop microvilli (fig. 8d).
resulting in the formation of nuclear (karyoplast) and cytoplasmic (cytoplast) parts. Mild centrifugation without the presence of CB does not result in cell enucleation, and CB treatment without centrifugation results in the nuclear outpocketing but very limited enucleation (<0.5%) [l, 191. Electron microscopical studies indicate that the karyoplasts, though enclosed by a small amount of cytoplasm (10-l 5 %) and an intact plasma membrane, are not capable of regenerating the missing cytoplasm and within 36-72 h degenerate [24]. When replated in fresh medium most karyoplasts can attach to the substrate but are incapable of motion and retain a relatively
DISCUSSION Recent studies [33, 34, 351 have reported that the drug cytochalasin B (CB), either directly or indirectly, disassembles the cortical 50-70 A actin-like microfilaments of most mammalian cells. When such cells growing in monolayer culture are incubated in CB, each nucleus segregates into an outpocketing on the cell surface probably due to a drop in the viscosity of the cortical layer of cytoplasm. If CB treatment is combined with mild centrlfugation the nucleus separates from the main body of each cell
Fig. 8. Distorted cytoplasts recover rapidly after being
placed in fresh medium. (u) 10 mm recovery in fresh medium. Note the bits of cytoplasmic material left behind (arrows). x2 500; (b) 20 min recovery. X2 200; (c) 40 min recovery. The overall morphology is similar to that of the whole cells. x 1500; (d) Microvigi develop on the cell surfaces by 2 h of recovery. x 1500.
Topography
of cell enucleation
53
Exptl Cell Rev 94 (1975)
54
Shay, Gershenhaum
and Porter
spherical form (fail to spread) as though lacking a cytoskeletal form control mechanism [24]. Previous ultrastructural studies have reported that these relatively isometric karyoplasts do not contain microfilaments, microtubules, or the centrosphere-associated cytoplasm but do contain fragments of endoplasmic reticulum and numerous free ribosomes [24, 291. The fact that the karyoplasts fail to survive is an interesting phenomenon and one is lead to assume that the organization of certain cytoplasmic elements is essential to the viability of cells [24]. These karyoplasts, however, have been recombined with cytoplasts using inactivated Sendai virus and viable hybrids have been formed [6, 281. This indicates, that at least for a short period, the karyoplasts are functional and can be rescued if fused with another cytoplasm. As confirmed by light and electron microscopy, the cytoplasts, though greatly distorted at the end of the enucleation procedure, are able to recover a morphology similar to whole cells when placed in fresh medium. Thin section analysis reveals that the cytoplasts contain all organelles with the exception of the nucleus [24, 291. Cytoplasts do not incorporate 3H-thymidine but for at least 12 h continue to synthesize protein as judged by incorporation of 3Hleucine [19, 201. Cytoplasts, when trypsinized and replated on coverslips, contain alI the information necessary for cell attachment, spreading, shape formation, and locomotion. It has also been reported that cytoplasts contain precursors necessary for the assembly of microtubules [21, 22, 251. However, as would be expected, the cytoplasts survive only 24-48 h. Since the cytoplasts are capable of locomotion, spreading, and shape formation it would appear that the cytoplasm during its short period
of viability has functions independent of direct nuclear control. The studies presented here are reminiscent of the earlier works of Cole [38] and of Danielli & Harvey [39] which indicated that tension at the cell surface is primarily related to the structure of the plasma membrane and without reference to the underlying cortical layer of cytoplasm. Present studies-and-those of others [33, 34, 351 would indicate that the role of the cortical layer of cytoplasm in relation to the plasma membrane is complex and that one should, from observations reported here, think of the structure of the cortical microfilament network as an important factor in determining tension at the cell surface. This research was supported in part by a fellowship (Jerry W. Shay) and a grant (Keith R. Porter) from the Muscular Dystrophy Association of America, Inc.
REFERENCES 1. Carter, S B, Nature 213 (1%7) 261. 2. Croce, C M & Koprowski, H, Virology 51 (1973) 3. Croce, C M, Tomassine, N & Koprowski, H, Methods in cell biology (ed D M Prescott) vol. 8, p. 145. Academic Press, New York (1974). 4. Ege, T, Hamberg, H, Krondahl, U, Erickson, J & Ringer& N R, Exp cell res 87 (1974) 365. 5. Eae. T & Rinaertz. N R, EXD cell res 87 (1974) 378. 6. Ege, T, Krondahl, U & Ringertz, N R, Exp cell res 88 (1974) 428. 7. Follett, E A C, Exp cell res 84 (1974) 72. 8. Goldman, R D, Berg, G, Bushnell, A, Chang, C, Dicker-man, L, Hopkin, H, Miller, M, Pollack, R & Wang, E, Locomotion of tissue cells (Ciba found. symp. 14) p. 83. Elsevier Excerpta Medica, North Holland, Amsterdam (1973). 9. Goldman. R D. Pollack. R & Honkin. N H. Proc natlacad’sci US 71 (1973) 750. L 10. Goldman. R D & Pollack. R. Methods in cell biology (ed D M Prescott) vol.‘8, p. 123. Academic Press, New York (1974). 11. Ladda, R L & Estensen, R D, Proc natl acad sci US 67 (1970) 1528. 12. I’$?, R A & Ruddle, F H, J cell biol 63 (1974) 13. Pollack, R & Goldman, R, Science 179 (1973) 915. 14. Pollack, R, Goldman, R D, Conlon, S & Chang, C, Cell 3 (1974) 51. 15. Poste, G, Exp cell res 73 (1972) 273.
55
Topography of cell enucleation 16. Poste. G & Reeve. P. Exo cell res 73 (1972) 287. 17. Paste; G, Methods in ceil biology (ed D M Prescott) vol. 7. D. 211. Academic Press, New York ji973). 18. Prescott, D M, Kates, J & Kirkpatrick,
J B, J mol biol59 (1971) 505. 19. Prescott, D M. Mverson. D &Wallace, J, EXD cell res 71 (1972) 480.20. Prescott. D M & Kirkpatrick, J B, Methods in cell biology (ed D M Prescott) vol. 7, p. 189. Academic Press, New York (1973). 21. Schroder, C H & Hsie, A W, Nature new bio1246 (1974) 58. 22. Sethi, K K & Brandis, H, Nature 250 (1974) 225. 23. Shay, J W, Porter, K R & Prescott, D M, J cell biol
59(1973)311a.
24. - Proc natl acad sci US 71 (1974) 3059. 25. Shay, J W & Hammond, K J, In vitro 9 (1974) 358 a. 26. Shay, J W & Gershenbaum, M R, Electron
microscopy 1974, vol. 2, Proc 8th int. congr. electron microscopy (ed J V Sander & D J Goodchild) p. 336. Australia Academy of Science, Canberra (1974). 77 -,. Shay, J W & Krueger, T C, J cell biol 63 (1974) 311a. 28. Veomett, G, Prescott, D M, Shay, J W &Porter, K R, Proc natl acad sci US 71 (1974) 1999.
29. Wise, G E & Prescott, D M, Exp cell res 81 (1973) 65. 30. - Anat ret 175 (1973) 472. 31. Wright, W E & Hayflick, L, Exp cell res 74 (1972)
187. 32. Wright, W E, Methods in cell biology (ed D M
Prescott) vol. 7, p. 203. Academic Press, New York (1973). 33. Gershenbaum, M R, Shay, J W & Porter, K R,
34. 35. 36.
Scanning electron microscopy 1971. Proc 7th ann scanning electron microscope symp (ed 0 Johari & I Corvin) p. 590. IIT Research Institute, Chicago, 111.(1974). Spooner, B S, Yamada, K M & Wessell, N K, J cell biol49 (1971) 595. Schroeder, T E, Zellforsch Z mikrosk Anat 109 (1970)431. Porter, K R, Kelley, D & Andrews, PM, Proc 5th ann stereoscan colloa. II. 1. Kent Cambridge Scientitic Co., Chicago, Bl: (1972). Porter, K, Prescott, D M & Frve, J, J cell biol 57 Y
37.
(1973) 815. 38. Cole, K S. J cell coma ohvsiol 1 (1932) 1.
39. Danielli, J F & Harvey, E-N, J cell camp physiol5 (1935) 483.
Received January 20, 1975
Exptl Cell Res 94 (1975)
ENUCLEATION OF CYTOCHALASIN THE
OF CHO B AND
TOPOGRAPHY
CELLS
BY MEANS
CENTRIFUGATION:
OF ENUCLEATION
J. W. SHAY,’ M. R. GERSHENBAUM2and
K. R. PORTER2
‘Department of Cell Biology, University of Texas Health Science Center, Dallas, TX 75235 and 2Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80302, USA
SUMMARY Scanning electron microscopy has been used to observe the changes in morphology associated with the enucleation of Chinese hamster ovary cells using cytochalasin B and centrifugal force. Cells grown in monolayer culture on glass coverslips were inserted into centrifuge tubes with growth medium or growth medium containing cytochalasin B and subsequently centrifuged for various periods up to 60 min at 3 000 g in a prewarmed RC-2B centrifuge containing an SS-34 rotor. The cells spun without cytochalasin B were not enucleated and, when fixed and examined, were found not to differ in their morphology from cells of control cultures not subjected to centrifugal force. In contrast, cells centrifuged in growth medium containing cytochalasin B immediately show gross morphological changes. Initially the nucleus of each cell protrudes as an outpacketing on the cell’s free surface. As the outpacketing extends from the cell body, a stalk of cytoplasm is formed, which gradually decreases in diameter. Finally, the stalk breaks and the nucleus and small amounts of associated cytoplasm (karyoplast) sediments to the bottom of the centrifuge tube. When placed in fresh growth medium without cytochalasin B the greatly distorted enucleated cells (cytoplasts) which remain attached to the glass coverslip, rapidly recover a morphology similar to that of the normal nucleated cells.
Cytochalasin B (CB), a drug isolated from the fungus Helminthosporium dematiodeum, has been used to achieve the enucleation of populations of mammalian cells growing in monolayer culture [l-32]. A few cells in populations exposed to CB for extended periods of time release their nuclei spontaneously [l, 11, 15, 16, 181.However, the percentage of cells enucleated can be increased enormously by using centrifugal force concurrently with CB treatment [19, 311. Thus, up to 99% of the cells of a given population can be separated into nuclear and cytoplasmic parts [23, 24, 28, 29, 301. The products of the procedure include a 4-751811
pellet of nuclei (karyoplasts) and a population of greatly distorted enucleated cells (cytoplasts) which are adherent to a coverslip [23, 241. Transmission electron microscopy reveals that the karyoplasts consist of a nucleus surrounded by a thin sheath of cytoplasm limited by an intact plasma membrane. Ribosomes, fragments of the endoplasmic reticulum and a few mitochondria can be identitied in the cytoplasm. The cytoplasts, which appear similar to whole cells without nuclei [23, 24, 29, 301 contain centrioles, microtubules, microfilaments, Golgi, endoplasmic reticulum, mitochondria and ribosomes. Cytoplasts apparExptl Cell Res 94 (1975)
48
Shay, Gershrnbaum and Porter
Fig. I. Phase contrast light micrographs of centrifuged
CHO cells and recovering cytoplasts. (a) 20 min centrifugation in CB-one cell still contains a centrally located nucleus (urron-). x500; (b) after 40 min centrifugation cells begin to lose their nuclei. x500; (c)
ently contain the mechanisms necessary for attachment to a substrate, spreading, anisometric shape formation [23, 241, pinocytosis, contact inhibition, and cell locomotion [8,9, lo]. The karyoplasts, on the other hand, remain essentially isometric and lack the capacity to move [23, 24, 281. Previous reports have described the ultrastructure, the viability and some of the uses of the cytoplasts and karyoplasts [l-32], and recently the reconstruction of viable whole cells from the two parts has been reported
[6,231. The effects of cytochalasin in this process are puzzling. It has been reported that CB induces the disassembly of the finer filamentous (50-70 A microfilaments) components of the cytoplasm [33, 34, 351 especially in the cell cortex and one may assume that this is accompanied by a drop in viscosity. Possibly it is this latter change that permits the nucleus to be centrifuged out of the cell. As a first step in a further study of this interesting phenomenon we decided to observe the response of Chinese hamster fiptl
Cell Res 94 (1975)
by 60 min of centrifugation most cells have lost their nucleus. These distorted cytoplasts sometimes reach over 100 pm long. x500; (d) after 20 min incubation in fresh medium, the cytoplasts begin to recover a somewhat normal morphology. x500.
ovary (CHO) cells to cytochalasin B and centrifugal force and to compare this to the response of cells subjected only to centrifugal force. Using the scanning electron microscope we will illustrate the actual processes of enucleation and describe the form and behavior of karyoplasts and cytoplasts following separation.
MATERIALS
AND METHODS
Chinese hamster ovarv cells were mown on round glass (18 mm) coverslibs at 37°C (5% C0.J in Ham’s F-12 medium containing 10% fetal calf serum and 1% antibiotics (50 III/ml) penicillin and 50 pglml streutomvcin) GIBCo.. Grand Island. NY.). A stock cytochalasin ’ B (Aldrich Chemical Co., Inc., Milwaukee, Wise.) solution was made up in absolute ethanol (I mglml) and added to the growth medium to give a final concentration of 10 pg/ml. Cells were
2. Scanning electron micrograph of nonsynchronous CHO cells in logarithmic growth. These cells were spun at 3 000 g for 40 min in growth medium. x800. Fig. 3. One minute centrifugation in 10 pg/ml CB. Note the filooodia knnws~ which extend out to the original celh& attachment points X950. Fia. 4. After 5 min centrifuaation in CB. the nucleus is segregated into a cytoplaimic outpocketing on the cell’s free surface. X900. Fig.
Topography
of cell enucleation
49
Exptl Cell Res 94 (1975)
50
Shay, Gershenbaum
and Porter
grown to semiconfluency in normal growth medium on coverglasses which were then inserted cell side down into centrifuge tubes (30 ml corex no. 8445) containing cytochalasin-B as previously described [8, 9, IO, 13, 19; 23-301. These tubes were then spun for various periods up to 60 min at 3000 g in a prewarmed (37°C) RC-2B centrifuge containing an SS-34 rotor. Examination of various-&ages of the enucleation processes on living material was accomplished by removing the glass inserts at various intervals and placing them cell side down on depression slides containing growth medium. Observations were‘ made with a Zeiss light microscope equipped with phase contrast optics. The cells or parts of cells were fixed in glutaraldehyde for scanning electron microscopy and subsequently prepared as described earlier [36]. They were examined in a Kent Cambridge S-4 scanning electron microscope operated at 20 kV.
OBSERVATIONS Phase contrast light microscopy
Within 20 min after beginning centrifugation in medium containing CB the nuclei of most CHO cells appear segregated into small outpacketings of cytoplasm that bulge out from the main body of the cell (fig. la). From one of these cells (fig. 1a, arrow) there extends a thin strand of cytoplasm but the nucleus is still centrally located. After 40 min centrifugation in the presence of CB (fig. lb) most cells have lost their nuclei. Occasionally cells are observed with a thin strand or stalk of cytoplasm connected to its nucleus. By 60 min centrifugation in CB (fig. 1c) usually 95-99 % of the cells still attached to the substrate are enucleated resulting in greatly distorted cytoplasts which frequently reach over 100 pm long and 0.5 pm in diameter. After the nucleus is lost many of these distorted cytoplasts also show evidence of blebbing along their slender stalk (fig. 1b, c). If the coverslips containing these distorted cytoplasts are removed from the centrifuge tubes and placed into fresh medium, the cytoplasts quickly (20 min) recover from this distortion and regain a morphology similar to the original cell (fig. 1d). Exptl Cell Res 94 (1975)
Scanning electron microscopy
Scanning electron microscopy can be a valuable tool in the study of ultrastructural changes in the surfaces of cells. Fig. 2 is a scanning electron micrograph of a culture of nonsynchronous CHO cells spun at 3 000 g for 40 min in growth medium. The variations of cell shape in this micrograph correspond to variations in the cell cycle [37] and do not reflect differences due to the centrifugation. Fig. 3 illustrates a similar culture that was spun at 3 000 g for 1 min in the presence of 10 pg/ml CB and then prepared for scanning electron microscopy. Centrifugation and CB causes the cytoplasm to rapidly draw up into a relatively compact mass leaving thin strands of cytoplasm (filopodia) extending out to the original cellular attachment points on the glass coverslip. After 5 min centrifugation in CB (fig. 4) the nucleus of each cell appears to protrude from an outpocketing at the cell’s free surface and at higher magnification (fig. 5a) these effects are observed more clearly. (CB treatment alone will also produce these changes.) With increasing periods of centrifugation in the presence of CB (fig. .5b, 10 min; fig. 5c, 20 min; fig. 5d, 40 min) the nuclei appear to extend from a thick stalk of cytoplasm (fig. 5 6) which with time become increasingly longer and thinner (fig. 5c, 5d) until the nuclei are removed by a breaking of this cytoplasmic stalk. After the nucleus is lost the cytoplasmic stalk of each distorted cytoplast frequently shows evidence of blebbing along its length especially at the end where the nucleus was severed (fig. 50 While still attached to the cytoplasmic stalk the surface membranes around the nucleus usually appear smooth (fig. 6), but as soon as detached, they develop surface microvilli or blebs but rarely both (fig. 7). Groups of karyoplasts are frequently observed in
Topography
of cell enucleation
51
Fig. 5. With increasing lengths of centrifugation in the presence of CB, the nuclei appear to extend out from the cell body on a stalk of cytoplasm. This stalk becomes increasingly longer and thinner with time. (a) 5
min centrifugation. X2 000; (b) 10 min centrifugation. X2 Ooo; (c) 20 min centrifugation. X 1 100; (d) 40 min centrifugation. X 1 100.
clumps (fig. 7) and do not show the effects of contact. Most karyoplasts have the ability to attach to a substrate but do not spread or move about. The cytoplasts when placed in fresh
medium begin rapidly to recover from the CB treatment (fig. 8a), occasionally leaving some cytoplasmic material behind (fig. 8a, arrows). Some of this cytoplasmic material left on the coverslip results from cells Exptl Cell Res 94 (1975)
52
Shay, Gershenbaum
and Porter
Fig. 6. The surface membrane around the nucleus usually appears smooth when still attached to the cell by a cytoplasmic stalk (arrow). ~6 500.
Fig. 7. Clump of karyoplasts after being replated in fresh medium. Note that blebs and microvilh rarely appear together. x2 650.
that detached during centrifugation. After 20 min recovery, the cytoplasts are usually slightly elongate (fig. 8b), but by 40 min, they appear similar to the overall morphology of whole cells (fig. 8~). The cytoplasts at this time usually show few surface details but by 2 h recovery in fresh medium most develop microvilli (fig. 8d).
resulting in the formation of nuclear (karyoplast) and cytoplasmic (cytoplast) parts. Mild centrifugation without the presence of CB does not result in cell enucleation, and CB treatment without centrifugation results in the nuclear outpocketing but very limited enucleation (<0.5%) [l, 191. Electron microscopical studies indicate that the karyoplasts, though enclosed by a small amount of cytoplasm (10-l 5 %) and an intact plasma membrane, are not capable of regenerating the missing cytoplasm and within 36-72 h degenerate [24]. When replated in fresh medium most karyoplasts can attach to the substrate but are incapable of motion and retain a relatively
DISCUSSION Recent studies [33, 34, 351 have reported that the drug cytochalasin B (CB), either directly or indirectly, disassembles the cortical 50-70 A actin-like microfilaments of most mammalian cells. When such cells growing in monolayer culture are incubated in CB, each nucleus segregates into an outpocketing on the cell surface probably due to a drop in the viscosity of the cortical layer of cytoplasm. If CB treatment is combined with mild centrlfugation the nucleus separates from the main body of each cell
Fig. 8. Distorted cytoplasts recover rapidly after being
placed in fresh medium. (u) 10 mm recovery in fresh medium. Note the bits of cytoplasmic material left behind (arrows). x2 500; (b) 20 min recovery. X2 200; (c) 40 min recovery. The overall morphology is similar to that of the whole cells. x 1500; (d) Microvigi develop on the cell surfaces by 2 h of recovery. x 1500.
Topography
of cell enucleation
53
Exptl Cell Rev 94 (1975)
54
Shay, Gershenhaum
and Porter
spherical form (fail to spread) as though lacking a cytoskeletal form control mechanism [24]. Previous ultrastructural studies have reported that these relatively isometric karyoplasts do not contain microfilaments, microtubules, or the centrosphere-associated cytoplasm but do contain fragments of endoplasmic reticulum and numerous free ribosomes [24, 291. The fact that the karyoplasts fail to survive is an interesting phenomenon and one is lead to assume that the organization of certain cytoplasmic elements is essential to the viability of cells [24]. These karyoplasts, however, have been recombined with cytoplasts using inactivated Sendai virus and viable hybrids have been formed [6, 281. This indicates, that at least for a short period, the karyoplasts are functional and can be rescued if fused with another cytoplasm. As confirmed by light and electron microscopy, the cytoplasts, though greatly distorted at the end of the enucleation procedure, are able to recover a morphology similar to whole cells when placed in fresh medium. Thin section analysis reveals that the cytoplasts contain all organelles with the exception of the nucleus [24, 291. Cytoplasts do not incorporate 3H-thymidine but for at least 12 h continue to synthesize protein as judged by incorporation of 3Hleucine [19, 201. Cytoplasts, when trypsinized and replated on coverslips, contain alI the information necessary for cell attachment, spreading, shape formation, and locomotion. It has also been reported that cytoplasts contain precursors necessary for the assembly of microtubules [21, 22, 251. However, as would be expected, the cytoplasts survive only 24-48 h. Since the cytoplasts are capable of locomotion, spreading, and shape formation it would appear that the cytoplasm during its short period
of viability has functions independent of direct nuclear control. The studies presented here are reminiscent of the earlier works of Cole [38] and of Danielli & Harvey [39] which indicated that tension at the cell surface is primarily related to the structure of the plasma membrane and without reference to the underlying cortical layer of cytoplasm. Present studies-and-those of others [33, 34, 351 would indicate that the role of the cortical layer of cytoplasm in relation to the plasma membrane is complex and that one should, from observations reported here, think of the structure of the cortical microfilament network as an important factor in determining tension at the cell surface. This research was supported in part by a fellowship (Jerry W. Shay) and a grant (Keith R. Porter) from the Muscular Dystrophy Association of America, Inc.
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Received January 20, 1975
Exptl Cell Res 94 (1975)