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Cell Surface Morphology After Trypsinisation Depends on Initial Cell Shape
Differentiation
Differentiation 15, 61-66 (1979)
0 Springer-Verlag I979
Cell Surface Morphology After Trypsinisation Depends on Initial Cell Shape C. J. HARRISON’ and T. D. ALLEN Department of Ultrastructure, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, England
Cells growing in tissue culture exhibit constant variation in shape and surface morphology, particularly during the process of mitosis, where the cell rounds up exhibiting an intensely microvillous surface prior to cytokinesis. During routine subculturing, cells are induced to round up and relinquish contact with the substratum. Although the cells retain their viability throughout trypsinisation, their surface morphology demonstrates a variety of changes between finger-like microvillous projections, and spherical protruberances termed blebs. The reaction of individual cells to cell rounding, in the presence of trypsin appears to be dependent on cell shape, which may be modulated naturally or altered by experimental agents. Cells of bipolar morphology, termed fibroblasts, produce a blebbed surface morphology in response to trypsin, whereas isometric, ‘epithelioid’ cells respond by the formation of a microvillous cell surface. Blebbed cells subsequently undergo membrane reorganisation towards a more organised, and more permanent microvillous cell surface, even in the continued presence of trypsin. Naturally occurring spherical cells, for example, mitotic or suspension cultures, are microvillous and trypsin has no effect on their surface morphology. It would appear that blebs are the cells response to experimentally induced rapid change of shape of well spread cells, and thus represent a pathological response for prevention of membrane loss in conditions which produce a rapid assumption of a minimum surface area configuration, i.e. a sphere, which occurs too quickly for membrane resorption, or normal storage in the form of microvilli.
Microvilli and blebs are the most commonly observed surface features of cultured mammalian cells. Their density and distribution depends on several factors, particularly conditions which produce alterations in cell shape, such as morphological changes during the cell cycle [l-41; and in vitro transformation [5-91. Attachment of cells after plating results in a decrease in the density of surface protruberances during spreading 1101. Trypsinisation produces cell rounding, with an associated effect on surface morphology [ll-131. This normally rapid series of changes has been sequentially monitored by the use of low concentrations of trypsin on CHO 1 Present address and addressfor reprint requests: Department of Medical Genetics, St. Marys’ Hospital, Hathersage Road, Manchester MI3 OJH, England
populations, which exhibit cells of both typical bipolar ‘fibroblastic’ morphology, and isometric ‘epithelioid’ morphology in the logarithmic growth phase (Fig. la). Exposure of ‘fibroblastic’ CHO cells to 0.01% trypsin at room temperature produced an initial reaction (approximately 2 min) in which lateral cell-substratum attachment was reduced, resulting in a more cylindrical cell profile. This was followed by the release of one of the poles of the cell, clearly visible in phase contrast microscopy, which then ‘recoiled‘ rapidly towards the cell body (2-5 min). Preparations for SEM at this stage revealed semi-flattened cells with a blebbed surface morphology in the areas of ‘recoil’ (Fig. 1b). This series of events then occurred at the opposite pole, resulting in the formation of spherical cells of overall blebbed mor030 1-4681/79/00 15/006 1/30 1.20
62
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
a
b
Fig. 1. a SEM of asynchronous logarithmic CHO population. The majority of cells display the characteristic ‘fibroblastic’ morphology intermixed with spherical mitotic cells and ‘epitheloid’ cells at various stages of cell spreading. Scale bar = 20 pm. b SEM of CHO cells exposed to 0.01% trypsin at RT for 4 min. The cells are in a semi-flattened state with a blebbed surface in the area of recoil of one of the polar regions upon its detachment from the substratum. The other process remains in position, attached to the substratum. Scale bar = 5 pm. c SEM of a blebbed CHO cell detached from the substratum by 0.01% trypsin. The blebs are distributed over the entire cell surface and are of variable diameter. Scale bar = 1 pm
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
63
Table 1. Distribution of surface morphology of CHO cells during cell rounding and detachment in the presence of 0.01% trypsin at room temperatures Time
Cells in situ Flat cells (fibroblastic)
Semi-flattened cells
.
microvillous
blebbed %
%
0
microvillous %
Total blebbed cells
Spherical cells
blebbed micro% villous
%
blebbed %
%
13.8
-
1.2
12.1
12.9
54.3 24.5 1.3
-
1.6 3.0 2.0
32.1 59.5 2.1
5.1 4.5 14.8 31.1
-
12.1
(Control)
2 4 6 8 10 20
5.1 8.5 19.2
68.3
31.0 61.7 12.3 63.9 5 1.6 43.3
At time 0 the majority of cells are fibroblastic and of microvillous surface morphology, and represent the S phase cells within the asynchronous logarithmic population. The semi-flattened blebbed cells at this time are of ‘epithelioid’ outline and represent the G, phase [ 141. In the presence of 0.01% trypsin the percentage of blebbed cells increases up to 6 min as cell rounding occurs. At 4 min the majority of cells are blebbed. The semiflattened blebbed cells represent the fibroblasts in which recoil of one pole has occurred. This event occurred between approximately 2 to 5 min. At 6 min the majority of cells are spherical with a blebbed surface morphology, resulting from the recoil of the second pole. The cells then become detached from the substratum by the action of trypsin and the total percentage of blebbed cells subsequently decreases as the cells in suspension reorganise their cell surface to become microvillous
phology, which subsequently became detached (Fig. 1c). The blebbed surface morphology so formed was transient, for if these cells were maintained in suspension, even in the continued presence of trypsin, the surface became reorganised to a microvillous morphology, within 30 min. This series of events is quantitated in Table 1. The separation of cells into those possessing a microvillous or blebbed surface morphology was feasible, as blebs and microvilli were almost completely exclusive. In all preparations examined, cells with both blebs and microvilli consistently amounted to less than 5% of the total sample. Average figures for the numbers of blebdcell were between 200-400, whereas estimates of microvilli were between 750 and 1,250 microvilli per cell. Sections cut through blebbed surfaces revealed a typical bleb structure, namely an absence of cytoplasmic organelles other than ribosomes and the continuation of the peripheral 60A cytoplasmic filaments across the neck of the bleb. Sections through microvilli revealed a central core of microfilaments, but not, however, as well-developed as those in typical in vivo epithelial microvilli.
The response of ‘epithelioid’ cells (Fig. 2a) to trypsinisation differs from the bipolar ‘fibroblastic’ cells. In response to 0.01% trypsin at room temperature, there was uniform peripheral cytoplasmic retraction into a ‘hump’ over the central region as the cells assumed a spherical morphology. Throughout the rounding up process the cells maintained an ‘epithelioid’ outline and a microvillous cell surface. Contact was maintained with the substratum by retraction fibrils, which demarcated the outline of the originally flattened ‘epithelioid’ cell (Fig. 2b). The microvilli persisted throughout the cell detachment (Fig. 2c). Similar observations were also characteristic of HeLa cells and other ‘epithelioid’ cell types examined [141. Thus it appears that the original cell shape influences both the method of cell rounding and the resultant surface morphology of the detached cells. Culture conditions may be manipulated to produce changes in the overall morphology of CHO cultures. Increasing the serum concentration from the normal 10% by steps to 50% induces an increasingly ‘epithelioid’ population. Trypsinisation of such populations
64
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
b
a
d
C
Fig. 2. a SEM of CHO cells of ‘epithelioid’ outline. They are attached to the substratum around the entire cell periphery. Scale bar = 10 pm. b SEM of an ‘epithelioid’ cell after 2 min exposure to 0.01% trypsin at RT. The cell has maintained a microvillous cell surface whilst the bulk of the cytoplasm has retracted into a hump over the central region of the cell. Radial contact is maintained with the substratum by retraction fibrils, which demarcate the outline of the originally flattened ‘epithelioid‘ cell. Scale bar = 5 pm. c Spherical cell with microvillous morphology resulting from epithelioid cell detachment by trypsinisation. Scale bar = 2 pm. d SEM of an HU synchronised CHO population of S phase cells exposed to 0.01%trypsin for 2 rnin.The peripheral region shows numerous retraction fibrils which have severed at the edge resulting in a recoil of the peripheral cytoplasm and the production of blebs. Scale bar = 10 pm
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
65
Table 2. The distribution of surface morphology of CHO cells in suspension after 24 h incubation in medium containing various concentrations of serum followed by total cell detachment by 0.01% trypsin at room temperature
SE
Serum concentration of medium
Detached cells Microvillous
Blebbed
%
%
%
10
39.4
60.6
f 0.11
20
29.0
71.0
k 0.13
30
66.7
33.3
f 0.20
40
64.5 87.8
35.5 12.2
k 0.06
50
f 0.23
The typical response of the ‘fibroblastic’ population is shown at the normal serum concentrations (10% and 20%). Incubation in higher serum concentrations produced an increasingly ‘epithelioid‘ morphology which resulted in a decreasing percentage: of blebbed cells upon detachment
Fig. 3. Schematic representation of surface morphology associated with CHO cells throughout inherent and induced shape changes. The response of extended bipolar cells to trypsinisation is indicated at the top, leading to a blebbed spherical cell which may reorganise to a microvillous surface. Cell cycle changes are indicated by broken arrows, the cells exhibiting well spread bipolar morphology in ‘S’ phase, epithelioid morphology in G1 and G2, and spherical microvillous morphology in mitosis. Trypsinisation of epithelioid cells results in microvillous morphology, except where an epithelioid morphology has been induced by colcemid or extreme spreading (super flattened) by HU synchronisation in CHO cells
showed a parallel increase in the number of detached cells of microvillous morphology (Table 2). If an ‘epithelioid’ morphology is induced in CHO fibroblasts by colcemid treatment [15, 16, 17, 181, then the majority of detached cells possess a blebbed surface (71% after 8 min 0.01% trypsin at room temperature), In this situation it appears that cells may be dependent on the integrity of the cytoskeleton for the maintenance and production of a microvillous cell surface during rounding 1191. Fibroblast populations in the S phase of the cell cycle are usually of extended bipolar morphology 111. Synchronisation in the S phase by hydroxyurea (HU) [201 produces a change to a largely ‘epithelioid’ population. Chinese hamster lung fibroblasts synchronised in this way produce a microvillous surface morphology on detachment by trypsin. HU synchronised CHO fibroblasts also assume an ‘epithelioid’cell shape but become ‘superflattened’ to the extent that their diameter becomes equivalent to the length of a normal S phase fibroblast. Such extreme flatness is never observed in the ‘epithelioid’cells of a normal population. Trypsinisation of the ‘superflattened’ cells results in a similar dynamic recoil at the peripheral regions as observed at the poles of trypsinised fibroblasts, which also results in the for-
mation of a blebbed surface morphology (Fig. 2d). Thus blebs and microvilli may function as a method of storage for excess cell membrane during cell rounding 1211. Blebs represent a response to experimentally induced cell rounding and may provide a transient membrane storage as an alternative to large scale membrane loss. Microvilli are formed during the organised cell rounding process which occurs during mitosis and are the characteristic feature of spherical cells in suspension culture. Also trypsinised cells of blebbed morphology reorganise their cell surface to a microvillous morphology. Therefore, microvilli may represent a more permanent membrane storage function. CHO mitotic cells and P388F mouse lymphoma cells in suspension culture possess a microvillous cell surface and are morphologically resistant to the direct action of trypsin [141. They showed no alteration in surface morphology even when subjected to extended trypsinisation (0.05% at 37O C for 30 min). Thus the direct effect of trypsin appears to be one of cell detachment with the resultant detached cell surface morphology being a secondary effect dependent on the original cell shape, and extent of spreading. Acknowledgements: This work was supported by grants from the Cancer Research Campaign and the Medical Research Council.
66
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
References 1. Porter, K. R., Prescott, D., Frye, J.: The changes in surface morphology of CHO cells during the cell cycle. J. Cell Biol. 57, 815 (1973) 2. Follett, E. A. C., Goldman, R. D.: The occurrence of microvilli during spreading and growth of BHK 21/C13 fibroblasts. Exptl. Cell Res. 59, 124 (1970) 3. Sauk, J. J., White, J. G., Witkop, C. J., Jr.: Alterations in the surface morphology of synchronised B- 16 melanoma cells during the cell cycle. Acta Dermatol. Venereol (Stockh.) 55, 331 (1975) 4. Lundgren, E., Roos, G.: Cell surface changes in HeLa cells as an indication of cell cycle events. Cancer Res. 36, 4044 (1976) 5. Allen, T. D., Iype, P. T., Murphy, M. J., Jr.: The surface morphology of normal and malignant rat liver epithelial cells in culture. In Vitro 12, 837 (1976) 6. Boyde, A., Weiss, R. A., Vesely, P.: Scanning electron microscopy of cells in culture. Exptl. Cell Res. 71, 313 (1972) 7. Porter, K. R., Todaro, G. J., Fonte, V. J.: A scanning electron microscope study of surface features of vital and spontaneous transformants of mouse Balb/3T3 cells. J. Cell Biol. 59, 633 (1973) 8. Malick, L. E., Langenbach, R.: Scanning electron microscopy of in vitro chemically transformed mouse embryo cells. J. Cell Biol. 68, 654 (1974) 9. Borek, C., Fengolio, M.: Scanning electron microscopy of surface features of hamster embryo cells transformed in vitro by X irradiation. Cancer Res. 36, 1325 (1976) 10. O’Neill, C. H., Follett, E. A. C.: An inverse relation between cell density and the number of microvilli in cultures of BHK 21 hamster fibroblasts. J. Cell Sci. 7, 697 (1970) 11. Furcht, L. T., Wendelschafer-Crabb, 0.: Trypsin induced coordinate alterations in cell shape, cytoskeleton and intrinsic
membrane structure of contact inhibited cells. Exptl. Cell Res. 114, 1 (1978) 12. Revel, J., Hoch, P., Ho, D.: Adhesion of cultured cells to their substratum. Exptl. Cell Res. 84, 207 (1973) 13. Dalen, H., Todd, P. W.: Surface morphology of trypsinised human cells in vitro. Exptl. Cell Res. 66, 353 (1971) 14. Harrison, C. J.: A study of the surface topography of mammalian cells in culture. PhD. Thesis, University of Manchester (1978) 15. Goldman,R. D.: The role of three cytoplasmic fibres in BHK-21 cell motility. I. Microtubules and the effects of colchicine. J. Cell Biol. 51, 752 (1971) 16. Vasiliev, J. M., Gelfand, I. M., Domnina, L. V., Ivanova, 0. Y., Komm, S. G., Olshevskaja, L. V.: Effects of colcemid on the locomotory behaviour of fibroblasts. J. Embryol. Exptl. Morph. 24, 625 (1970) 17. Weisenberg, R. C., Borisy, G. G., Taylor, E. W.: The colchicine binding properties of mammalian brain and its relation to microtubules. Biochemistry 7, 4460 (1968) 18. Owellen, R.J., Owens, A. H., Jr., Donigian, C.: The binding of vincristine, vinblastine and colchicine to tubulin. Biochem. Biophys. Res. Commun. 47, 685 (1972) 19. Nicolson, G. L.: Transmembrane control of the receptors on normal and tumour cells. Biochem. Biophys. Acta 457, 57 (1976) 20. Tobey, R. A., Crissman, H. A.: Preparation of large quantities of synchronised mammalian cells in late G1 in the pre-DNA replication phase of the cell cycle. Exptl. Cell Res. 75, 460 (1972) 21. Erickson, C. A., Trinkaus, J. P.: Microvilli and blebs as sources of reserve surface membrane during cell spreading. Exptl. Cell Res. 99, 375 (1976)
Received April 1979/Accepted May 1979
Differentiation 15, 61-66 (1979)
0 Springer-Verlag I979
Cell Surface Morphology After Trypsinisation Depends on Initial Cell Shape C. J. HARRISON’ and T. D. ALLEN Department of Ultrastructure, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, England
Cells growing in tissue culture exhibit constant variation in shape and surface morphology, particularly during the process of mitosis, where the cell rounds up exhibiting an intensely microvillous surface prior to cytokinesis. During routine subculturing, cells are induced to round up and relinquish contact with the substratum. Although the cells retain their viability throughout trypsinisation, their surface morphology demonstrates a variety of changes between finger-like microvillous projections, and spherical protruberances termed blebs. The reaction of individual cells to cell rounding, in the presence of trypsin appears to be dependent on cell shape, which may be modulated naturally or altered by experimental agents. Cells of bipolar morphology, termed fibroblasts, produce a blebbed surface morphology in response to trypsin, whereas isometric, ‘epithelioid’ cells respond by the formation of a microvillous cell surface. Blebbed cells subsequently undergo membrane reorganisation towards a more organised, and more permanent microvillous cell surface, even in the continued presence of trypsin. Naturally occurring spherical cells, for example, mitotic or suspension cultures, are microvillous and trypsin has no effect on their surface morphology. It would appear that blebs are the cells response to experimentally induced rapid change of shape of well spread cells, and thus represent a pathological response for prevention of membrane loss in conditions which produce a rapid assumption of a minimum surface area configuration, i.e. a sphere, which occurs too quickly for membrane resorption, or normal storage in the form of microvilli.
Microvilli and blebs are the most commonly observed surface features of cultured mammalian cells. Their density and distribution depends on several factors, particularly conditions which produce alterations in cell shape, such as morphological changes during the cell cycle [l-41; and in vitro transformation [5-91. Attachment of cells after plating results in a decrease in the density of surface protruberances during spreading 1101. Trypsinisation produces cell rounding, with an associated effect on surface morphology [ll-131. This normally rapid series of changes has been sequentially monitored by the use of low concentrations of trypsin on CHO 1 Present address and addressfor reprint requests: Department of Medical Genetics, St. Marys’ Hospital, Hathersage Road, Manchester MI3 OJH, England
populations, which exhibit cells of both typical bipolar ‘fibroblastic’ morphology, and isometric ‘epithelioid’ morphology in the logarithmic growth phase (Fig. la). Exposure of ‘fibroblastic’ CHO cells to 0.01% trypsin at room temperature produced an initial reaction (approximately 2 min) in which lateral cell-substratum attachment was reduced, resulting in a more cylindrical cell profile. This was followed by the release of one of the poles of the cell, clearly visible in phase contrast microscopy, which then ‘recoiled‘ rapidly towards the cell body (2-5 min). Preparations for SEM at this stage revealed semi-flattened cells with a blebbed surface morphology in the areas of ‘recoil’ (Fig. 1b). This series of events then occurred at the opposite pole, resulting in the formation of spherical cells of overall blebbed mor030 1-4681/79/00 15/006 1/30 1.20
62
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
a
b
Fig. 1. a SEM of asynchronous logarithmic CHO population. The majority of cells display the characteristic ‘fibroblastic’ morphology intermixed with spherical mitotic cells and ‘epitheloid’ cells at various stages of cell spreading. Scale bar = 20 pm. b SEM of CHO cells exposed to 0.01% trypsin at RT for 4 min. The cells are in a semi-flattened state with a blebbed surface in the area of recoil of one of the polar regions upon its detachment from the substratum. The other process remains in position, attached to the substratum. Scale bar = 5 pm. c SEM of a blebbed CHO cell detached from the substratum by 0.01% trypsin. The blebs are distributed over the entire cell surface and are of variable diameter. Scale bar = 1 pm
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
63
Table 1. Distribution of surface morphology of CHO cells during cell rounding and detachment in the presence of 0.01% trypsin at room temperatures Time
Cells in situ Flat cells (fibroblastic)
Semi-flattened cells
.
microvillous
blebbed %
%
0
microvillous %
Total blebbed cells
Spherical cells
blebbed micro% villous
%
blebbed %
%
13.8
-
1.2
12.1
12.9
54.3 24.5 1.3
-
1.6 3.0 2.0
32.1 59.5 2.1
5.1 4.5 14.8 31.1
-
12.1
(Control)
2 4 6 8 10 20
5.1 8.5 19.2
68.3
31.0 61.7 12.3 63.9 5 1.6 43.3
At time 0 the majority of cells are fibroblastic and of microvillous surface morphology, and represent the S phase cells within the asynchronous logarithmic population. The semi-flattened blebbed cells at this time are of ‘epithelioid’ outline and represent the G, phase [ 141. In the presence of 0.01% trypsin the percentage of blebbed cells increases up to 6 min as cell rounding occurs. At 4 min the majority of cells are blebbed. The semiflattened blebbed cells represent the fibroblasts in which recoil of one pole has occurred. This event occurred between approximately 2 to 5 min. At 6 min the majority of cells are spherical with a blebbed surface morphology, resulting from the recoil of the second pole. The cells then become detached from the substratum by the action of trypsin and the total percentage of blebbed cells subsequently decreases as the cells in suspension reorganise their cell surface to become microvillous
phology, which subsequently became detached (Fig. 1c). The blebbed surface morphology so formed was transient, for if these cells were maintained in suspension, even in the continued presence of trypsin, the surface became reorganised to a microvillous morphology, within 30 min. This series of events is quantitated in Table 1. The separation of cells into those possessing a microvillous or blebbed surface morphology was feasible, as blebs and microvilli were almost completely exclusive. In all preparations examined, cells with both blebs and microvilli consistently amounted to less than 5% of the total sample. Average figures for the numbers of blebdcell were between 200-400, whereas estimates of microvilli were between 750 and 1,250 microvilli per cell. Sections cut through blebbed surfaces revealed a typical bleb structure, namely an absence of cytoplasmic organelles other than ribosomes and the continuation of the peripheral 60A cytoplasmic filaments across the neck of the bleb. Sections through microvilli revealed a central core of microfilaments, but not, however, as well-developed as those in typical in vivo epithelial microvilli.
The response of ‘epithelioid’ cells (Fig. 2a) to trypsinisation differs from the bipolar ‘fibroblastic’ cells. In response to 0.01% trypsin at room temperature, there was uniform peripheral cytoplasmic retraction into a ‘hump’ over the central region as the cells assumed a spherical morphology. Throughout the rounding up process the cells maintained an ‘epithelioid’ outline and a microvillous cell surface. Contact was maintained with the substratum by retraction fibrils, which demarcated the outline of the originally flattened ‘epithelioid’ cell (Fig. 2b). The microvilli persisted throughout the cell detachment (Fig. 2c). Similar observations were also characteristic of HeLa cells and other ‘epithelioid’ cell types examined [141. Thus it appears that the original cell shape influences both the method of cell rounding and the resultant surface morphology of the detached cells. Culture conditions may be manipulated to produce changes in the overall morphology of CHO cultures. Increasing the serum concentration from the normal 10% by steps to 50% induces an increasingly ‘epithelioid’ population. Trypsinisation of such populations
64
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
b
a
d
C
Fig. 2. a SEM of CHO cells of ‘epithelioid’ outline. They are attached to the substratum around the entire cell periphery. Scale bar = 10 pm. b SEM of an ‘epithelioid’ cell after 2 min exposure to 0.01% trypsin at RT. The cell has maintained a microvillous cell surface whilst the bulk of the cytoplasm has retracted into a hump over the central region of the cell. Radial contact is maintained with the substratum by retraction fibrils, which demarcate the outline of the originally flattened ‘epithelioid‘ cell. Scale bar = 5 pm. c Spherical cell with microvillous morphology resulting from epithelioid cell detachment by trypsinisation. Scale bar = 2 pm. d SEM of an HU synchronised CHO population of S phase cells exposed to 0.01%trypsin for 2 rnin.The peripheral region shows numerous retraction fibrils which have severed at the edge resulting in a recoil of the peripheral cytoplasm and the production of blebs. Scale bar = 10 pm
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
65
Table 2. The distribution of surface morphology of CHO cells in suspension after 24 h incubation in medium containing various concentrations of serum followed by total cell detachment by 0.01% trypsin at room temperature
SE
Serum concentration of medium
Detached cells Microvillous
Blebbed
%
%
%
10
39.4
60.6
f 0.11
20
29.0
71.0
k 0.13
30
66.7
33.3
f 0.20
40
64.5 87.8
35.5 12.2
k 0.06
50
f 0.23
The typical response of the ‘fibroblastic’ population is shown at the normal serum concentrations (10% and 20%). Incubation in higher serum concentrations produced an increasingly ‘epithelioid‘ morphology which resulted in a decreasing percentage: of blebbed cells upon detachment
Fig. 3. Schematic representation of surface morphology associated with CHO cells throughout inherent and induced shape changes. The response of extended bipolar cells to trypsinisation is indicated at the top, leading to a blebbed spherical cell which may reorganise to a microvillous surface. Cell cycle changes are indicated by broken arrows, the cells exhibiting well spread bipolar morphology in ‘S’ phase, epithelioid morphology in G1 and G2, and spherical microvillous morphology in mitosis. Trypsinisation of epithelioid cells results in microvillous morphology, except where an epithelioid morphology has been induced by colcemid or extreme spreading (super flattened) by HU synchronisation in CHO cells
showed a parallel increase in the number of detached cells of microvillous morphology (Table 2). If an ‘epithelioid’ morphology is induced in CHO fibroblasts by colcemid treatment [15, 16, 17, 181, then the majority of detached cells possess a blebbed surface (71% after 8 min 0.01% trypsin at room temperature), In this situation it appears that cells may be dependent on the integrity of the cytoskeleton for the maintenance and production of a microvillous cell surface during rounding 1191. Fibroblast populations in the S phase of the cell cycle are usually of extended bipolar morphology 111. Synchronisation in the S phase by hydroxyurea (HU) [201 produces a change to a largely ‘epithelioid’ population. Chinese hamster lung fibroblasts synchronised in this way produce a microvillous surface morphology on detachment by trypsin. HU synchronised CHO fibroblasts also assume an ‘epithelioid’cell shape but become ‘superflattened’ to the extent that their diameter becomes equivalent to the length of a normal S phase fibroblast. Such extreme flatness is never observed in the ‘epithelioid’cells of a normal population. Trypsinisation of the ‘superflattened’ cells results in a similar dynamic recoil at the peripheral regions as observed at the poles of trypsinised fibroblasts, which also results in the for-
mation of a blebbed surface morphology (Fig. 2d). Thus blebs and microvilli may function as a method of storage for excess cell membrane during cell rounding 1211. Blebs represent a response to experimentally induced cell rounding and may provide a transient membrane storage as an alternative to large scale membrane loss. Microvilli are formed during the organised cell rounding process which occurs during mitosis and are the characteristic feature of spherical cells in suspension culture. Also trypsinised cells of blebbed morphology reorganise their cell surface to a microvillous morphology. Therefore, microvilli may represent a more permanent membrane storage function. CHO mitotic cells and P388F mouse lymphoma cells in suspension culture possess a microvillous cell surface and are morphologically resistant to the direct action of trypsin [141. They showed no alteration in surface morphology even when subjected to extended trypsinisation (0.05% at 37O C for 30 min). Thus the direct effect of trypsin appears to be one of cell detachment with the resultant detached cell surface morphology being a secondary effect dependent on the original cell shape, and extent of spreading. Acknowledgements: This work was supported by grants from the Cancer Research Campaign and the Medical Research Council.
66
C. J. Harrison and T. D. Allen: Cell Surface Morphology After Trypsinisation
References 1. Porter, K. R., Prescott, D., Frye, J.: The changes in surface morphology of CHO cells during the cell cycle. J. Cell Biol. 57, 815 (1973) 2. Follett, E. A. C., Goldman, R. D.: The occurrence of microvilli during spreading and growth of BHK 21/C13 fibroblasts. Exptl. Cell Res. 59, 124 (1970) 3. Sauk, J. J., White, J. G., Witkop, C. J., Jr.: Alterations in the surface morphology of synchronised B- 16 melanoma cells during the cell cycle. Acta Dermatol. Venereol (Stockh.) 55, 331 (1975) 4. Lundgren, E., Roos, G.: Cell surface changes in HeLa cells as an indication of cell cycle events. Cancer Res. 36, 4044 (1976) 5. Allen, T. D., Iype, P. T., Murphy, M. J., Jr.: The surface morphology of normal and malignant rat liver epithelial cells in culture. In Vitro 12, 837 (1976) 6. Boyde, A., Weiss, R. A., Vesely, P.: Scanning electron microscopy of cells in culture. Exptl. Cell Res. 71, 313 (1972) 7. Porter, K. R., Todaro, G. J., Fonte, V. J.: A scanning electron microscope study of surface features of vital and spontaneous transformants of mouse Balb/3T3 cells. J. Cell Biol. 59, 633 (1973) 8. Malick, L. E., Langenbach, R.: Scanning electron microscopy of in vitro chemically transformed mouse embryo cells. J. Cell Biol. 68, 654 (1974) 9. Borek, C., Fengolio, M.: Scanning electron microscopy of surface features of hamster embryo cells transformed in vitro by X irradiation. Cancer Res. 36, 1325 (1976) 10. O’Neill, C. H., Follett, E. A. C.: An inverse relation between cell density and the number of microvilli in cultures of BHK 21 hamster fibroblasts. J. Cell Sci. 7, 697 (1970) 11. Furcht, L. T., Wendelschafer-Crabb, 0.: Trypsin induced coordinate alterations in cell shape, cytoskeleton and intrinsic
membrane structure of contact inhibited cells. Exptl. Cell Res. 114, 1 (1978) 12. Revel, J., Hoch, P., Ho, D.: Adhesion of cultured cells to their substratum. Exptl. Cell Res. 84, 207 (1973) 13. Dalen, H., Todd, P. W.: Surface morphology of trypsinised human cells in vitro. Exptl. Cell Res. 66, 353 (1971) 14. Harrison, C. J.: A study of the surface topography of mammalian cells in culture. PhD. Thesis, University of Manchester (1978) 15. Goldman,R. D.: The role of three cytoplasmic fibres in BHK-21 cell motility. I. Microtubules and the effects of colchicine. J. Cell Biol. 51, 752 (1971) 16. Vasiliev, J. M., Gelfand, I. M., Domnina, L. V., Ivanova, 0. Y., Komm, S. G., Olshevskaja, L. V.: Effects of colcemid on the locomotory behaviour of fibroblasts. J. Embryol. Exptl. Morph. 24, 625 (1970) 17. Weisenberg, R. C., Borisy, G. G., Taylor, E. W.: The colchicine binding properties of mammalian brain and its relation to microtubules. Biochemistry 7, 4460 (1968) 18. Owellen, R.J., Owens, A. H., Jr., Donigian, C.: The binding of vincristine, vinblastine and colchicine to tubulin. Biochem. Biophys. Res. Commun. 47, 685 (1972) 19. Nicolson, G. L.: Transmembrane control of the receptors on normal and tumour cells. Biochem. Biophys. Acta 457, 57 (1976) 20. Tobey, R. A., Crissman, H. A.: Preparation of large quantities of synchronised mammalian cells in late G1 in the pre-DNA replication phase of the cell cycle. Exptl. Cell Res. 75, 460 (1972) 21. Erickson, C. A., Trinkaus, J. P.: Microvilli and blebs as sources of reserve surface membrane during cell spreading. Exptl. Cell Res. 99, 375 (1976)
Received April 1979/Accepted May 1979