ULTRASTRUCTURAL STUDIES RELATING TO THE SURFACE MORPHOLOGY OF CULTURED CELLS

Transcription

1 J. Cell Set. 6, (1970) 477 Printed in Great Britain ULTRASTRUCTURAL STUDIES RELATING TO THE SURFACE MORPHOLOGY OF CULTURED CELLS R. G. P. PUGH-HUMPHREYS AND W. SINCLAIR Department of Zoology, Electron Microscopy Utut, U.C.W. Aberystwyth, Penglais, Aberystwyth, Wales, U.K. SUMMARY Scanning electron microscopy and a surface-replica technique for transmission electron microscopy have been used to study the ultrastructural features of cultured-cell surfaces The presence of microvilh measuring fim in diameter by up to 5 ftm in length has been noted as a regular feature of Landschutz ascites, ' fibroblastic' HeLa, and canine kidney, cells. The surfaces of chick mesenchyme cells were notably almost devoid of microvilh. The presence of microvilh at the cell surface is discussed briefly. INTRODUCTION The cell surface is considered to play an important part in cell-contact behaviour in vitro. Attempts to study the nature of the cell surface have concentrated mainly on its chemistry rather than its three-dimensional topography. The surface replica technique of Fisher & Cooper (1967), and observations on whole cells in culture using the Stereoscan electron microscope (Boyde, Grainger & James, 1969; Clarke, Salsbury & Rowland, 1968; Carr, Clarke & Salsbury, 1968), have facilitated surveys of cell-surface features. Because studies of cell interaction in vitro are of interest in this laboratory, and because of the possible influence of surface microanatomy on cell contact phenomena, it was decided to examine the surfaces of those cell types currently in use, using both replica and scanning electron microscopy. MATERIALS AND METHODS The cell types investigated were, 'fibroblastic' HeLa cells; canine Madin kidney cells; Landschutz ascites tumour cells (originally obtained from the Chester Beatty Research Institute and passaged through CBJ51 mice); and mesenchyme cells obtained after trypsin treatment of g.day-old chick embryo muscle tissue by the technique of Kemp, Jones, Cunningham & James (1967) The chick mesenchyme cells and HeLa cells were cultured in Eagle's Minimal Essential Medium (MEM) containing 10 % calf serum, the canine kidney cells in HBME (Flow Laboratories) + 10% fructose phosphate +10% foetal bovine serum, for h in Leighton tubes gassed with a 5 % CO,-air mixture and kept at 37 C. The ascites cells were aspirated from the peritoneal cavity of CB/ji mice on the 5th day after inoculation of the tumour; the cells were washed by centnfugation in MEM several times, resuspended in fresh medium and cultured for 3-4 h at 37 C C. 31-2

2 478 R. G. P. Pugh-Humphreys and W. Sinclair The cell cultures were washed in synthetic medium, fixed in 5 % glutaraldehyde for min and rinsed in several changes of cacodylate buffer, then once in 30 % alcohol. They were drained of excess liquid, placed on a brass block precooled in liquid nitrogen, and transferred to an evaporation unit where freeze drying was completed under vacuum. To obtain the surface replicas, freeze-dried cultures were shadowed with gold-palladium at an angle of 45, followed by a carbon coat at Overnight immersion of the shadowed preparations in 10 % KOH followed by careful immersion in 4 % acetic acid caused the replicas to lift clear of the covershps, so that they could be picked up on collodion-coated grids and examined in an AEI EM6B electron microscope A Cambridge Stereoscan electron microscope was used for making observations on freezedned preparations coated with gold-palladium to give them a conducting surface. Light-microscope studies were made on May-Grunwald-Giemsa stained cell cultures. RESULTS Of the two fibroblastic cell species studied, namely the HeLa (Fig. 1) and chick mesenchyme cells (Fig. 2), only the HeLa cells bore numerous surface projections or microvilli (Fig. 3). These microvilli were observed on the upper surface and on the flattened edges of the cells (Fig. 4), and measured /tm in width by up to 4-5 in length. Table 1. An estimation of the number of cells bearing microvilli and the relative quantities of microvilli borne on individual cell surfaces at time of fixation Cell type Landschutz ascites Chick mesenchyme HeLa Canine Madin kidney Morphology adopted in vitro Estimated % of cells observed bearing microvilli Estimate of microvillus coverage upon individual cell surfaces Spheroid Fibroblastic Fibroblastic Epithelial % = 10% > 9 % 9 % Many None few Many Many By comparison, the chick fibroblasts had relatively few microvilli (Fig. 5), and those present were usually confined to the edges of the cells, where they appeared to radiate out over the glass substratum (Fig. 6). Membrane-like flaps were occasionally observed on the upper surfaces of the chick fibroblasts (Fig. 5) and it is possible that these correspond with the surface 'ruffles' observed by Ambrose (1961) and Ingram (1969). Both the HeLa and chick mesenchyme cells displayed a cellular polarity, with a leading edge or 'ruffled membrane' and a 'tail' region. The canine Madin kidney cells, which adopt an epithelial facies, grow in confluent cell sheets in culture (Fig. 7). Most of the cells carried microvilli distributed evenly over their visible surfaces (Fig. 8) with some microvilli appearing to extend from the edge of the cell over the glass substratum (Fig. 9). Those cells not bearing many microvilli presented a rather smooth surface. The ascites cells attach to, but do not spread on, glass and maintain a spheroid

3 Ultrastructure of cell surfaces 479 morphology after many hours in culture. The surfaces of these cells are covered with a profusion of microvilli which gives them a distinctly hairy appearance (Fig. 10). In this respect they resemble other free tumour cells present in malignant effusions (Spriggs & Meek, 1961). Table 1 summarizes the observations made upon individual cells within the populations. For any one cell type the number of cells bearing microvilli varies through the range shown in column 3. There is also a range of variation among the cell types observed regarding the number of microvilli found on individual cell surfaces. An arbitrary score of ' few' or ' many' has been given to indicate the numbers of microvilli found. DISCUSSION A variation in the distribution of microvilli on cultured BHK2i\C 13 fibroblasts has been reported by Follet & O'Neill (1969). The variations reported in this communication may well find a ready explanation in Follett & O'Neill's (1969) suggestion that the presence of microvilli is a reflexion of a particular physiological state of the individuals within a cell population. The observation of Willoch (1967), that a reduction in glucose concentration within the culture medium caused a reduction in the numbers of HeLa cell-surface microvilli, reinforces the suggestion of a connexion between the physiological state of a cell and the presence of microvilli on its surface. However, this idea would have to be tested by, for example, a study of synchronized cell cultures to determine whether the phase of the cell cycle is correlated with the production of microvilli. The presence of microvilli on isolated epithelial and fibroblast cell surfaces was reported by Overman & Eiring (1961). They noted that HeLa cells had numerous microvilli whereas chick fibroblasts had very few, an observation confirmed in these studies. The observations of Fisher & Cooper (1967) of the microvilli on the surfaces of HeLa cells differ somewhat from the present findings- However, judging by their published pictures, Fisher & Cooper used an epithelial type of HeLa cell whereas the kind used in this study adopted a fibroblastic facies. The possible difference in cell type may account for the discrepancy between the two sets of observations. The role of microvilli at the surface of isolated cells is very speculative. Suggestions that they are concerned in the selective attachment of cells (Taylor & Robbins, 1963) and cell aggregation (Lesseps, 1963; Pethica, 1961; Weiss, 1967), have been made. In the latter respect, Weiss (1967) considers that the ability of cells to put out low radius of curvature probes, with dimensions similar to those of the microvilli described in this report, may be an important factor in the initiation of intercellular contacts during cell aggregation. This hypothesis has been subjected to experimental analysis and the results will be reported in due course.

4 480 R. G. P. Pugh-Humphreys and W. Sinclair We are indebted to the Department of Metallurgy, Sheffield University for the opportunity to use their Stereoscan and for the instruction given to one of us (R. G. P. P-H.) in its use. R. G P. P-H. is in tenure of an S.R.C. grant. REFERENCES AMBROSE, E J. (1961). The movements of fibrocytes. Expl Cell Res. (Suppl) 8, BOYDE, A., GRAINGER, F. & JAMES, D. W. (1969). Scanning electron microscopic observations of chick embryo fibroblasts in vitro with particular reference to the movement of cells under others. Z. Zellforsch. mikrosk. Anat. 94, CARR, I., CLARKE, J. A. & SALSBURY, A. J. (1968). The surface structure of mouse peritoneal cells a study with the scanning electron microscope. J. Microscopy 89, 105-m. CLARKE, J. A., SALSBURY, A. J. & ROWLAND, G. F. (1968) Surface ultrastructure of human leucocytes, mouse macrophages and rat liver cells, and of isolated nuclei and nucleoh. Br J. Haemat. 14, FISHER, H. W. & COOPER, T W. (1967). Electron microscope studies of the microvilh of HeLa cells. J. Cell Biol. 34, FOLLETT, E. A. C. & O'NEILL, C. H. (1969). The distribution of microvilh on BHK21IC13 fibroblasts. Expl Cell Res. 55, INGRAM, V. M. (1969). A side view of moving fibroblasts. Nature, Lond. 222, KEMP, R. B., JONES, B. M., CUNNINGHAM, I. & JAMES, M. C. M. (1967). Quantitative investigation on the effect of puromycin on the aggregation of trypsin- and versene-dissociated chick fibroblast cells. J. Cell Set 2, LESSEPS, R. J. (1963). Cell surface projections: their role in the aggregation of embryonic chick cells as revealed by electron microscopy. J. exp. Zool 153, OVERMAN, J R. & EIRING, A. G. (1961). Electron microscope studies of intact epithelial and fibroblast cell surfaces. Proc. Soc. exp. Biol Med. 107, PETHICA, B. A. (1961). The physical chemistry of cell adhesion. Expl Cell Res. (Suppl.) 8, SPRIGGS, A. I. & MEEK, G. A. (1961). Surface specialisations of free tumour cells in effusions. J. Path Bact. 82, TAYLOR, A. C. & ROBBINS, E. (1963). Observations on microextensions from the surface of isolated vertebrate cells. Devi Biol. 7, WEISS, L. (1967). The Cell Periphery, Metastasis, and Other Contact Phenomena. Amsterdam: North-Holland Publishing Co. WILLOCH, M. (1967). Changes in HeLa cell ultrastructure under conditions of reduced glucose supply. Acta path, microbtol. scand. 71, {Received 20 June 1969)

5 Ultrastructwe of cell surfaces Fig. 1. Fibroblastic HeLa cells in culture. May-Grunwald-Giemsa. Fig. 2. Nine-day-old chick embryo mesenchyme cells. May-Grunwald-Giemsa. Fig. 3. Microvilli visible on the surface of a fibroblastic HeLa cell (surface replica), x

6 482 R. G. P. Pugh-Humphreys and W. Sinclair Fig. 4. Microvilh and what appears to be a membrane flap on the flattened leading edge of a HeLa cell (surface replica), x Fig. 5. A surface view of a 9-day-old chick embryo mesenchyme cell showing membranous 'flaps'. The surface appears to be pitted and bears very few microvilh (surface replica), x

7 Ultrastructure of cell surfaces 483 Fig. 6. Microvillus projecting from the side of a cultured chick embryo mesenchyme cell. Note that the adjacent cell surface is free of microvilli (surface replica), x Fig. 7. Canine Madin kidney cells in culture. May-Grunwald-Giemsa. Fig. 8. Surface replica of canine kidney cell showing microvilli distributed fairly evenly over the cell surface. A ridge on the surface appears on the left of the picture, x

8 48 4 R. G. P. Pugh-Humphreys and W. Sinclair Fig. 9. The free edge of a canine kidney cell showing microvilli abutting on to the substratum (surface replica), x Fig. 10. A scanning electron micrograph of 5th day LandschQtz ascites tumour cells showing microvilli present at the cell surface, x approximately.