SURFACE EXTENSIONS ON BHK CELLS GROWN IN MONOLAYERS AND AGAR SUSPENSION

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1 J. Cell Sci. io, (1972) Printed in Great Britain SURFACE EXTENSIONS ON BHK CELLS GROWN IN MONOLAYERS AND AGAR SUSPENSION A. A. TUFFERY* Department of Microbiology, University of Western Australia, School of Medicine Victoria Square, Perth, Western Australia, 6000 SUMMARY Three types of surface extensions found on a subline of BHK cells capable of growing in agar are described. These are: 1. Microvilli - borne on the cell surface, lacking rigid cores, found on trypsinized cells and on cells growing in solid tissue type growth; 2. Microspikes - borne on the cell margins, containing rigid cores, found on spread, attached cells, but not trypsinized cells or cells growing in solid tissues; 3. Retraction fibrils - coreless 'passive' cell extensions drawn out distally as motile cells move across a surface. Microvilli also seem to be associated with micropinocytic vesicles. It is suggested that microvilli on tissue cells in vitro may be homologous with the cell membrane interdigitations found in, for example, gut epithelium, and possibly also with the microvilli of brush borders. Their association with these structures suggests they may play a role in producing a local environment near the cell membrane in which micropinocytosis may function more effectively. INTRODUCTION A prominent feature of mammalian cells cultured in vitro is the possession of fine filamentous surface extensions. These have been figured and described by many writers, and given a variety of names, of which 'microvilli' seems to be most widely used. This term has been applied to the long filaments radiating from the margin of attached, spread cells, and to shorter, surface-borne structures. Judging from the appearance of these structures in published micrographs, especially those illustiating replica and scanning-microscope preparations, it is possible that these cell surface structures may comprise 2 quite different cell organelles. The acquisition of a line of BHK 21 cells which grew in monolayers and agar suspension, and produced both types of structure, offered an opportunity for the investigation of this question. It was hoped to observe the behaviour of these structures in spread, attached cells and in solid, colonial, masses of cells suspended in agar. Possibly one might then be better able to define the nature of these structures. Present address: Department of Biological Sciences, Ewell County Technical College, Reigate Road, Ewell, Surrey, England.

2 124 A. A. Tuffery MATERIALS AND METHODS The BHK21 cells were originally obtained by Dr P. J. Simons from the Western Australia Public Health Service Virology Laboratories. It was found that, when suspended in soft agar (Macphereon & Montagnier, 1964) a high proportion of the cells grew to form large colonies. A number of individual colonies were picked and grown to mass culture as monolayers. Electron microscopy Replica preparations of glutaraldehyde-fixed cells grown on coverslips were made as described by Fisher & Cooper (1967). Sections of cell monolayers were prepared as described elsewhere (A. A. Tuffery & R. S. U. Baker, in preparation). Colonies of cells growing in agar were picked out in small plugs of agar with a pasteur pipette, ejected gently into 6-5 % glutaraldehyde buffer in 0-2 M cacodylate buffer (ph 7-4) and fixed for 1 h or overnight at 4 C. After a very thorough washing with several changes of buffer, these colonies were postfixed in 1 % osmium tetroxide in buffer (3 h at 4 C), stained with 5 % aqueous uranyl acetate (1 h room temperature), dehydrated through ethanol and propylene oxide and embedded in Araldite. Some colonies were fixed with glutaraldehyde and osmium tetroxide containing 3 mg per ml ruthenium red; in this case, the uranyl acetate stage was omitted. Observations were made with a JEM model T6 electron microscope or a Philips EM 300 microscope. RESULTS Monolayers A high proportion of fully spread cells bore surface microextensions as well as marginal structures. In replicas, the surface structures (microvilli) were smooth, parallel sided with bluntly rounded ends, and more or less flattened to the cell surface (Fig. 2). They measured about 1190 x 139 nm (measurements include the Cr). Replicas also demonstrated marginal microextensions which lay close to or along the coverslip surface, and frequently showed evidence of an apparently rigid core which could be seen extending back as a root for some distance into the body of the cell (Fig. 3). The broader extensions often contained multiple cores. These extensions were of variable length from o-6 to 15 /tm (most over 6 /tm) with a width of o-32-i-i4/tm. They did not taper. Between these extensions the cell margin was regularly scalloped, as if the cytoplasmic membrane was drawing back over these structures at the time of fixation. The extensions were fully enclosed by this membrane and frequently they overlay or abutted on similar extensions or processes from adjacent cells, but never were there signs of specialized structure or orientation at these areas of meeting. Some of the marginal microextensions had a 'soft' appearance (i.e. coreless, irregularly bent, and flattened) and showed local swellings (or collapsed empty sacs) at intervals along their length or at their tips. Sometimes ridges or folds were seen which, in section, would be almost indistinguishable from sectioned microvilli. Coverslip-grown cells showed numerous fingerlike, sometimes slightly clubbed microvilli on all cell surfaces. These microvilli intermingled with those of adjacent cells and sometimes lay as close together as in agar-grown cells (i.e nm). Microvilli on the substratum side of cells were often flattened between the cell body and carbon layer and restricted to the peripheral zones

3 Surface extensions on BHK cells 125 of the cell (Fig. 4) but some lay less confined in small local spaces underneath the cell. Often fairly large limbs of adjacent cells or processes overlapped one another, but only marginal microextensions overlay adjacent cells in other areas. Generally, monolayer cells were not very closely packed together, but 'close' and 'tight' junctions were sometimes seen (Figs. 5, 6). 'Membranous bodies' were sometimes found (Figs. 7, 8) arising from the cytoplasmic membrane of some cells. They appeared to be stalked, bubble-like, complex masses of membranous material. Their appearance suggested an origin from microvilli, but they could not be identified in replicas. Sections also demonstrated large numbers of cytoplasmic fibrils which tended to run in bundles or bands and were especially prominent in some cells just beneath the cytoplasmic membrane. Some fibrils could be traced into the microvilli, where they formed loose open bundles or meshworks. Such fibrils, and a few ribosomes, constituted the only recognizable structures found inside these organelles. Much more prominent fibrillar bundles could be found in the marginal microextensions in a few fortunate sections. Agar suspension colonies Sections through whole colonies showed numbers of close-packed polygonal cells, with no intercellular spaces in the colony centre, but irregular spaces towards the edge of the colony. At low magnifications one got an impression of sections taken through parenchyma-like tissue, e.g. liver (Figs. 10, 12, 13). Cells at the edges of the colonies also preserved a polygonal shape, with no tendency to spread or 'flow' out into the agar. Cells ranged from about 2ixio/im to 25x15/4111 and contained large nuclei (15-19 x 6-10 /im) which filled the bulk of the cell although the cytoplasm width was variable. Numerous mitochondria were found, together with variable numbers of empty vesicles, and cytoplasmic fibrils lay scattered throughout many cells in more or less parallel arrays which tended to be particularly prominent just beneath the cytoplasmic membrane. These bands were less sharply delineated than those seen in the monolayer cultures. The only other inclusions seen worthy of remark were 'myelin bodies' which were very intensely stained in cells fixefl overnight with osmium/ruthenium red. Most cells contained quite large numbers, for example, more than 170 were seen in one section of one cell. At first sight, the cell border contacts appeared to be simply abutments of one cell with another, with an occasional infolding, but higher magnifications and especially observation of ruthenium red-stained preparations, showed a very extensive infolding pattern. This pattern was especially marked at the 'corners' where 3 or more cells met. A few simple infoldings were found on the long straight edges of cells, but in the corners, the infoldings became highly interdigitated, and the cell connexions quite impossible to interpret in single sections (Figs ). The cytoplasmic membranes of adjacent cells, both along the straight portions and in the complex folds, lay parallel at a uniform distance apart. In ruthenium red preparations, the stain filled this intercellular cleft in a patchy manner, giving the cell

4 126 A. A. Tuffery borders a ladder-like appearance (Fig. 16). Oblique sections through such zones suggested that stain deposition was in minute patches throughout. Micropinocytic vesicles were numerous in most cells and their distribution at cell boundaries was especially distinct in ruthenium red preparations where their uptake of stain betrayed their connexion with the intercellular cleft (Figs. 14, 15). Few such Fig. 1. Diagram of a BHK cell growing in agar, illustrating the types of links formed with adjacent cells, micropinocytic vesicle distribution, and vesicle formation. Each cytoplasmic membrane is shown as a solid line. Types a and b are less frequently seen, but types h and ; are never seen. All other forms of infolding and interdigitation are seen frequently. Except for type «, these are most often seen at the corners of cells rather than along the straight sides. vesicles occurred along the straight cell edges except in zones where the cleft was locally widened, but large numbers were seen in the regions where adjacent cell membranes interfolded one with another. Both adjacent cells formed pinocytic vesicles but these vesicles were never formed on the thin protrusions of one cell where it was enclosed by the adjacent cell, i.e. pinocytic vesicles were formed only by the 'outside' cell. Some folds or incursions of a cell's own membrane were lined by pinocytic vesicles (Figs. 1, 14). Occasionally one could find a row of vesicles (which were not stained with ruthenium red) arising from the apex of an infolding (Fig. 18) or from a small notch in the cytoplasmic membrane; these were seen at the corners of the cells where the interdigitations were very complex. They gave the impression of having arisen by successive invagination from the cell membrane, each one subsequently moving more deeply into the cell (Figs ). Alternatively, they could represent

5 Surface extensions on BHK cells 127 the remnants of membrane material left when a cell protrusion (microvillus) having folded back against the cell boundary, proceeded to fuse with the body of the cell. The boundaries of cells in the outermost parts of agar colonies where the cells were not quite so tightly packed, bore numerous microvilli, which interdigitated with those of neighbouring cells (cf. Figs. 9 and 13). Where the intercellular spaces became quite wide, longer microvilli were found stretching across the gap to lie close and parallel to the neighbouring cell membrane or microvillus. These microvilli were clearly not randomly oriented, but seemed to be specifically oriented to make contact with the neighbouring cells (Figs. 22, 23). The outermost borders of cells on the rim of the agar colonies were either smooth, moderately endowed with microvilli or (less often) thrown into extensive folds, wrinkles or club-shaped extrusions. DISCUSSION The surface-borne structures found on many cells in tissue culture are clearly of a different nature from the marginal extensions seen in almost all spread (monolayer) cells in culture. The former type I shall refer to as 'microvilli', the latter as 'microspikes '. ' Retraction fibrils' as described by Taylor & Robbins (1963) constitute a third class of cellular surface extension. The term microspikes was first used by Weiss (1962) to refer to the long straight filamentous projections arising from the edge or margin of spread cells and containing an apparently rigid core. As reported by Buckley & Porter (1967), and in this paper, the core consists of 8-nm microfibrils. They are characteristically associated with a scolloped cell margin and are retracted during detachment by trypsinization. 'Retraction fibrils' have not been discussed in this paper, but it is very likely that some of the coreless, bent microextensions seen on some cells were of this type. Taylor & Robbins (1963) distinguish such structures from microspikes on the basis of their passive mode of formation, their lack of any core, their freely mobile nature moving with medium currents when unattached at the distal end and their frequently greater length. The long processes formed during the division of attached cells, e.g. Chang liver cells (Dalen & Scheie, 1969) and mouse embryo cells (Cornell, 1969) would also appear to be retraction fibrils. The term ' microvilli' may perhaps be best reserved for those fingerlike extensions borne on the surface (not the margin) of the cell. Except in the matter of permanence, the microvilli seen on fully spread HeLa cells (Cooper & Fisher, 1968) and the BHK cell subline described here may be considered identical with the microvilli which arise temporarily on fully rounded cells (Follett & O'Neill, 1969; Follett & Goldman, 1970). Table 1 lists the characteristic features of these organelles and contrasts them with microspikes and retraction fibrils. They are particularly characterized by their appearance over the whole cell surface, their lack of rigidity (though a central fibrillar network is often seen in sections) and their association with the micropinocytosis.

6 128 A. A. Tuffery In closely packed monolayer cells, one can see in fortuitous sections lines of what appear to be micropinocytic vesicles arising near the bases of microvilli, and similar rows are more easily demonstrated in the corners of extremely close-packed cells in agar-suspended colonies. These also arise from what might be interpreted as the base of the cleft formed-between a microvillus and its parent cell. BHK cells suspended in agar clearly possessed numerous microvilli, which seemed almost to deliberately stretch or grow across intervening spaces to make contact with neighbouring cells. A role in establishing intercellular contacts has been suggested for microvilli by Trelstad, Hay & Revel (1967). As the intercellular spaces grow smaller, the microvilli interdigitations grow closer and tighter. When all space has been occupied, it is easy Table 1. Characteristics of microvilli, microspikes and retraction fibrils Microvilli Microspikes _? Retraction fibrils Distribution: surface margin of spread cells Presence: on detached trypsinized cells on attached dividing cells on established cell lines Association with cell movement (i.e. distribution on trailing edges) Presence of ' roots' and rigid cores Core structure: dense fibrillar bundles loose fibrillar mesh Cell border: curved or straight frequently scalloped Comparative sizes Width Association with micropinocytosis Presence in solid-tissue type growth Variable Shorter Uniform Longer Variable? Longer Thin to imagine these interdigitating microextensions producing the complex links seen at the corners of close-packed cells. Such complex links are well known in many tissues, e.g. gut epithelium, and it may be remarked that the smallest intercellular spaces among the agar-grown cells bear a marked resemblance to say, the acinar caniculi in pancreas, or the bile caniculi in liver. The interpretation of these links as microvilli trapped between adjacent cells is also supported by the fact that micropinocytic vesicles are found only on the 'outside' of such structures (Fig. 1, p. 126). Micropinocytic vesicles are never seen on free microvilli. Although the microvilli found on intestinal epithelial cells (and others) are not immediately comparable with the microvilli of tissue culture cells (they are more numerous, more regularly arranged, and more permanent than those on most cells in culture) there are some analogies worth noting. Fawcett (1966) describes a sort of loose framework of cytoplasmic fibrils inside such microvilli, which derive from or intermesh with a sub-plasmalemmal fibrillar array, exactly comparable with the situation

7 Surface extensions on BHK cells 129 in the cells described here. And at the bases of these microvilli are micropinocytic vesicles, capable of taking up materials held or trapped in the microvilli interstices. In the BHK cells, it was noted that micropinocytic vesicles were located largely on cell membrane abutting complex links, i.e. opening into the intercellular space where it is highly contorted. Possibly ions or other cell metabolites may be conserved or accummulated here in a situation comparable with that in the brush border of intestinal epithelial cells. Perhaps microspikes are a wholly laboratory artifact, formed by growing, moving, cells on hard surfaces in vitro, but microvilli may well be genuine organelles associated with the uptake of materials by direct absorption (increased cell membrane area) or by micropinocytosis from parts of the immediate local environment confined by microvilli (brush borders, complex links in agar-suspended cell colonies or cells in certain tissues). REFERENCES BUCKLEY, I. K. & PORTER, K. R. (1967). Cytoplasmic fibrils in living cultured cells. A light and electron microscope study. Protoplasma 64, COOPER, T. W. & FISHER, H. W. (1968). Electron microscopic survey of the presence of microvilli on cultured mammalian cells. J. natn. Cancer Inst. 41, CORNELL, R. (1969). In situ observations on the surface projections of mouse embryo cell strains. Expl Cell Res. 57, DALEN, H. & SCHEIE, P. (1969). Microextensions on Chang's liver cells as observed throughout their division cycle. Expl Cell Res. 57, FAWCETT, D. W. (1966). The Cell, Its Organelles and Inclusions. Philadelphia and London: Saunders. FISHER, H. W. & COOPER, T. W. (1967). Electron microscope studies of the microvilli of HeLa cells. J. Cell Biol. 34, FOLLETT, E. A. C. & GOLDMAN, R. D. (1970). The occurrence of microvilli during spreading and growth of BHK21/C13 fibroblasts. Expl Cell Res. 59, FOLLETT, E. A. C. & O'NEILL, C. H. (1969). The distribution of microvilli on BHK21/C13 fibroblasts. Expl Cell Res. 55, MACPHERSON, I. & MONTAGNIER, L. (1964). Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, TAYLOR, A. C. & ROBBINS, E. (1963). Observations on micro-extensions from the surface of isolated vertebrate cells. Devi Biol. 7, TRELSTAD, R. L., HAY, E. D. & REVEL, J. P. (1967). Cell contact during early morphogenesis in the chick embryo. Devi Biol. 16, WEISS, P. (1962). From cell to molecule. In The Molecular Control of Cellular Activity (ed. J. M. Allen), pp New York: McGraw-Hill. (Received 24 June 1971)

8 130 A. A. Tuffery All figures are of BHK cells in culture, fixed with glutaraldehyde and osmium tetroxide, except Figs. 2 and 3 (fixed with glutaraldehyde alone), and Figs. 12 and (fixed in the presence of ruthenium red). Fig. 2. Replica preparation of part of the surface of a fully spread cell, showing numerous surface-borne microvilli. x Fig. 3. Replica preparation of the edge of a cell growing on a coverslip, showing cored microspikes. x Fig. 4. Vertical section through cells growing on a block of Araldite. Peripherally located microvilli may be seen with the lower ones trapped beneath the body of the cell, x Fig. 5. 'Close junctions' between 2 cells, x Fig. 6. 'Tight junction' between 2 cells, x Figs. 7, 8. Examples of complex membranous bodies seen on some cultured cells. They appear to arise from microvilli. Fig. 7, x 12000; Fig. 8, x

9 Surface extensions on BHK cells \ 0-2

10 132 A. A. Tuffery Fig. 9. Section parallel to the coverslip of a monolayer of confluent cells. Compare with the agar-grown cells in Fig. 13. Note how the microvilli show varying degrees of compression between adjacent cells as the space between is occluded, x Fig. 10. Section of cells growing in agar. The intercellular links are similar to those in Fig. 12, but less obvious because there is no ruthenium red present. Numbers of intracellular vesicles of unknown significance may be seen in these cells, x Fig. 11. An example of the complex links found between agar-grown cells. The arrow indicates a microvillus with a microfibrillar cored structure, x Fig. 12. Section of cells growing in agar. The ruthenium red filling the intracellular cleft demonstrates very clearly the complex links found between these cells. Various types can be identified in the diagram Fig. 1 (p. 126). x 2200.

11 Surface extensions on BHK cells

12 i 34 A. A. Tuffery Fig. 13. Cells growing in agar. The complex links in areas of close packing are clearly comparable with the microvilh seen in areas where there are distinct gaps between the cells. Compare with the monolayer cells in Fig. 9. x Fig. 14. Complex links between cells growing in agar. Note their association with micropinocytic vesicles, which also take up the ruthenium red. x Fig. 15. An intracellular link which has been cut tangentially. The uptake of stain by micropinocytic vesicles in communication with the intracellular space at the time of fixation clearly demonstrates their association with the link, x Fig. 16. Note the ladder-like appearance of the ruthenium red stain between closely apposed microvilli. x Fig. 17. Part of the intercellular space. Note the presence of micropinocytic vesicles lining a locally widened space, x Fig. 18. Links between cells growing in agar. The arrow indicates a line of vesicles associated with these complex links, x

13 Surface extensions on BHK cells

14 136 A. A. Tuffery Figs Lines of vesicles associated with complex links. Suggestions of vesicle fusion (or formation) may be seen at the sites indicated by arrows, x Figs. 22, 23. Microvilli on cells growing in agar. They appear to be deliberately extending across the spaces between cells to make contact with distant cells, x