Cell locomotion involves several structural-functional activities: membrane extensibility, microfilament regulation and adhesive interactions. There is evidence for Ca21- requirement in all of these. Our data may clarify the role of Ca2+ in locomotion and adhesion. Morphologic and spreading responses of isolated blastula-late gastrula Rana pipiens germ layer cells to varying molar concentrations of Ca2+: 0-Ca2+, Standard Ca2+ (Barth’s Xsolution), 1·5 × and 2·0 × Std Ca2+ were viewed by S.E.M. after 1 h in culture. Ionic strength and pH were constant. All cells showed quantitative relationships between Ca2+ concentration and surface extensibility, projection formation and presumably adhesion, but with tissue- and stagespecific variations. Cells in Ca2+-free medium fail to adhere (50%), flatten or form surface projections. Cells in media with increasing Ca2+ generally formed more numerous and extensive surface projections, spread and adhered to a greater extent. In some cases there were no quantitative differences in response between 1·5 × and 2·0×standard Ca2+. Cells in suspension for 1 h in standard solution remained spherical, forming no projections. We infer from these results that both Ca2+ and contact with a physical substratum, cell-cell or cellglass are required for mobilization of the various systems involved in locomotion and adhesion. In addition, components of these systems are quantitatively activated by increased availability of Ca2+.

Cell locomotion requires participation of several structural-functional cell activities. These include (1) plasmalemma extensibility, (2) organization and regulation of a submembrane microfilament and microtubule apparatus to form locomotor organelles, (3) cell periphery adhesive interactions with a substratum and (4) energy availability. There is considerable evidence that Ca2+ is involved in the operation of all these activities (Weiss, 1970; Durham, 1974; Spooner, 1975). Experiments reported here provide data which may clarify some aspects of Ca2+ participation in cell movement and adhesion.

Cells move most frequently by forming projections of varied kinds which attach to a substratum. This has been seen in vivo in many embryos (Gustafson, 1964; Brick, Schaeffer, Schaeffer & Gennaro, 1974; Spiegelman & Bennett, 1974; Nakatsuji, 1976). Cell microprojections are also involved in the initial process of intercellular association and in making focal adhesions in cell attachment to a substratum (Abercrombie, Heaysman & Pegrum, 1971; Izzard & Lochner, 1976; LeBlanc & Brick, 1981).

Microprojection formation is associated with microfilaments as observed in many species (Spiegelman & Bennett, 1974; Nakatsuji, 1976). Concentrations of cortical microfilaments are seen at sites of close apposition to the substratum and at intercellular contacts (Abercrombie et al., 1971; Heaysman & Pegrum, 1973). The relationships and deployment of microfilaments suggest that microfilament Ca2+-dependent contractile activity is involved in formation and withdrawal of cell protrusions and probably related to cell extensibility and, therefore, locomotion.

Cell periphery adhesive interactions are also related to Ca2+ availability. Ca2+-free medium aids embryo disaggregation (Jones & Elsdale, 1963), and reaggregation of amphibian blastula and gastrula cells can occur by restoration of Ca2+ (Steinberg, 1962; Jones & Elsdale, 1963). Weiss, 1960, has shown that Ca2+ is essential for attachment of cells to glass and Taylor, 1961, showed that Ca2+-free medium inhibits cell spreading. Curtis, 1962, suggested that Ca2+ decreases cell surface charge thus promoting adhesion. Schaeffer, 1972, showed that Ca2+ reduced electrokinetic mobility of various amphibian embryo cells. We propose that Ca2+ has other, indirect roles in cell adhesion by virtue of the suggested relationship of Ca2+ to cell projections and formation of cell-cell contacts commonly achieved via cell projections.

Two categories of experiments are reported. In the first series Ca2+ concentrations were varied for germ-layer cells of amphibian blastulae and gastrulae. Our objectives were to determine (1) the role of Ca2+ in cell extension over the substratum, (2) the Ca2+ requirement for initial adhesion and/or subsequent projection formation in cell spreading and (3) if varying Ca2+ concentration would alter the form and number of projections extended onto the substratum. LeBlanc & Brick (1981) have demonstrated stage and presumptive (Pr) tissuespecific variations in spreading and adhesive behaviour of Rana pipiens blastula and gastrula cells. We have here used the same array of cells in order to evaluate the role of Ca2+ in the various cell behaviours.

The second series of experiments evaluated the substrate-projection relationship. Inasmuch as spreading cells invariably adhere and conform to their substrata, the influence of such direct surface contacts on cell form must be considerable (Elsdale & Bard, 1972). We considered two possibilities; (1) the substratum might bring about a redistribution of projections already present on the surface of a cell settling on the substratum, or (2) that a substratum might be required for actual projection formation. To test these hypotheses, cells from one Pr tissue type, at one stage were maintained in suspension under the same environmental conditions as spreading cells (Le Blanc & Brick, 1981), the only difference was lack of a substratum.

Induced ovulation and fertilization of Rana pipiens were according to Rugh (1962). Embryos were maintained at 13°C in spring water. Blastula, early-midlate gastrula stages were used (St 9–11 1/2, Shumway, 1940).

Embryonic regions utilized were: (1) Blastula (St 9) Pr head endoderm (Pr dorsal lip); Pr notochord; Pr inner neural ectoderm; Pr inner epidermal ectoderm. (2)Early gastrula (St 10) Pr head endoderm (dorsal lip); Pr notochord; Pr inner neural ectoderm; Pr inner epidermal ectoderm. (3) Mid-gastrula (St 11) Pr head endoderm (leading edge of the invaginating fold); Pr notochord (dorsal lip); Pr inner neural ectoderm; Pr inner epidermal ectoderm. (4) Late gastrula (St 11 1 /2) Pr head endoderm (leading edge of the invaginating fold); Pr notochord (chordamesoderm midway between anterior and posterior ends); Pr inner neural ectoderm; Pr inner epidermal ectoderm.

Methods for dissection, disaggregation and culturing have been previously reported (LeBlanc & Brick, 1981). In the present experiments, all cells spread for 60 min on glass under sterile conditions at 23–25°C, the time required for marked spreading to occur.

Modifications of the standard solution, Barth’s X solution, were used for the spreading studies (Barth & Barth, 1959), pH 7·5, ionic strength 0·09797. Barth’s solution is prepared in three parts, A, B, and C; parts B and C consist of buffers. We, therefore, varied only part A in subsequent test solutions. Ionic strength and pH were kept constant. Only part A with varied Ca2+ concentrations is listed.

When changes in Ca2+ concentration in solutions 1–3, or Ca2+ and Mg2+ in solution 4 were made, ionic strength was maintained by adjusting the concentration of NaCl. Solutions 1–4 were constructed to view the effect of Ca2+ on cell spreading behaviour. Mg2+ was not adjusted in solutions 1–3 because it is a cofactor in membrane enzyme reactions and because it is a divalent cation associated with cell adhesiveness. Any membrane changes observed should be attributable specifically to Ca2+ and not to a change in concentration of polyvalent cations in general. NaCl was chosen to be adjusted because it is readily diffusible. The largest change in NaCl molarity was in solution 4 where an additional 0·0047 mole was added. In solutions 1–3, the largest change in NaCl was 0·0022 mole.

Osmolality of the incubation media was measured on a Fiske osmatic automatic osmometer, model 130; the readings were as follows: Barth’s X solution = 171 milliosmoles; solution 1 (Ca2+-free) = 174 milliosmoles; solution 2 (1·5 × molarity of Ca2+) = 171 milliosmoles; solution 3 (2 × molarity of Ca2+) = 167 milliosmoles; solution 4 (Ca2+-Mg2+-free) = 177 milliosmoles.

Preparation of cells on coverslips for S.E.M. has been reported previously (LeBlanc & Brick, 1981). Cells were examined in an AMR 1000 Scanning Electron Microscope at 20 kV.

For each Pr cell type at each stage and in the various media, at least two cultures were made, and in most cases, more than two. In each culture, cells from all areas on the substratum were observed, the number of examined cells ranging from 20–60. Description of a Pr cell type at a particular stage and in a particular medium will be characteristic of typical surface behaviour for those cells, unless otherwise noted.

Cells for the suspension study were prepared as for the cell spreading observations. These cells, in Barth’s X medium, were drawn up into a 0-575 mm diameter polyethylene tubing fitted to a microinjector. The polyethylene tubing was mounted on the base of a dissecting microscope so cells could be observed during the 30 or 60 min suspension. The cells were manually manipulated back and forth for 30 to 60 min, during which they remained positioned in the slug of moving medium and were not observed to form contacts with each other or with the tubing. Cells were fixed immediately at the end of 30 min or 1 h, placed on a flotronic membrane and dehydrated as previously reported (LeBlanc & Brick, 1981) and examined by S.E.M.

(1) Attachment and spreading of cells in media lacking (7) Ca2+ and (2) Ca2+ and Mg2+

All Pr tissues at all stages studied attached to the substratum without forming extensions or spreading (Figs. 1-6). Generally, about half of the cells on a coverslip adhered. This was determined by adding fixative to the coverslip while observing the cells with a dissecting microscope. The unattached surfaces are virtually devoid of projections, even if these were usually present on the freshly disaggregated cells, and surface infolding and delineation of underlying cortical structures are very evident. The morphologic features displayed by each cell type at each stage are uniform. There are no subpopulations as was the case in several of the cell types in Standard Barth’s X solution (LeBlanc & Brick, 1981) and in media with increased calcium.

(2) Attachment and spreading of cells in media with 1·5 × and 2·0 × molarity of Ca2+

With the exception of a few isolated cells, most cells adhere to the glass in these media. All observations are summarized in Table 1. For ready comparison, the morphologic behaviour of each cell type at each stage in Standard Barth’s X solution (LeBlanc & Brick, 1981) is included in the table.

(A) Pr inner neural ectoderm

At most stages, the major process of extension is the lamellipodium. Late blastula cells adhere and spread by forming lamellipodia which often extended from the entire periphery (Fig. 7). In media with 2 x Ca2+ some cells assume an elongate U-shaped configuration with a broad lamellipodium extending to the glass from one peripheral region (Fig. 8). Early gastrula cells spread via broad membranous lamellipodia which often projected filopodia to the glass similar to mid-gastrula cells in which the lamellipodia radiated filopodia or microspikes (Fig. 9). Some mid-gastrula cells, similar to late gastrula cells, projected stem-like protrusions to the glass. In 2× Ca2+ a number of mid-gastrula cells exhibit branching, relatively broad, ‘rope-like’ extensions to the glass and to surfaces of neighbouring cells across an intervening space (Fig. 10).

(B) Pr inner epidermal ectoderm

These cells spread and adhered via membranous lamellipodia at all stages (Figs. 11-16). Late blastula cells in 2 × Ca2+ (Fig. 11) tend to have broader lamellipodia than when in 1·5 ×Ca2+. Figure 14 shows an early gastrula cell projecting a relatively broad lamellipodium onto the upper surface of an adjacent cell. Note the ruffling of portions of the periphery. These cells can also project long filopodia across the upper surfaces of cells which they are contacting. Mid-gastrula cells often exhibit an elongate morphology and tend to be markedly flattened against the glass. In 2 × Ca2+ some cells produced membranous extensions with a tortuous folding and component regions resembling a lobopodium (Fig. 15). Projections to the substratum rarely developed from the entire cell periphery. Most late gastrula cells bear filopodia at the periphery of their lamellipodia (Fig. 16). There is also extensive spreading of cells onto surfaces of neighbouring cells.

(C) Pr Head endoderm

Late blastula cells in 1·5 ×Ca2+ produce short stemlike adhesions, while in 2 × Ca2+ they exhibit short stems, but with an encircling membranous extension with peripheral filopodia radiating from the stem. Early gastrula cells of the dorsal lip region in media with enhanced Ca2+ extended membranous protrusions to the glass (Fig. 17) which may be relatively flat and smooth, or undulating with filopodia extending from their periphery (Fig. 17). Cells of the leading edge of the invaginating fold (mid-gastrula) adhere to the glass via stem projections in these media. Cells either exhibit a very elongate often convoluted stem (Fig. 18), or a broader, shorter bulbous stem (Fig. 19), the latter more common in medium with 2 × Ca2+ and the former more common in medium with 1·5 ×Ca2+. Late gastrula cells of the leading edge of the invaginating fold exhibit long, stem-like adhesions in medium with 1·5 × Ca2+ and broader stem adhesions with extending membranous protrusions in medium with 2 × Ca2+. Cells in 1·5 × Ca2+, with long convoluted projections were seen adhering to the glass and also to the surface of other cells (Fig. 20).

(D) Pr notochord

These cells, at late blastula project filopodia to the glass in medium with 1·5 × Ca2+, but in medium with 2 × Ca2+, although filopodia are produced there are also points on the cell periphery with membranous projections. Early gastrula cells produce filopodia in both media and unattached surfaces have numerous filopodia and bulbous microvilli-like projections (Figs. 21, 22). The behaviour of these cells changes at mid- and late-gastrula. At mid-gastrula, cells from the dorsal lip in both media produce broad stem adhesions and lamellipodia, the latter are more common in medium with 2 x Ca2+ and in this concentration, long, finger-like protrusions from the unattached surfaces are present, Fig. 23. Cells taken from the chordamesoderm mid-way along the anterior-posterior axis at late gastrula in l·5×Ca2+, form broad stem-adhesions with peripheral filopodia, while these cells in 2 ×Ca2+ form broad lamellipodia with peripheral radiating filopodia (Fig. 24).

(3) Suspension of dissociated Pr inner neural ectoderm cells, late gastrula, for 30 to 60 min

These experiments were done to determine whether surface projections would be produced in the absence of contact with a substratum, either living or non-living. Pr late gastrula inner neural ectoderm cells after 30 min or 1 h of suspension in Barth’s X solution were spherical, with relatively smooth surfaces (Fig. 25).

There are three noteworthy aspects of the data which clarify and provide additional insight into the relationship of Ca2+ to plasma membrane extensibility, surface projection formation, cell adhesion and spreading, at least for amphibian embryo cells during gastrulation. These are (1) failure in Ca2+-free medium of cells to adhere, flatten and form surface projections; (2) the enhanced ability of these cells in media with additional Ca2+, compared to these cells in standard medium (LeBlanc & Brick, 1981), to form more numerous, more extensive surface projections, increased spreading and presumably increased adhesiveness; and (3) the requirement for a solid substratum, even in the presence of Ca2+, for plasma membrane extensibility and surface projection formation.

There is a quantitative relationship between Ca2+ availability and cell surface morphologic expression. While there were some variations in the specific features of surface extensibility and projection formation among the various cell types in media with Ca2+ and within each cell type in the two media with increased calcium and the standard medium (LeBlanc & Brick, 1981), all cells responded quantitatively with respect to Ca2+ concentration. This is clearly evident in the absence of spreading and projection formation in all cells in the absence of Ca2+ and the enhanced response in these respects by all cells in the several Ca2+ concentrations. Although some cell types, i.e. early and late gastrula Pr inner neural ectoderm, early and late gastrula Pr inner epidermal ectoderm, early gastrula Pr head endoderm and early gastrula Pr notochord cells responded similarly in 1·5 × and 2×Ca2+, others nevertheless, demonstrated enhanced extensibility and projection formation in 2 × Ca2+ compared to 1·5×Ca2+, i.e. blastula and mid-gastrula Pr inner neural ectoderm, blastula and mid-gastrula Pr inner epidermal ectoderm, blastula, mid- and late-gastrula Pr head endoderm and blastula, mid- and late-gastrula Pr notochord cells.

We have shown that external Ca2+ is not essential for initial adhesion of amphibian embryo cells to glass. However, half the cells did not adhere in contrast to cells in standard medium (LeBlanc & Brick, 1981) or in the media with increased Ca2+ in which almost all cells adhered. Ca2+ or Mg2+ effect on initial adhesion of cells cannot be excluded since intracellular sources of these ions may provide low, but sufficient concentrations. In both ion deficient media cell surface morphologies were similar and the cells remained spherical suggestive of a low degree of adhesiveness. These coinciding observations suggest that these effects may be primarily due to absence of Ca2+.

The importance of Ca2+ for cell-cell and cell-substratum adhesion has long been recognized (Feldman, 1955; Steinberg, 1962; Jones & Elsdale, 1963). Steinberg (1962) demonstrated that Ca2+ binds to embryonic cell surfaces. Tissue culture studies indicate that Ca2+ is essential for attachment of cells to glass (Weiss, 1960). Taylor (1961), however, did not find Ca2+ essential forcell attachment to glass, but in media containing serum, omission of Ca2+ resulted in spreading inhibition.

All cells are probably negatively charged at physiological pH with many cation-binding groups. Curtis (1962) postulated that Ca2+ might decrease negative surface charge of cells, and thus by decreasing repulsive forces allow cells to come into close approximation where Van der Waals-London forces might promote adhesion. Ca2+ significantly reduces surface charge density of various amphibian gastrula cells, presumably thereby promoting adhesion (Schaeffer, 1972).

Cell spreading may be dependent on sequential formation of new adherent plaques. Only focal points of adhesion may be produced in cell substratum attachment and cell microprojections appear involved in early intercellular association (Abercrombie et al., 1971; Izzard & Lochner, 1976). Abercrombie et al. (1971) and Heaysman & Pegrum (1973) demonstrated concentrated areas of microfilaments in cortical cytoplasm beneath adhesive plaques. Microfilaments are also located beneath cell-cell contacts (Heaysman & Pegrum, 1973); close appositions with concentrations of subplasmalemma microfilaments being formed within 20 sec of the first visible contact. This points to extremely rapid mobilization of microfilament assembly. In fibroblasts, the entire lamella region of spreading cells is filled with a meshwork of microfilaments which appear to insert on the cell membrane inner surface (Abercrombie et al., 1971; Spooner, Yamada & Wessells, 1971; Di Pasquale, 1975). Spooner et al. (1971), using Cytochalasin B, concluded that this network was indispensable for locomotion, particularly in the extension phase (Luduena & Wessells, 1973). It, therefore, seems a likely hypothesis that contractile activity of these microfilaments may be involved in protrusion formation and their withdrawal. There is evidence that such cytoplasmic microfilaments may be f-actin, and therefore, contractile (Pollard, 1972; Spooner, Ash, Wrenn, Frater & Wessells, 1973), and apparently insert on the plasma-membrane (Pollard & Korn, 1973). Spooner (1975) suggests microfilaments might be regulated by uptake or release of Ca2+.

The present study indicates that Ca2+ levels affect surface morphology by being related to protrusion formation. Microfilaments may be involved in cell protrusion formation and the activity of microfilaments in turn regulated by Ca2+. Evidence for the presence of microfilaments in protrusion is extensive (Baker 1965; Betchaku & Trinkaus, 1974; Di Pasquale, 1975; Nakatsuji, 1976).

Ben-Shaul, Ophir, Cohen & Moscona (1977), from freeze-fracture studies on chick embryonic retinal cells, observed pits frequently arranged as a rim at bases of blebs or lobopodia. They suggest these might represent anchoring sites for contractile structures, such as microfilaments and speculated that contraction of such a system with an accompanying cytoplasmic flow might result in projection of small blebs or lobopodia.

There is evidence which suggests that cell membranes have Ca2+ pumping activity that may be analogous to that of sarcoplasmic reticulum (Perdue, 1971; Hurwitz, Fitzpatrick, Debbas & Landon, 1973). Letourneau & Wessells (1974) have speculated that Ca2+ pumping may be a component of cell locomotion.

Huxley (1973) noted a number of motile cells, which contained an actin-like protein frequently identified with filaments, and that many of the same cells contained myosin-like proteins. He suggested that the actin filament-myosin head assembly may be a basic motile mechanism. Durham (1974) suggested that all nonmuscle movements, if they involve actin and myosin, are controlled by Ca2+ flows across the membranes, which, in turn, are determined by chemical and electrical processes at those membranes.

It appears, therefore, that a submembranal contractile system may be involved in cell extensibility and that it may be regulated by Ca2+. Without Ca2+ in the medium, in vitro cells in this study were not able to form protrusions. In addition, even if disaggregated cells of a Pr tissue type at a particular stage initially exhibited surface projections (LeBlanc & Brick, 1981) these cells in Ca2+-free medium were almost completely devoid of projections. Ca2+-free and Ca2+-Mg2+-free media could not maintain surface projections. It would appear that a certain extracellular level of Ca2+ may be required for maintaining the structure of a surface projection.

Increases in Ca2+ concentrations used in experimental media are well within the physiological range of tolerance determined by Barth & Barth (1974) for amphibian embryo cells. In media with l·5×Ca2+ and 2·0×Ca2+ of standard molarity the various Pr cells at the four stages, exhibited alterations in forms of protrusions extended onto the surrounding glass; cellular projections were visibly different from those formed in Barth’s X solution (LeBlanc & Brick, 1981). In some cases there were obvious form differences in the cell extensions between cells incubated in medium with 1·5×Ca2+ and in medium with 2·0 × Ca2+. However, in other instances there were no detectable differences, although there was always a change in spreading behaviour from that seen in standard medium. There may well exist a maximum extracellular Ca2+ concentration, dependent to a degree on Pr tissue type and stage, above which there would be no further structural alterations in protrusions. In addition to changes in protrusion form, in media with increased molar concentration of Ca2+, there was also exhibited, in some instances, a change in distribution of projections onto the substratum. Generally, limited areas of the periphery became dominant regions of extension rather than the entire periphery. Alterations in overall cellular morphology were also observed in certain tissue types at a particular stage (e.g. mid-gastrula Pr notochord cells) in media with increased Ca2+.

Cells from all Pr tissue types adhered in approximately the same frequency as in Barth’s X solution. This is in direct contrast to the frequency of adhesion in media deficient in Ca2+. Both Pr neural and epidermal ectoderm, at each stage, in solutions with increased Ca2+, generally exhibited membranous, lamellipodial extensions rather than filopodia. Lamellipodia in other cells exhibit a microfilamentous network (Spooner et al., 1971; Luduena & Wessells, 1973; Di Pasquale, 1975). This network is extensive and may involve more active cellular mobilization than microfilaments in filopodia, with a higher Ca2+ dependency. A second possibility, which is suggested by the results of longer incubation of the two Pr ectodermal tissues (LeBlanc & Brick, in preparation), where after 1 h filopodia were observed, and after 5 h lamellipodia, is that the spreading process may have been accelerated by the presence of increased Ca2+, perhaps by a more rapid mobilization of underlying cortical structures, such as microfilaments. Filopodia may be extended in greater numbers and undergo fusion into a membranous structure during the 1 h period. Filopodia which are coalescing and with intervening cytoplasmic webbing are not unusual. In addition, ectodermal cells, in increased Ca2+, show more flattening and more intercellular contacts.

Pr head endoderm and Pr notochord cells also exhibit alterations in protrusions related to extracellular Ca2+ levels. These tend to be numerically increased or thicker filopodia, extensive stem adhesions and lamellipodia. These cells in increased Ca2+ also can exhibit highly convoluted stem or fingerlike regions which may be markedly elongated. This morphological alteration may require extensive membrane and underlyingcortical structure rearrangements. These two Pr tissues in increased Ca2+ also exhibit a higher incidence of intercellular contact.

Since spreading cells invariably adhere and conform to their substrate, the influence of such direct surface contacts on cell form must be considerable. North (1970) suggested that phagocytosis is basically the same as the spreading of a cell on a surface; the cell on a flat surface is, in effect, attempting to phagocytose a sphere of infinite diameter. The membrane is responding to contact with the external structure by the adjustment of its adhesiveness and possibly the tension in an actomyosin network by utilizing a mechanism which may involve Ca2+ (Durham 1974). Wolpert & Gingell (1968) argued that inducers of endocytosis bring about a response by direct electrical effect on the membrane potential difference, and Gingell (1970) showed how a reduction in membrane potential would lead to entry of Ca2+ and a resulting contractile response. Contact of the cell membrane with a substratum may result in membrane potential and conformational changes which result in an entry of Ca2+.

We theorized that a substratum, living or non-living, would influence the initial mechanisms for protrusion of cell projections. We considered two possibilities. First, that the surface of cells suspended in Barth’s X solution for 30 min or 1 h would develop an even distribution of cellular projections, such as filopodia. If this were the case, it was reasoned that the concentrated areas of projections seen on the surface adjacent to the glass (LeBlanc & Brick, 1981) were the result of redistribution of projections in response to contact with a substratum. The second alternative was that after 30 min or 1 h of suspension, during which the cells were allowed no lasting contact with a substratum, living or non-living, cells would be devoid of projections. They might, however, have a few surface blebs. Small blebs have the appearance of ‘empty’ vesicles emerging from the cell surface and may be the result of changes in membrane fluidity only (Ben-Shaul et al., 1977), and, therefore, possibly do not require the mobilization of such cellular structures as microfilaments.

Our observations fit the second possibility; cell surfaces of Pr inner neural ectoderm cells were relatively smooth and devoid of projections. Areas of slight folding and elevations observed are in no way comparable to the blebbing seen in freshly disaggregated cells (LeBlanc & Brick, 1981). It may be, following removal of contact with other cells in disaggregation, that cell membranes experience an immediate fluidity change in Ca2+-Mg2+-free disaggregation medium. Whereas after 30 min or 1 h in medium with both Ca2+ and Mg2+ and a reduced pH (7·5) this is no longer evident. Holtfreter (1943) observed that a pH of 9·6, or more, resulted in the entire surface of isolated amphibian embryo cells breaking forth in a number of hyaline blisters which resembled rapidly protruding and retracting ‘bubbles’. In the present study, disaggregation medium pH was 8·9. A substratum, living or non-living, appears required for formation of protrusions, as well as a medium which, from our observations, must contain Ca2+.

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