Electrokinetic properties of dissociated chick embryo cells. I. Ph-surface charge relationships and the effect of calcium ions

Electrokinetic Properties of Dissociated Chick Embryo Cells I. PH-SURFACE CHARGE RELATIONSHIPS AND THE

EFFECT OF CALCIUM IONS

MICHAEL COLLINS l Department of Biology, The Johns Hopkins University, Baltimore, Maryland

ABSTRACT Since the electrokinetic properties of a particle depend entirely upon the particle's surface, microscopic electrophoresis of dissociated cells can yield information about the cellular surfaces. Dissociated heart ventricle and liver cells from five-day chick embryos as well as neural retinal cells from seven-day embryos have been examined electrophoretically over a wide pH range and the pH-net surface charge density relation for each type of cell has been determined. Under the conditions selected the cells show differences with respect to these relations which appear to reflect differences in the molecular composition of the cells' surfaces.

Since intercellular adhesion may occur through bivalent cation bridges with calcium being the cation most likely to be involved, the suppression of surface charge density by means of calcium ions at physiological concentration has been determined at physiological pH for heart ventricle, liver, neural retinal, and back epidermal cells. The degree of charge suppression, which varies among cells of different types, can be correlated with the apparent cellular adhesiveness as judged from sorting out behavior in some but not all cases.

Dye exclusion, the ability of cells removed from the eIectrophoresis apparatus to reaggregate, and the reversibility of pH effects and calcium effects have been used to try to detect damage to the cells caused by experimental conditions. The results of these tests have seemed satisfactory.

A severe impediment to understanding the varied forms of animals has been ignorance of the means by which these complex forms are generated from seem- ingly simple masses of cells. To correct this ignorance of the physical bases and the mechanics of morphogenesis, experi- menters have shown that considerable mor- phogenetic capabilities reside within individual tissue masses and even within the individual cells. Embryonic cells can be dissociated and cells of dissimilar types mixed together. Such cells can sort out one type from another. They establish specific mutual relations like those which are established when the same combinations of solid tissues confront each other in vitro or in the organism (Holtfreter, '39; '44; Steinberg, '62c; Weiss and Taylor, '60).

The ability of cells to sort out apparently depends in large measure upon their motility and their adhesiveness to each other. While other means of driving the sorting out process have been suggested,

J. ExP. ZOOL., 163: 23-38.

(Curtis, '61; Townes and Holtfreter, '55) , none has been supported by compelling evidence and most can be ruled out on the basis of work by Steinberg ('62b, e) and by Trinkaus and Lentz ('64). Consequently, the most promising view is that differences in adhesiveness between cells, com- bined with the motility of those cells, drive the sorting out process. Cellular motility is well known and is implicit in sorting out experiments. Marking experiments would be required to prove that cells do move about within aggregates.

Adhesive differences between cells, and the physical bases of adhesion, have not been explained. The literature contains a number of proposals, but in every case the supporting evidence lacks the weight of proof (Curtis, '60, '64; Loeb, '22; Moscana, '60; L. Weiss, '62; P. Weiss, '41, '47). One possibility (Coman, '54; Steinberg, '58), argued most cogently by Pethica ('61), is that cells are bound together through biva-

1 Present address: Department of Zoology, The University of Texas, Austin, Texas.

23

24 MICHAEL COLLINS

lent cation bridges. Such bridges would exist by virtue of the ability of a bivalent ion, such as calcium, to complex simul- taneously with two anionic groups form- ing a salt of slight solubility. If these two anionic groups were on the surface of two different cells, complex formation would yield a bivalent cation bridge between the two cells. Such bridges would tend to hold the cells together. The number of such bridges per unit surface-contact area would determine how strongly the two cells adhere.

If this explanation were correct, one wOu1d expect that the more strongly adhesive of two types of cell would bind more calcium at the cellular surface and might have a higher negative surface charge density. To explore that possibility, one must determine (1) adhesiveness and (2) charge density and calcium binding capacity for cells of several types. The best information on relative cellular adhesiveness presently available comes from the work of Steinberg ('63a, b, c). That author has ordered a series of embryonic tissues on the basis of sorting out behavior. When cells of any type in this series are dissociated and mixed in culture with cells of any other given type, they will consistently sort out internally or externally. For example, when dissociated heart and liver cells are allowed to aggregate together, the result will be mixed aggregates which can attain the following specific structure. The liver tissue forms a confluent external phase with one or more masses of heart tissue embedded within the liver. At the extremes of the series are cells from the basal ,layer of the epidermis which sort out internally with respect to any other member of the series and liver cells which sort out externally to any other member. This hierarchy probably reflects quantitative adhesive differences among cells, with cells of a more adhesive type, always sorting out internally to those of a less adhesive type.

Cells from that series have been examined electrophoretically. Their mobilities and net surface charge densities have been determined and the effects of calcium ion on those properties have been measured. This was done in order to determine to

what extent cellular surface properties would correlate with sorting out behavior.

MATERIALS AND METHODS

Electrophoresis apparatus The microscopic electrophoresis appara-

tus used in these experiments is essentially a copy of one designed by Bangham and others specifically for measuring mobilities of cells (Bangham et al., '58). The electrophoresis tube is simple and has no end chambers containing strong electrolyte solutions which could either damage the cells being studied or alter the suspending medium. The apparatus used is illustrated in figure 1.

The use of this apparatus is simple. It is washed in situ with dichromate clean- ing solution and rinsed with distilled water and then with the suspending liquid. The tube is filled with up to 5 ml of cell suspension and brought up to volume with suspending liquid. The electrodes are in- serted in the end sockets, forcing out excess liquid. The microscope is focused on the stationary layer where net water flow due to endosmosis is zero. The electrodes are connected to the power supply and the.current is turned on.

The eyepiece of the microscope contains an ocular micrometer to measure the distance covered by a cell in the electric field. The cell is timed with a stopwatch and the electrophoretic mobility is later calculated from the field strength, time, and distance. By reversing the direction of current one can observe a cell going first left and then right. This eliminates the effect of polari- zation of the electrodes due to a sustained current in one direction.

Preparation of human erythrocytes Erythrocytes were obtained fresh by

finger puncture before each mobility determination. A cubic millimeter of blood diluted directly into 15 ml of M/15 phosphate buffer, pH 7.4, showed the same mobility as washed erythroctyes and so blood was simply drawn up in a blood dilu- tion pipette and diluted 1500-fold in a beaker. The cell suspensions were poured directly into the electrophoresis tube and the mobility determinations were made as described above.

EMBRYONIC CELL SURFACE PROPERTIES 25

Fig. 1 Diagrammatic drawing of the microscopic electrophoresis apparatus used for this work.

Preparation of chick cells for electrcrphoresis

Cells from the livers and hearts of White Leghorn chick embryos incubated for five days, from eight-day back epidermis, and from seven-day neural retinas were used.

All tissues were dissected out in cold Tyrode’s solution adjusted to physiological pH by aeration with a 5% C02-95% air mixture. Eight-day back epidermis was removed from the subjacent dermis after a three-hour treatment at 5°C with the crude trypsin solution described below (see Rawles, ’63) .

The tissues were divided into small pieces by knives made from steel needles or by passage through a small bore glass pipette. After this initial fragmentation, the tissues were prepared for final dissociation by soaking in appropriate solutions, described below. Liver tissue was incubated at 37°C for 30 minutes in a crude trypsin solution which was 3% trypsin 1:250 (Difco) and 1% pancreatin 4 X USP (Nutritional Biochemicals) in calcium- and magnesium-free Tyrode’s solution. In its preparation this mixture stood on ice about 30 minutes with stirring; this

was followed by 20 minutes of centrifuga- tion at 1090 X g, which left a clear super- natant solution which was adjusted to pH 7.6 and stored frozen until use. Neural retinal and heart ventricle tissue were incubated for 20 minutes in identical crude trypsin solution. Neural retinal tissue was also dissociated by soaking for 30 minutes in calcium- and magnesium-free Tyrode’s solution adjusted to pH 8.4; this was done at room temperature. Epidermis was kept in calcium- and magnesium-free Tyrode’s at pH 10.0 for about five minutes.

After the tissues had been appropriately soaked, they were dissociated by repeated pipetting with a fine glass pipette followed by shearing according to the method of Auerbach and Grobstein (’58). The neural retinal cells, dissociated in larger quantities, were sheared in centrifuge tubes agi- tated by a Cyclomixer (Clay-Adams, Inc.). Following the final dissociation step, cells were washed and resuspended in a solution which was in most cases 0.145 M sodium chloride plus 3 X M sodium bicarbonate with pH adjusted with 0.145 M hydrochloric acid and 0.145 M sodium hydroxide. This h a 1 suspension was made

26 MICHAEL COLLINS

in a hypodermic syringe from which it was introduced into the electrophoresis apparatus.

Solutions used as suspension media for mobility determinations

The major ionic components in most solutions were sodium and chloride. The ionic strength was 0.145 in all cases except for some mobility determinations done with erythrocytes. Sodium bicarbonate was present in concentration of 3 X M; jts contribution to the ionic strength was negligible at pH values of ten or less.

In solutions containing calcium chloride, the ionic strength was held constant by diminution of the sodium chloride concentration. This required the addition of sucrose in appropriate amounts in order to maintain a constant osmotic pressure and prevent cytolysis.

mie viscosities of these solutions were determined by means of an Ostwald vis- cosimeter used in a thermostated water bath and they were included in all surface charge density calculations. The pH values were determined in the presence of cells,

RESULTS AND DISCUSSION

Sirtce the electrophoretic mobility of a particle suspended in liquid depends entirely on the properties of that particle's surface and is independent of properties, for example density, of the internal bulk of the particle, the results of the mobility determinations reported below can be taken as evidence bearing strictly on the nature of the cells' surfaces. They are influenced by ot.her factors only indirectly, through modification of cellular surface electrokinetic properties. Nearly all of the experiments involve the determination of electrophoretic mobility and net surface charge density for different types of cells under different conditions of pH or calcium concentration.

Calibration of apparatus with human erythrocytes

Many consistent mobility data for human erythrocytes are in the literature. The reliability of data from the apparatus used here is supported by the following obser- vations. First, three groups of mobility de-

terminations in M/15 phosphate buffer at pH 7.4 produced these values: 1.31 rt 0.0058; 1.33 2 0.0156; 1.30 2 0.017 X lo-* cm/sec/volt/cm. These values agree with previous reports (Abramson, '29; Ada and Stone, '50; Hanig, '58; Hartman et al., '52; Woernley et al., '61). Second, determination of pH-mobility relationships in sodium chloride solutions at three ionic strengths produced results similar to those reported by Seaman and Heard ('60). The results were virtually identical at 0.145 M and quite similar at other ionic strengths.

pll-mobility relationships for dissaci- ated chick embryo cells

In figure 2 the electrophoretic mobility and the net surface charge density of trypsin-dissociated chick heart ventricle cells are plotted as functions of the pII of the suspending medium. These curves are representative, after this type of treatment, for a number of kinds of cells. Over a wide range of pH the movement of the cells is toward the anode as a consequence of a negative net surface charge. That is true for all types of cell examined.

These curves have the general appear- ance of typical acid-base titration curves, with decrease in negative surface charge as the hydrogen ion concentration is increased. They are similar to curves obtained for purified proteins and may in fact reflect protein in the surface of the heart cell. That would be consistent with the Danielli-Davson ('35) model of the plasma membrane which is widely accepted in one or another modification. The steeper sections of the curves at the lower and higher pH values could very well represent the dissociation of carboxyl and amino groups respectively. Between pH 4 and pH 9, the curves are less steep, but they do maintain positive slopes which might be attributed to the presence (in smaller quantities) of groups with pKs in that range. For example, the imidazole group in histidine has a pK of 5.97 and the sulfhydryl group of cysteine has a pK of 8.33 (Greenstein and Winitz, '61).

Surface charge densities were calculated according to the equation of Gouy and Gorin (Abramson et al., '42; Brinton and Laufer, '59; Thompson and McLees, '61).


- -60

E 2 -.40 4- - P

9 .oo

2 -20 al

E u

t.20

+.40

$60

PH Fig. 2 Electrophoretic mobility (cm/sec/volt/cm X 104) and net surface charge density (esu/cm2

x 10-3) as functions of pH for trypsin-dissociated embryonic heart ventricle cells. Standard error of the mean is indicated for mobilities here and in figures below.

.='-VT 1 + KRi 2000T

(1) v'X!i [exp (- Zi e(/kT) - 11

In Equation 1, the net surface charge density in esu cm-z, has the same sign as the electrophoretic mobility; Ri is the radius of the counter ion in centimeters; N is Avagadro's number; D is the dielectric constant of the suspending medium; k is Boltzmann's constant in ergs/degree; T is the absolute temperature; Ci is the concentration in moles per liter of ions of the ith species: Zi is the charge (with the correct sign) of the ions of the ith species; e is the charge of the electron in esu; I is the zeta potential obtained from the electrophoretic mobility by means of Equation (3 ) of Helmholtz and Smoluchowski. K, the reciprocal of the Debye length, is given by:

- - K = 4- 8 r Ne2 4;

1000 DkT In Equation 2, r/2 is the ionic strength.

is : The Helmholtz-Smoluchowski equation

(3)

In it, n is the viscosity of the suspending medium in poises and ct is the mobility.

p = - I D 4 r v

In using this equation one assumes that the radius of curvature at the shear boundary is relatively large. This is not known. If the radius of curvature were considered to be less than 10 A, however, the following equation would apply :

(4)

In that most extreme, and unlikely, case the error introduced by calculating I from the Helmholtz-Smoluchowski equation would be only a factor of 0.67 (Brinton and Laufer, '59).

The following values were used in these equations: Ri = 2.5 X cm; N = 6.023 X loa; D = 78.54; k = 1.380 X lo-'' erg/ deg/mole; T = 298"; e = 4.8 X lo-'' esu;

Inspection of figures 2 through 4 reveals, not unexpectedly, that the shapes of all these plots of mobility or net surface charge density against pH are similar. It also reveals that they are all different, something which could not have been anticipated with equal confidence. Com- paring heart and liver cell data in figure 5, we see that above pH 4 the liver cells have an appreciably greater negative surface charge and that at lower pH values, the two curves are practically the same. It

ID 6 r s

p =-

r/2 = 0.145.

28 MICHAEL COLLINS

-1.40

-120

-0 -1.00 E Q -230 .- ; ' - M E -.40

-20

. c o t '

P H

-

?!+ -,.:f- -

-

' ' I I I I I I ,

PH

Fig. 3 Electrophoretic mobility and net surface charge density as functions of pH for trypsin-dissociated embryonic liver cells.

PH

Fig. 4 Electrophoretic mobility and net surface charge density as functions of pH for trypsindis- sociated embryonic neural retinal cells.

appears that the chemical nature of the charged groups on these two types of cell is the same, but that these cells differ in the relative quantities of different groups. The mobility of a cell in an electric field reflects the balance of negative and positive charges. From an examination of the two curves in figure 5, it appears that the higher net negative surface charge of the

liver cells is due to an excess of anionic sites rather than a deficiency of cationic sites. Similar data for trypsin-dissociated neural retinal cells appear in figure 4. A comparison of these cells with heart cells in figure 6, for example, reveals an even greater difference between the surface properties of two types of cell. First of all, the net negative surface charge den-


-3.0

-2 .5

-2.0

" 2 - 1.5

- 1.0

f -0.5 \ 13

N

g 0.0

+0.5

+ 1.0

+ 1.5

-3.5

-3.0

- -

-2.5

-2.0 -

-

f.3 I

0 -1.5

E -1.0 -

2 -0.5 -

0.0

+0.5 -

CI.0

- Y N

0 \

0

-

-

+1.5 -

- - - - - - - - - -

LIVER CELLS

t2.0 2 3 4 5 6 7 8 9 10

PH

Fig. 5 Comparison of the net sui.face charge density-pH relationships for embryonic heart and liver cells.

sity of the neural retinal cells is appreciably greater over a wide range of pH. This indicates that the excess of negative over positive sites is greater for the neural retinal cells. The difference in shape of the two curves is quite striking. In the lower pH range, however, the shapes appear to be the same and there the two curves differ in that the neural retinal curve is displaced upward on the negative charge density scale. That seems to show that the surfaces are qualitatively similar with respect to the type of acidic group present at the surface and that they differ either in the number of such groups or in the number of positively charged groups exposed at low pH. The really striking difference between the two types of cell appears in the pH range 5-7.5 where the curve for neural retinal cells has a negative slope. That section is unusual, although it is certainly real. One possible circum- stance which could account for it would be a pH-dependent configurational change in the surface material, exposing more negative sites - or retracting positive sites - below neutrality and thereby increasing the net negative charge in this pH range.

Whatever the explanation may be, it is clear that the two curves differ and that the differences reflect molecular differences between the surfaces of the two types of cell.

Effects of calcium in physiological concentration on the mobility of dissociated neural retinal cells

In all cases mentioned thus far, cells have been suspended in solutions containing no bivalent cations. The pH-mobility relation of trypsin-dissociated neural retinal cells has been determined in the presence of a physiological concentration of calcium ions. The concentration selected was 1.8 X M, as found for example in the balanced salt solution of Earle, among others. The ionic strength was held constant by reducing the sodium chloride concentration by an appropriate amount while maintaining isotonicity with sucrose. Figures 7, 8 and 9 show the results of these determinations. They are as one would expect if calcium were reducing mobility by binding to negatively charged sites. A physiological calcium ion concentration brings about a clear diminution of net negative charge in the middle

-3.51

+ * . o r 2 3 4 5 6 7 8 9 10

PH Fig. 6 Comparison of the net surface charge

density-pH relationships for embryonic heart and neural retinal cells.

30 MICHAEL COLLINS

-1.40

-1.20 -

-'20L .oo 1 1 1 , 1 1 1

L 3 4 5 6 7 8 9 10

PH PH

Fig. 7 Electrophoretic mobility and net surface charge density as functions of pH for trypsin-dissociated neural retinal cells, determined in the presence of a physiological concentration of calcium ions (1.8 x M).

-4.0

-3.5

-3.0

-2.0

3 -1.5

NO CALCIUM

1.8" CALCIUM

-0.5 t 2 3 4 5 6 7 8 9 I0

SURFACE p H

Fig. 8 Comparison of the net surface charge densjty-surface pH relationships for trypsin-dissociated neural retinal cells determined in the presence and absence of calcium ions. Surface pH has been calculated after Hartley and Roe ('40) and is very close to the bulk pH.

S U R F A C E pH

Fig. 9 Difference between the two curves in figure 8 a5 a function of surface pH. This curve presumably relates calcium ion binding to surface ]pH.

pH range where most of the anionic sites are dissociated and hence free to bind a cation such as calcium. At lower pH values, where the sites become increasingly protonated, calcium reduced the net charge considerably less. One would expect the curves to be parallel at higher pH values since all anionic sites would be dissociated. A possible explanation for the re- duction of calcium binding capacity apparently caused by high pH and well illustrated in figure 9 would be the involve- ment of charged cationic sites in calcium binding in a synergistic way. If that were so, higher pH could reduce their effect and inhibit calcium binding. Another possible explanation would be a pH-dependent configurational change in the cell surface as mentioned above. That this effect is due totally to calcium hydroxide's low solubility seems unlikely since the solubility of calcium hydroxide at 25°C is slightly greater than 1.8 X M. The negative slopes of both the pH-charge density curve (fig. 7) and the curve which presumably relates calcium complexing to pH (fig. 9) both start at pH 5. This could mean that pH values below neutrality do cause a configurational change which exposes new anionic sites which bind calcium.

Whatever the explanation, these results are similar to results obtained by Stein- berg using radioactive isotopes and amphibian material (Steinberg, '62a). The

EMBRYONIC CELL SURFACE PROPERTIES

fact that these similar phenomena have been observed ( 1 ) by means of determination of surface charge and (2) by direct measurement of the binding of radioactive metal ions tends to reinforce both methods of observation. The isotope studies sup- port the interpretation of surface charge diminution in terms of calcium ion binding while the electrophoretic studies indicate that the radiosotopes were in fact bound at the surfaces rather than within the cells.

Surface pH was calculated for figures 15 and 16 according to Hartley and Roe ('40); McLaren and Babcock ('59). The difference between surface and bulk pH here is low.

Effects of calcium in physiological concentration on the mobility of

dissociated cells of four types The Introduction refers to the hierach-

ical ordering of different types of embryonic cells, apparently on the basis of strength of cohesiveness, reported by Stein- berg ('63a, b, c). If one assumed that the cells in the reaggregating system adhere primarily by means of bivalent cation bridges, it would appear that the strength of cellular cohesion would reflect the density of such bridges per unit area of con-

31

tact surface. Also, the more strongly co- hering cells would exhibit concomitantly a greater ion-binding capacity and perhaps a higher charge density as well. Heart cells segregate internally when cultured with liver cells (see table 1). Figure 5 shows, however, that the liver cells have more highly charged surfaces. Additional information on this point appears in table 2. Mobility determinations made in the presence of 1.8 X M calcium were compared with determinations made in calcium-free solutions. In all cases, the addition of calcium, with ionic strength held constant, depressed surface charge and mobility, as would be expected. This depression was about twice as great for

TABLE 1

Four tissues used in this paper. They are arranged according to their sorting out behavior

Tissue Behavior when dissociated cells

of two types are mixed

Epidermis Internal to the others

Heart ventricle

Liver Internal to neural retina

Neural retina

Internal to liver and neural retina

External to the others

TABLE 2 Depression of electrophoretic mobility and net surface charge density by calcium at a

concentration of 1.8 x M

Mobility (cm/sec/v/cm x 101) rt standard error of the mean x 101

Without calcium With calcium Difference Cell type

Epidermis -0.811 - 0.70 0.11 k 0.0256 k 0.0171

Heart -0.84 - 0.65 0.19 k 0.0374 rt: 0.0245

- 0.67 0.17 2 0.0390

Liver - 0.96 -0.86 0.10

-0.85 0.11 c 0.0290

( trypsin-dissociated) - 0.91 -0.78 0.13

t: 0.0254 k 0.327

Neural retina

c 0.01 18 rt: 0.0182

Net surface charge density (esu/cm2 X Epidermis -2.12 - 1.90 0.22 Heart - 2.20 - 1.76 0.44 Liver - 2.52 - 2.34 0.18 Neural retina - 2.39 - 2.02 0.37

32 MICHAEL COLLINS

heart cells as for liver cells. That suggested that, whiIe the liver cells showed a geater charge density, the apparently more cohesive heart cells had greater calcium binding capacities which might account for their greater cohesiveness.

Experiments with other types of cells soon called that simple explanation into question. For example, dissociated cells from eight-day chick embryo back epidermis sorted out internally with respect to all other tissues in the hierarchy mentioned above. So, to be consistent with the results for heart and liver, epidermis would have to exhibit a surface charge depression, as a result of the addition of calcium, even greater than that of heart cells. Reference to table 2 shows, however, that this depression €or epidermis was really about the same as that observed for liver.

Trypsin-dissociated neural retinal cells were studied also. Results presented in table 2 show that they responded to 1.8 X loT3 M calcium to about the same extent as liver cells. Steinberg has shown that dissociated heart and neural retinal cells mixed in culture form reaggregates which sort out with the retinal cells external to the heart cells (Steinberg, '63a, c, '64). In this respect the neural retinal cells behave the same as the liver cells. Moscona has reported that dissociated neural retinal cells and liver cells, when mixed in culture, reaggregate but fail to sort out (Moscona, '60). In this work, however, dissociated seven-day neural retinal cells were mixed with dissociated five-day liver cells in suspension culture at 80 gyrations per minute on a New Brunswick Scientific Company Gyratory Shaker (Steinberg, '62b). The cells reaggregated well and reaggregates fixed after a day in culture showed that the two types of cell can sort out. ' f ie liver cells were internal with respect to the retinal cells. The culture medium used for this reaggregation experiment was the horse serum-Tyrode's solution-eimbryo extract medium described above The reaggregates were fixed with the fixative use for glycogen staining by Lison and Vokaer and stained for glycogen according to the periodic acid-Schif€'s rea- gent method using dimedone to block aldehyde groups which could interfere with

the glycogen stain (Pearse, '60). Counter staining was with celestine blue and hema- lum. Since glycogen is present abundantly in liver cells, but not detectable in neural retinal cells, this staining provides a clear distinction between cells of the two types.

The results in this section are not in good accord with the cation bridge hypothesis. Epidermis shows a disparity between its segregative and calcium-binding properties. Epidermis behaves as though it were very strongly cohesive, and yet its calcium-binding capacity seems to be about the same as that of the apparently much less cohesive liver. These cells of the epidermis are, however, epithelial cells. The surface on one side of such a cell might be presumed normally to be different from the surface on the other side. Also means of the adhesion of epithelial cells to one another is perhaps not typical. In 1925 Chambers and Renyi noticed, in the course of microdissection of human epidermis, what appear to be points of protoplasmic connection between cells which were being pulled apart (Chambers and Renyi, '25). It was impossible for them to determine the exact nature of this uneveness of adhesion between epidermal cells, but it now seems likely that it is due to the specialized structures associated with ap- posed plasma membranes seen in a wide variety of epithelial tissues, for example the "junctional complexes" found in various epithelia by Farquhar and Palade ('63).

It may be that the epithelial cell is specialized and differs to some extent from other types of cell in its means of adhesion. This possibility can be investigated by ex- panding the number of types of cell studied in this way - including other epithelial cells to see whether they too behave atypically, Thyroid cells may be desirable since they are epithelial and could be obtained in relatively large quantity. In any event, it is clear that the hypothesis which suggested these experiments has not been fully substantiated.

Effects of suspension media on chick embryo cells

Cells typically flourish only in environments where conditions are regulated within narrow ranges; alien environments can


cause damage and even death. It is well known that acidic conditions, for example, affect cells adversely. Erythrocyte lysis in- creases rapidly as the pH is lowered. Since this is so, and since all of the electrophoretic determinations reported here are con- ducted in protein-free solutions with widely varying calcium and hydrogen ion concentrations, it is important to have some check upon the condition of the cells studied and upon the damage which they sus- tain during a determination.

Unfortunately, there is no perfectly satisfactory control for this type of experiment. While it seems certain that, as in any experiment, cells do undergo changes, it is not possible to be sure whether or not such changes include modification of cell surface properties which would influence mobility and charge density determinations. Within these limits, these experiments have been controlled very carefully. Three methods have been used in order to look in three ways for changes occurring during determinations.

The standard methods for viability determination rely on the ability of viable

cells to exclude dyes present in their en- vironment - an ability absent from mori- bund cells (Papenheimer, '17). A number of dyes can be used, but nigrosin was selected because of its own low toxicity (Kaltenbach et al., '58). To determine the dye-excluding capacities of cells which have been dissociated and treated with an appropriate solution for over ten minutes (most mobility measurements were made within about five minutes after suspension of the cells), cells were washed and suspended in a 0.5% solution of nigrosin in Eagle's medium. Counts of cells stained and not stained were made with a mechanical counter as a drop of nigrosin solution with cells on a slide with coverslip was observed under the microscope. Table 3 contains the results of the dye exclusion tests of cells treated with the 0.145 M saline solution adjusted to various pHs with 0.145 N HC1 or NaOH. In the case of the liver and heart cells the results show a surprisingly high per- centage of cells excluding dye at low pH values. The results for trypsin-dissociated neural retinal cells are roughly compar-

TABLE 3 Results of nigrosin- exclusion tests with cells exposed to various suspending media. These

numbers represent the per cents of cells excluding the dye. Each number represents a separate count

.-

Neural Neural retinal, retinal, in Ca- and trypsin Mg-free

tvrodes

PH Liver Heart dissociated

2.0 2.5 3.0 3.5 4.0 5.0 5.5 6.0 7.3 8.4 8.9 9.0 9.5 9.7 10.0 10.5 10.8 Control not exposed

to any suspending medium

%

89,78,76

86,90

94

89,92

93,87

93,92

98.97

%

79,78 86

94,91 92 89

97 93

94

93

96.95

% % 48,44 52,36

83,84 64,71

96,95 80,85 91,91

98,96 86,89

97,96 86,83

loo, 100 92,90,90

34 MICHAEL COLLINS

able. Even the results for the neural retinal cells dissociated by calcium- and magnesium-free Tyrode’s solution are bet- ter than expected. These results seem to indicate that the experimental conditions do cause changes in the membranes of solme cells which increase those membranes’ permeability. The effects of exposure to experimental conditions, however, do not appear great enough to destroy the utility of the data presented above. While solme cells do show permeability changes, such changes need not cause changes in surface charge density. And most of the celils fail to show permeability changes demonstrable by this technique.

A second test which detects changes be- caming apparent later, is determination of the ability of cells to reaggregate normally after exposure to experimental conditions. The procedure here is to (1) dissociate and suspend cells as if to introduce them into the electrophoresis apparatus, (2) let them stand for over six minutes, ( 3 ) centrifuge them out of suspension, (4) resuspend them in the standard culture medium and (5) let them reaggregate on the shaker at 80 rpm and 37°C. Table 4 shows that the cells tended to retain the ability to reaggregate in a normal fashion when exposed to a wide range of conditions. As with the dye exclusion tests, results were less favorable at the extremes. Thiese data suggest about the same situa- tion as do the dye exclusion data.

A third test has been the reversibility of the measured effects of the experi-

mental conditions. For example, if lower- ing the pH of the suspending medium from 7.4 to 3.0 lowers the mobility of cells suspended in the medium, then raising the pH back up to 7.4 may restore the cells’ original mobility. If the mobility is re- stored, that reversibility indicates that the surface examined at pH 3.0 is the same surface as was examined at pH 7.4. Once again, this offers no guarantee, but it has been done to provide maximum information on cellular changes during mobility determinations.

In the experiments, summarized in table 5 , cells were dissociated and suspended in appropriate solutions as if for introduction into the electrophoresis apparatus. After standing for about six minutes, cells were centrifuged out of suspension, washed and suspended in 0.145 M saline at physiological pH. The cells were then introduced into the electrophoresis tube for mobility determinations.

Since mobility data on dissociated cells are quite variable and the variability in- creases with prolonged exposure of cells to saline, it is rarely possible in these experiments to duplicate exactly a value obtained at physiological pH. Since that is so, the danger of subjective appraisals of reversibility must be avoided. That was done by comparing, by means of t tests, data from cells exposed to extreme conditions with data obtained under standard conditions. In table 5, p represents the probability that the two samples compared are from identical populations. In many

TABLE 4 Ability o f cells to reaggregate normally after treatment with various suspending media

Cell type

Neural retinal.

dissociated Neural with retinal, calcium-

Heart trypsin and. dissociated magnesium.

free Tyrode’s solution

PH Liver

2.5 poor poor poor fair 3.0 fair f air/poor - - 3.5 good - good good 4.5 good good good - 7.3 - - good 9.0 good good good good

10.0 good good good good

-


TABLE 5 Reversal of p H effects

Cell type and treatment Values of p

PH

9.0 4.0 P > 0.5

Liver 9.9 0.5 > p > 0.2 0.5 > p > 0.2

3.0 P > 0.5 Heart 10.0

9.1 4.9 4.8 4.0 3.9 3.0 2.5

0.2 > p > 0.1 P > 0.5 P > 0.5

P > 0.5 P > 0.5

0.5 > p > 0.2

0.5 > p > 0.2 p Y 0.1

Neural retinal trypsin dissociated 10.0 P > 0.5

2.5 P > 0.5

Tyrode’s solution 10.0 P > 0.5 2.5 P > 0.5

3.0 0.5 > p > 0.2

Neural retinal dissociated with calcium- and magnesium-free

Student’s t test has been used as an objective criterion for the reversal of pH effects. Different cells were exposed, for six minutes, to saline solutions with different pH values. They were returned then to pH 7.4 for surface charge density determinations. Those charge density values were compared with values obtained at pH 7.4 directly after dissoclabon. Comparisons were made by means of the t test. A value of P greater than 0.1 indicates no significant difference and hence reversal of the pH effect.

cases p is greater than 0.5 and in almost all other cases p is greater than 0.2. These seem to represent reversible effects, since much lower values of p are required to show significant differences between groups of data. Even the worst case, at pH 2.5, has a value of p of about 0.1. In this case, however, there appears to be a real difference due to exposure to the low pH. That indicates that near pH 2.5 there occurs some irreversible change in the cellular surface components which con- tribute to the surface charge.

General comments The cell surface cannot be viewed as

the solid surface of a rigid and impenetrable sphere. Seaman and Heard have pointed out that the erythrocyte surface is very likely a porous polyanion which exists in three dimensions with the inner regions of the surface exerting a limited but real effect on the properties of the surface electrokinetically detectable at the boundary of shear (Seaman and Heard, ’60). The extent to which materials deeper within a cell’s surface region influence the cell’s electrokinetic behavior depends upon

the ionic strength of the ambient medium. That is because as the ionic strength is increased the potential due to a charged surface decays with distance more steeply and is more likely to have declined to a negligible value at the shear boundary. So it is that groups with a given charge located for instance 10 A beneath the outermost boundary of a surface might, if the ionic strength were less than 0.01, have a large effect at that outermost boundary and beyond; the potential due to the same groups might, however, if the ionic strength were increased to 0.1, decay com- pletely within 10A and have no effect at that outermost boundary. Haydon has dis- cussed the consequences of cellular surface porosity for electrokinetic measurements (Haydon, ’61). He noted that the interaction of various planes of charged groups within a porous surface will influence the value of surface charge density apparent to the investigator. He states that the commonly used Gouy equation is really correct only for an impenetrable surface and included several equations for a number of conceivable permeable surfaces. He concludes that, “in all cases the

36 MICHAEL COLLINS

true surface charge of the cell is likely to be higher than calculated for an impenetrable surface." When considering the results of the present investigation, it is well to bear Haydon's conclusions in mind, but his paper indicates that one might expect a maximum error of twofold in the case of no restraint on the mobility of ions within the surface. The error is reduced as the mobility of the internal ions is restrained, and certainly in the plasma membrane that restraint is appreciable. While these considerations expressed by Haydon point up a source of error in de- termining absolute values of net surface charge density, they do not seem to im- pair the validity of the inferences based upon the data in this paper.

Evidence reported here shows that cells which seem to differ in adhesiveness have dSc:rent surface charge properties. These properties of the various types of cell re- spond differently to changes in hydrogen ion concentration and to calcium ions at physiological concentration. It is not possible at present to say that the more adhesive cells bind more calcium since there is an imperfect correlation between sorting lout behavior and surface charge suppression by calcium ion. While the data do not strictly fulfill the simplest expecta- tions from the bivalent cation bridge hy- pothjesis, they do not rule it out and they are 1.0 an extent consistent with it.

ACKNOWLEDGMENTS

".his work is from a dissertation sub- mitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy. It has been supported by a National Institutes of Health Predoctoral Fellowship and by a grant. to Dr. Malcolm Steinberg from the National Science Foundation. Thanks are due: Mr. Edward Kennedy for his assist- ance with the histological work; Mr. John Spurbeck for the art work; Mr. James Vitak who constructed the power supply unit; Mr. Howard Walton who constructed the electrophoresis apparatus; Dr. Thomas Thompson who read the manuscript and made several helpful suggestions; and, most of all, Dr. Malcolm Steinberg who suggested this problem and whose super-

vision and encouragement ensured its completion.

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Documents

Electrokinetic properties of dissociated chick embryo cells. I. Ph-surface charge relationships and the effect of calcium ions