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Chemical dissolution and in vitro reconstruction of sponge cell adhesions
I)E\'ELOPhZENTAL
BIOLOGY,
Chemical
8, 27-47
Dissolution
and
of Sponge I. Isolation
in Vitro
Reconstruction
Cell Adhesions
and Functional Components
TOM llepc~rtnmlt
(1963)
Demonstration Involved1
HUMPHREYS~.:~
of Zoolog!j, University of Chicngo, The Maine Biologicul Laboratory, Woods Hole, hfassachrisctfs Accepted
of the
May
and
1, 1963
INTRODUCTION
The mechanisms by which the individual cells of multicellular organisms are held in their various specific groupings and ordered arrays are not known (Weiss, 1958). Many aspects of this selective cell adhesion have been strikingly revealed by numerous studies dealing with developmental phenomena. Cell adhesion is species specific in aggregation of dissociated sponge cells (Wilson, 1910; Galtsoff, 1923) and individual slime mold amebas (Raper and Thorn, 1941; Bomier and Adams, 1958); and tissue specific in aggregation of dissociated cells from vertebrate embroys (Townes and Holtfreter, 1955; Moscona, 1957). Sorting of aggregating cells (Huxley, 1911; Galtsoff, 1925b; Moscona, 1952, 1956, 1957)) morphogenctic movements during normal development (Swift, 1914; DuShane, 1943; Weiss and Andres, 1952 ), tissue architecture ( Weiss and Moscona, 1959 ) , and perhaps ’ This work was submitted in partial fulfillment of the requirements for the tlegrw of Doctor of Philosophy in the Department of Zoology, The Universit) of Chicago, Dr. A. A. Moscuna, thc~is advisor. It was supported by grants C-4272 from the National Cancer Institute, U. S. P. H. S., and from the*Dr. Wallace C. and Clara A. Abbott Fund of the University of Chicago to Dr. A. A. Moscona. I’ During the course of this study the author held tenures of a National Science t;oundation Predoctoral Fellowship nncl The hlorris Miller Wells Fellowship of tlw Cencrd Biological Supply Housr. ” Present address: Department of Biology, hlnssachuwtts Institutes of Techuology, Cnmhridgc, Massachusetts. 27
28
TOM
HUMPHREYS
even tumor metastasis (Willis, 1952) may be directed by selective cell adhesion. Although selective cell adhesion has been well documented and has evoked much interest (Moscona, 1960; DeHaan, 1958; Weiss, 1958; Rinaldini, 1958; L. Weiss, 1960; and others), the fundamental question of the general morphological and chemical nature of the cell adhesion is itself unclear. Present views suggest five basically different models: (1) divalent cation stabilization of an intercellular cement (Ringer, 1890; Herbst, 1900; Gray, 1926; Chambers and Chambers, 1961) ; (2) bonding between sterically complementary surface groups (Tyler, 1947; Weiss, 1947); (3) calcium bridge bonding between cell surfaces (Coman, 1954; Steinberg, 1958); (4) longrange bonding between cell membranes (Curtis, 1960, 1962); and (5) function of specific cell products acting at the cell surface (Moscona, 1960, 1961b, 1962). These proposed models have unique features which might be distinguished experimentally by appropriate tests. An aggregating system involving simple, defined and controllable techniques was sought. Dissociated sponge cells, the classical material for the study of cell aggregation (Wilson, 1907, 1910), seemed highly suitable, since they aggregate well in simple, defined salt solutions ( Galtsoff, 1925a; delaubenfels, 1932). Previous studies on aggregation of dissociated sponge cells employed mechanical dissociation and self-aggregation techniques which are unsuitable for an analysis of the physical and chemical structure of a cell adhesion. The need to eliminate dependence on cell migration had already been recognized and met for embryonic vertebrate cells (Moscona, 1960, 1961a) and for slime mold amebas (Gerisch, 1959, 1960). The aggregating cells were maintained in suspension and brought randomly into contact by agitating the culture. Rotationmediated aggregation (Moscona, 1961a) was easily adaptable for the study of sponge adhesion (Humphreys et al., 1960b). Also a chemical procedure for dissociation which would break the intermolecular bonds involved in the cell adhesion, but which would not destroy macromolecules that might be involved, was sought. Dissociation by removal of divalent cations was the most promising (Steinberg, 1958), although previous reports discounted the role of divalent cations in sponge cell adhesion (Agrell, 1951; Spiegel, 1954a). Exploratory experiments showed that sponge tissue could be dissociated in calcium-
COMPONENTS
OF
SPOXGE
CELL
ADIIESIOSS
29
and magnesium-free sea water. Thus a method using cold calciumand magnesium-free sea water was developed which produced SLISpensions of single, viable cells capable of aggregation and development into sponges (Humplrrevsi ct (II., 196Oa). This method of dissociation, in fact, did preserve the functional integrity of the molecules involved and permitted the separation of the cell adhesion into three components: the cell surface (represented by whole cells), divalent cations, and an organic factor. When placed back together ill vitro these three components would spontaneously reassemble to reform apparently normal cell adhesions ( Humphreys, 1962). RIATERIALS
ASD
hlETIIODS
Sponges were collected near \\‘oods IIole during the summer and fall and maintained in running sea water. cbnly specimens which had been in the laboratory less than 4 days mere L~YI since ciiltrires of older material contained mrich cellular debris which interfered with aggregation. Four species of monaxonic sponges, Miclociona plifcw ( referred to as AI), Nnliclow~ occlhtrl ( I1 ), Halichondric~ panicc~r were utilized. The work was conducted and Climza cdutu (C), CD)> mainly on 3ficlocioncl, and the descriptions refer only to this species rmless stated otherwise. The compositions of the artificial sea water used in these experiments are listed in Table 1. For chemical dissociation of A/, 1 gm of blotted tissue free of foreign material was immersed in SO ml calcium- and magnesiumfree artificial sea water at pH 7.2 (C\J’F-SF\‘, see Table l), at O”C, cut into 3 mm,: fragments, and soaked for 30 minutes. These fragments were dissociated by pressing through no. 25 standard ctuality bolting cloth (Wilson, 1907) ) into a second 80 ml of cold CMF-SW. The concentration of the resulting suspension of cell clumps and single cells, estimated by hemacytometer counts, ranged between 10 and 40 x 10” cells per milliliter depending on the time of year. The suspension was sedimented for 2 minutes at 2000 rpm, and the cells were resuspended in fresh, cold CAIF-S’IT’ at a concentration of 20 X 10’; cells per milliliter. The suspension was agitated for 6-9 hours in order to complete dissociation and to maintain the cells in suspension. The loose cell clusters remaining after this treatment were easily disrupted by flushing the suspension through a Pasteur pipette with a l-mm orifice, The single cells were gently sedimented and resuspended
30
TOM
HUMPHREYS
TABLE COMPOSITION
OF
_~RTIF~xAL
1
AND
M~DIFIE:D
SEA
WATISR~
Salt (grams per liter glass-distilled water) Type
of seit water
MBL-SW CMF-SW Ca- and Mgfree MBL-SW C&substituted MBL-SW Mg-srtbstituted MBL-SW &-substituted MBL-SW Ca- and Mg-substituted M BL-SW Ca- and Sr-substituted MBL-SW Mg- and &-substituted MBL-SW
MgCln 6HzC)
Stal 6Hz0
1.0 -
4.7 ~ -
_ _ _
0.7
6.8
-
-
0.18
-
0.7
-
12.4
-
0.18
21.i
-
0.i
-
-
24.7
-
0.7
3
21.7
-
0.7
3.4
24.i
-
0.7
KC1
CaCh
0.7 0.8 0.7
-
21.7
NaCI
N&O4
24.f 27.0 24.7
1.0 -
24.7
_
‘I
6.2
IG.3
6 .:3
-
8.2
6.2
a.2
0.18 0.18
-
-
0.18 0.18 0.18
-
0.18 0.18
in about 1.5 ml cold Marine Biological Laboratory formula artificial sea water (MBL-SW, see Table 1) or in experimental medium by several more pipettings. The cell concentration was determined again and the suspension diluted as required and immediately placed in the aggregation vessels. Mechanically dissociated cells were obtained by pressing 1 gm sponge tissue through bolting cloth directly into 80 ml sea water (Wilson, 1907) at 0°C. The following variations were required for dissociation of the other species; use of 2 gm H, D, and C tissue, concentration of D and C cells to 40 X lo6 per milliliter for the 6- to g-hour treatment in cold CMF-SW and doubling sedimentation time for D and C cells. Standard conditions for rotation-mediated aggregation (Moscona, 1961a) were 30 X 10GM cells, 15 X 10GH cells, or 40 X 10” D or C cells suspended in 3 ml sea water in 25ml Erlenmeyer flasks rotated at 80 rpm on a gyratory shaker with a three-quarter inch diameter of rotation. The experiments on effects of cell concentration, frequency of rotation, and the other early tests of sponge aggregation were carried out at room temperature (usually 25°C); all other variables
besides the one being tested were maintained at the standard ~1~s. In all other experiments the shakers were maintained at 22” t 0.5’ or 5” 2 1’ as indicated. For interspecific mixtures, one-half the usual number of cells from each species was used. For preparation of the organic factor, 5 gm of blotted Al tissue or 20 gm of II tissue, were placed in 150 ml cold CMF-STY, cut into l-cm pieces, and soaked for 10 minutes each in four changes of 150 ml CPLIF-S\\’ at 0”. The tissue was collected and pressed through no. 14 standard clnality bolting clotll into 50 ml cold CMF-SW. The resulting suspension was shaken gently for 3 hours at 0°C. It was then centrifuged at 0°C in a Servall SS-1 angle-head centrifuge for 10 minutes at 7500 rpm. Thr supernatant, which was light red (Al) or blue (H) und slightly turbid, was decanted from the large celhllar pellet and recentrifuged at 11,000 rpm for 90 minutes at 0°C. The clear, \‘er> slightly colored supernatant was decanted, and its activity was stabilized by a d c1’mg one part 18.5 mill CaCl’ solution to 9 parts of the factor solution. Reconstruction of adhesions was accomplished by mixing the factor solution 1:l with a suspension of chemically dissociated cells in MBLS1V and subjecting the mixture to rotation-mctliatetl aggregation at
5 cc . E\‘AI,UA’lTOIi
Chemical
OF METHODS AND CENEKAI, OF SPONGE ACCHECATION
ASPECTS
Dissociation
Sponge tissue was chemically dissociated into single, viable cells (Fig. 1) by the described procedure for removal of divalent cations. Soaking the tissue in CMF-S\f’ for only 30 minutes before mechanically disrupting the tissue loosened the cells considerably but did not yet free the numerous spicules, which upon longer treatment would also come out of the collagenous matrix. Dissociating at S”, adjusting the cell concentration to 20 x 10” cells per milliliter, and shaking the suspension during dissociation minimized cell damage to the extent that no detectable loss of cells occurred between final and initial determinations and little cellular debris appeared in the cultures. In order to establish that dissociation was due to the removal of calcium and magnesium, various permutations of the variables (temperature, pH, medium changes, ionic composition, mechanical dis-
32
TOM
HUMPHREYS
persal, etc.) were tested. In all cases the cells dissociated only when calcium and magnesium were removed. The removal of these ions is therefore clearly the effective parameter of the dissociation procedure, The dissociation procedure was developed for Microciona, but it also proved effective and satisfactory for dissociation of Haliclona, Halichondria, and Cliom with the small changes indicated in the methods section. Chemically dissociated cells of all four of these species self-aggregated on glass at room temperature (Wilson, 1910) like mechanically dissociated cells of their species. Rotation-Mediated
Aggregation
Chemically dissociated cells placed in sea water in rotating flasks began to adhere immediately at 22”. Within 15 minutes many cells were in small irregular clusters (Fig. 2a). Examination of living aggregates and of histological sections showed that they consisted of randomly mixed, closely apposed, round cells (Fig. 3a). By 30 minutes the aggregates had become larger through further accretion of free cells and fusion of the small clusters (Fig. 2b). Subsequently, the clusters rounded up and the cells moved into closer association. After about 1 hour all cells capable of aggregating had been incorporated into cell masses (Fig. 2c), and between 3 and 6 hours the aggregates attained final size. At 12 hours the aggregates resembled s-hour aggregates (Fig. 3b) histologically except that a layer of flattened cells had appeared on the surfaces of the aggregates which were thus round and smooth. Thereafter the aggregates remained much the same for many hours. At this time there were about 2000 spherical, compact, smooth aggregates averaging about 0.14 mm in diameter in each flask (Fig. 2d). Occasionally, for unknown reasons, a ring of cells formed on the flask at the air-liquid interphase. Aggregates were maintained in rotating flasks, the sea water being
FIG. 1. Chemically dissociated cells. (a) Living preparation at low power. Line indicates 50 p. (b) Fixed and stained. Note wide range in cell size. Line indicates 10 p. FIG. 2. Time sequence of rotation-mediated aggregation of chemically disFociated sponge cells: (a) 15 minutes, (b) 30 minutes, (c) 1 hour, (d) 12 hours. Ragged aggregates quickly form, and these round up and become smooth. Line indicates 200 JJ.
COhfI’ONESTS
OF
Sl’ONClr
C:ELL
.\I~IIESIOiKS
:‘,:3
Histological sections of sponge aggregates in rotation-mediated aggreFIG. 3. gation. At (a) 15 minutes, (b) 3 hours. The cells at first adhere over small areas and then move closely together. Line indicates 10 p. Sample of single cells and small clumps from a culture at 5°C for FIG. 4. 24 haurs. Line indicates 50 G.
<:Oh11’0NENTS
OF
SI’OXGE
CELL
$5
ADHESIOKS
changed every 48 hours for over 2 months. After lo-14 days mlder these conditions translucent, canal-like areas could be seen in some aggregates. In histological sections some of the aggregates fixed on the 19th day showed canal-like structures, newly formed spicules, and spongin. These results indicated that the culture conditions were satisfactory for 111tissue. Cells or aggregates of all other species of sponges tested do not survive more than a few days under any conditions tested. Variables in Rotation-Mediated
Aggregation
The standard conditions for aggregation were established by tests to determine the effects of frequency of rotation, cell concentration, and temperature. Frequelmy of rotation. Suspended, compact sponge aggregates formed over a range of 70-110 rpm; at the higher frequencies there was much cellular damage. As with vertebrate cells, the size of aggregates varied with frequency (Moscona, 1961a). The most gentle frequency which maintained all cells in suspension (80 rpm) was chosen. Cell concentration. 1Vhen cell concentration was varied, the final size of aggregates increased with concentration asymptotically to a maximum over the range of 0.17 to 17 X 10’; cells per milliliter. The meaning of these cell concentration effects is unknown. The cell concentration of 10 X 10’; cells per milliliter was chosen since it was at a point where variation in cell concentration minimally affected aggregate size. Temperatz~e. Aggregation was studied at temperatures ranging from 5°C to 30°C. At 5°C the chemically dissociated cells did not adhere appreciably (Fig. 4) although they were brought together by the agitation of the medium. They adhered into many small aggregates at lO”C, and optimal aggregation occurred at 18-25°C. Cells which failed to adhere at 5°C would aggregate normally if the temperature was raised to the optimum within 6 hours. At 30°C the cells were -
FIG. 5. Aggregates formed when (a) calcium, (b) mngncsium, or (c) strontium is snbstitutcd for the divalent cations of sea water. Line indicates 200 ,p. Frc. 6. Histological section of aggregate from reconstruction procedure at 5°C. The cells are in the association typical of early stages of aggrrgation at 22°C. 1,inc indicates 10 p.
36
TOM
HUMPHREYS
adversely affected and loose and fuzzy cell masses resulted. Like vertebrate cells ( Moscona 1961a), the aggregation of sponge cells is inhibited by suboptimal temperature (Galtsoff, 1925a). The Role of Divalent Cations The divalent cation requirement for aggregation was determined by studying the effects of a number of artificial, modified sea waters (Table 1) substituted for MBL-SW in the standard aggregation procedure. As described before, round, compact aggregates form in MBL-SW at 25”. If CMF-SW or MBL-SW without its divalent cationic salts was substituted for MBL-SW, the cells remained almost completely separate and did not adhere although brought together by agitation of the medium. These results, along with the results of dissociation, indicate that sponge cells are unable to adhere in the absence of calcium and magnesium, or possibly, divalent cations in general. The specificity of this divalent cationic requirement was tested by using modifications of MBL-SW in which the divalent cationic salts were replaced by combinations of calcium, magnesium, or strontium chloride (Table 1). Normal aggregates formed when the divalent cations of MBL-SW were substituted with (1) CaCl, alone, (2) equimolar quantities of CaCl, and MgCl,, or (3) equimolar quantities of CaCl, and SrCl?. Figure 5a shows aggregates formed in MBL-SW with calcium only. When MgCl, or equimolar quantities of MgCl, and SrCl? were substituted, compact aggregates about one-half normal size were formed (Fig. 5b). Only very small, loose cell clusters formed with strontium alone (Fig. 5~). Other multivalent ions were not tested because normal aggregation can occur without them and because these results establish a preferential requirement for calcium. General
Aspects of Sponge
Cell Adhesion
In the light of these findings that divalent cations are necessary for sponge cell adhesion the observations of Spiegel (1954a,b) should be reinterpreted. Confirming previous observations (Galtsoff, 1925a), Spiegel found that dissociated 1M sponge cells failed to aggregate in isotonic NaCl or KC1 or in EDTA containing sea water. However, when cells maintained in these solutions for 24 hours were swirled, instantaneous clumping occurred. This clumping was considered to
COMPOXENTS
OF
SPONGE
CELL
ADHESIOXS
37
be the beginning of true aggregation and thus suggested that divalent cations were required only for sponge cell migration, not for cell adhesion. With the present evidence, however, this clumping can no longer be equated with normal aggregation, particularly since it is possible that many of these cells did not survive; according to Galtsoff (1925a) and in our experience, many do not participate in aggregation when returned to sea water. The divalent cationic requirement for cell adhesion appeared to be totally satisfied by calcium since normal aggregates formed when it was the only divalent cation present. The role of magnesium in producing smaller aggregates is not clear; it may play a unique role in cell adhesion or it may only partially substitute for calcium. Sponge cells aggregating on a stationary surface require both calcium and magnesium ( Galtsoff, 1925a; delaubenfels, 1932). When each was tested alone, magnesium allowed far more aggregation than calcium. Galtsoff therefore concluded that magnesium was probably specifically necessary for cell migration and both calcium and magnesium were important for cell adhesion. These results indicate clearly that both these divalent cations have functions in cell aggregation which cannot be fulfilled by strontium or by each other. It is possible that magnesium is required for cell migration and calcium for cell adhesion; but in our experiments the small aggregates formed in the presence of magnesium alone appeared to be as firmly cohesive as normal aggregates. A more complete understanding of the specific roles of these two ions must await further analysis. The chemically dissociated cells did not adhere at low temperatures even in the presence of divalent cations. Similar effects of low temperature on self-aggregation of mechanically dissociated cells was thought to be due to the failure of cells to meet because cell migration was inhibited ( Galtsoff, 1925a). Since in rotation-mediated aggregation the cells are brought together, the failure of the chemically dissociated cells to aggregate must evidently be due to their lack of adhesiveness. LOW temperatures prevent the aggregation of proteolytically dissociated embryonic vertebrate cells, This has been interpreted as being the inability of the cells to regenerate cell surface products because of inhibition of metabolic activities (Moscona, 1961b). The results with chemically dissociated sponge cells can also be SO interpreted. Although the chemical dissociation of the sponge cells did not involve treatments that would be expected to destrov
38
TOM
HUMPHREYS
macromolecules, the assumption that the surface of these nonadhering cells was indeed, functionally intact requires further proof before any conclusions concerning the nature of the cell adhesion can be supported. IN
VITRO
RECONSTRUCTION
Adhesion of Mechanically
OF SPONGE
Dissociated
CELL
ADHESIONS
Cells at Low Temperatures
To find out whether cells would adhere at low temperatures if all components of the cell adhesion were present, the aggregation of mechanically dissociated cells at 5” was studied, since it seemed likely that the mechanical dissociation would not destroy or remove any macromolecule involved in adhesion. It was found, indeed, that mechanically dissociated cells adhered very rapidly in the rotating flask at 5°C and formed compact O.l- to 0.2-mm aggregates. By their rough outlines and histological appearance they resembled early stages of aggregates produced at 22°C. At 5” they would not progress beyond this stage; but, if the temperature was raised, they quickly rounded up and continued to develop. This block to development was probably due to the low temperature inhibition of cell movement (Galtsoff, 1925a), which prevented the randomly adhering cells from proceeding with reorganization until the temperature was raised. It appeared that mechanically dissociated cells retained some factors which had been lost by the chemically dissociated cells. Reconstruction
of ‘Cell Adhesions after Ch.emical Dissociation
The inability of chemically dissociated cells to adhere at 5” even though calcium had been added back was strikingly abolished if the cell-free supernatants from chemical dissociation were also added; the cells adhered into compact aggregates of randomly mixed cells resembling closely those of mechanically dissociated cells at 5”. At 5°C they remained at this stage for days. If the temperature was raised within 3 days they rounded up and proceeded to develop. It appeared that the supernatants from chemical dissociation contained the missing factor which had been dissolved from the cells by the removal of divalent cations. Divalent
Cation Requirement
In the presence of the factor and divalent cations, chemically dissociated cells adhered into one large aggregate at 5OC. With only the
factor and no divalent cations, they did not adhere at all. The specificity of this cation requirement was examined, and calcium and magnesium were found equally effective, but strontium was completely ineffective. Just as divalent cations were necessary for aggregation, they were also required for the development of cell adhesions in the presence of the factor. It appears then that during dissociation the cell adhesion has been separated into three components; the cell surface, divalent cations, and an organic factor; which when placed back together in C&D spontaneously reassemble to form new cell adhesions. Qlrantitatice Assuy
of the Active Fuctor
In order to obtain supernatants with a higher concentration of the active factor, a modified dissociation procedure was adopted for extraction of the material (see Methods section). This yielded a very active solution. When it was tested in the reconstruction procedure at 5”, all the cells in the flask adhered into one large, rough, cylindrical aggregate about 1.5 mm in diameter and 5-6 mm long (Fig. 7a). This large aggregate consisted of smaller compact cell masses held loosely together by strands of cells. There were numerous unoccupied areas between (Fig. 6). F irm round clots and mucous strings of amorphous material with scattered cells also appeared at this high concentration. As the factor was diluted the aggregates became smaller (Fig. 7b) and the amorphous material did not develop. At the lowest effective concentration many rough, compact aggregates about 0.1 and 2.0 mm in diameter were formed (Fig, 7~). At greater dilutions the cells behaved as if they were in MBL-SW alone (Fig. 7d). This distinct end point provided a possibility of quantitating the activity of a preparation. The least effective concentration of factor was therefore defined as one unit activity per milliliter. The number of units per milliliter in a particular preparation was equal to the dilution required to reach the threshold minimal activity. lMost preparations of crude factor had an activity of 128 units/ml. The activit) of the solution was rapidly lost unless calcium was added to a concentration of 1.8 mM. Source of the Active Facto? Three experiments were done to determine the source of the material: (1) a mock extraction of the factor was conducted using 14BL-SW rather than CMF-S\fy; (2) sponge tissue was homogenized
40
TOM
HUMPHREYS
FIG. 7. Aggregates from reconstruction procedure at 5°C. (a) End of cylindrical aggregate formed at very high concentrations of factor. (b) Aggregates at intermediate concentration. (c) Aggregates at lowest effective concentration. (d) Cell adhesion which occurs below effective concentration. FIG. 8. Haliclonn aggregates. (a) At 22”C, (b) at 5°C.
CO1\lI’ONENTS
OF
SPOSGE
(:ELL
.\DHESIOXS
41
in MBL-SW; and (3) chemically dissociated cells were homogenized in CMF-SW. Examination of the homogenized cells showed that a majority were disrupted. None of the supernatants obtained from the extractions were active; none caused cell adhesion. Species
Specificity
Species specificity of aggregation in sponges has been repeatedly described (Wilson, 1910; Galtsoff, 1923; delaubenfels, 1932). The existence of this specificity provided a very useful and important possibility of demonstrating the specificity of the reactions involved in reassembly of the cell adhesion. Rotutiotz-),lediatetl aggregation of Halichu. Kotation-lnediated aggregation of a second species. Ifaliclonu occzrlutu (II), was studied. Chemically dissociated tl cells aggregated rapidly at 22°C to form compact, round O.l- to 0%mm aggregates (Fig. 8a). The aggregation process for N was very much like that for hl cells at 28” except that the aggregates were short lived. At 5” much smaller, 0.05 mm, round aggregates formed from chemically dissociated H cells (Fig. Bb). Mechanically dissociated cells at 5” formed aggregates with sizes intermediate between 5” and 23” H aggregates. Hulicrona factor. Supernatants from the extraction procedure applied to H tissue with the slight modifications specified in the Methods section caused chemically dissociated H cells to adhere into round aggregates about 0.3 mm in diameter at 5” (Fig. 9). This is about 200 times the cell mass of aggregates formed in MBL-SW at 5”. There was a tenuous halo of clear amorphous material around each aggregate, and numerous firm, round clots with scattered small aggregates in the culture. Serial dilution of the H crude factor resulted in smaller aggregates until at about L3, ’ o the concentration no effect could be detected. The H factor required divalent cations. Interspecific mixtuws in ~otatiofz-mFdi,utetl ugwgation. $1 is bright red-orange, and II is bluish purple. This color difference can be used to determine the species of small aggregates and even single cells of the larger cell types in living preparations. The specificitv of cell
42
TOM
HUMPHREYS
adhesion in mixtures of M and H cells was thus examined. Intermingled chemically dissociated M cells and H cells aggregated at 22” onIy with their own species. W’ithin 15 minutes any aggregate observed was distinctly colored like one or the other species. Cells of the one species were rarely found associated with aggregates of the opposite species. Thus in these mixed suspensions, aggregated by rotation, cells of each species aggregated independently of the other; the final size of aggregates was generally smaller than for each species separately. Aggregation of mechanically dissociated H and M cells at 5” was equally specific. Specificity of the active factor. When M factor was added to chemically dissociated H cells at 5” or H factor was added to chemically dissociated M cells at 5”, they had no effect; the cells of both species behaved as if they were in plain MBL-SW. M factor added to a mixture of M cells and H cells caused the M cells to adhere into a large aggregate, while the H cells behaved as if they were in plain MBL-SW at 5”. H factor tested on a mixture of cells from the two species caused the H cells to form large 0.3-mm aggregates while the M cells behaved as if they were in MBL-SW. Finally, if M factor and H factor were added to a mixture of M cells and H cells, the cells from each species adhered into completely separate aggregates. After 15 minutes the cells in such a mixture were in large, rather loose but separate aggregates. The cells continued to adhere separately and finally formed aggregates exactly as they would if each species had been in a separate flask (Fig. 10). The failure of any of these preparations to develop interspecific adhesions demonstrated that the spontaneous reassembly of the cell adhesions involved specific bonds. This specificity evidently lies in the reactions between the cell surface and the factor of the other species would have resulted in interspecific adhesions in the bispecific cell mixtures. DISCUSSION
The aggregation resulting upon recombination of cells, factors, and divalent cations could be due either to normal cell adhesions or to some nonfunctional agglutination of cells. Various evidence supports the conclusion that the separated components are involved normally in cell adhesion and that the reassembled adhesions are similar to normal, functional adhesions. The aggregates resemble early stages of normal aggregation and can proceed with development if the
COhl1’OSEIUTS
OF
SPONGE
CELL
ADHESIONS
43
temperature is raised to 22”. Divalent cations are required as in normal sponge cell aggregation. Removal of divalent cations is required to release the factor from the cells, as it is required to dissociate cells. The factor cannot be extracted from the cells by homogenization, indicating that it is not a product of cell destruction during the dissociation procedure. Calcium stabilization of the factor demonstrates that divalent cations may be closely associated with its function. The species specificity of the factor parallels that of normal sponge aggregation and rules out the possibility that its activity is clue to a nonfunctional agglutination of cells caused by some general physical or chemical characteristic of the substance such as charge ( Schmitt, 1941) . Th ese various considerations indicate that the facto] represents a dissolved intercellular matrix functioning in cell adhesion by binding the adjacent cell membranes together. This agrees with previously postulated role of cell surface materials in cell aggregation suggested by studies on embryonic cells (Moscona 1960, 1962). The factor demonstrated in the reconstruction of sponge cell adhesions fits historical expectations of an intercellular cement (Ringer, 1890; Herbst, 1900; Gray. 1926; and others); that is, it reacts with divalent cationic bridges bind the material to the cell surface. The adhesion similar to these early ideas but including certain elaborations necessitated by the present data is supported. Sponge cells appear to be held together by an intercellular material bound to each cell surface bt~ specij% ho?zds involving divalent cations. The divalent cations are thought to be involved in the bonds between the material and the cell surface because the factor comes off the cell only when these ions are removed. In this binding the ions apparently do not simply counteract the effects of the negative charge on the cell surface (Schmitt, 1941; Curtis, 1962), but must interact with the intercellular material since the ions are necessary for maintenance of the factor’s functional activity. A possible explanation for their flmction is that divalent cationic bridges bind the material to the cell surface, The cationic and species specificities demonstrate that the bonds in which these divalent cations might be involved could not be simple electrostatic attractions between charged groups, but rather specific complementary reactions between chemical groups on the cell surface, groups in the material, and the cations. Another possibility is that the divalent cations hold the molecules of the intercellular material in the functional configuration necessary for their specific interaction with
44
TOM
HUMPHREYS
the cell surface. The nature of the chemical linkages within the intercellular material itself cannot be deduced from the present data; past ideas that they are cross links of a precipated calcium salt are interpretations which are not critically supported by evidence. Various other morphological and experimental observations relating to cell adhesion agree with the above model. The importance of divalent cations in cell adhesion has been repeatedly confirmed (Steinberg, 1958). Many observations, such as morphological identification of intercellular materials (Gersh and Catchpole, 1949; Fawcett, 1958) or enzymatic dissociation of cells (Moscona, 1952), have indicated the presence of intercellular materials (Rinaldini, 1958; Moscona, 1960). Cell adhesion is specific in many instances (Wilson, 1910; Galtsoff, 1925a; Holtfreter, 1939; Raper and Thorn, 1941; Townes and Holtfreter, 1955; Moscona, 1957). Also, antigenically specific cell surface substances, which may be equivalent to the specific intercellular material of the present studies, have been demonstrated to be important in cell adhesion (Spiegel, 1954a,b; Gregg, 1956; Gregg and Trystad, 1958). The demonstration of a component other than the cell surface and divalent cations in cell adhesions does not lend support to the models of cell adhesion based on calcium bridges (Coman, 1954; Steinberg, 1958) and long-range forces (Curtis, 1960). The 100-200 A electronlucid separation commonly observed between membranes of adjacent cells viewed with the electron microscope is the main evidence for these theories. However, the interpretation of this electron-lucid area as an empty space (Coman, 1954; Curtis, 1960, 1962) has been questioned (Robertson, 1960) and must be reconsidered with the present experimental demonstration of an intercellular material involved in cell adhesion. SUMMARY
The purpose of this work was to study the mechanism of cell adhesion using aggregation of dissociated sponge cells as the experimental system. A method of chemical dissociation was developed for marine sponges using cold calcium- and magnesium-free sea water. The viable, single cells resulting from this dissociation were able to aggregate and develop into functional sponges. Rotation-mediated aggregation was used to study various aspects of the aggregation of sponge cells.
C;Olrfl'OXENTS
Ok- SPONGE CXLL
i\DHESIOSS
-45
Using these techniques, a specific requirement for the divalent cations calcium and magnesium was established for sponge cell adhesion. Low temperature was found to inhibit adhesion of chemically dissociated cells even when the divalent cations were added back to the cells. hIcchanicullv dissociated cells aggregated rapidly at low temperatures. This difference was sho\\*n to be due to a factor released into the supernatant during chemical dissociation of sponge tissue. \Vhen this factor was added back to the chemically dissociated cells along with tlivalent cations, they adhered rapidly at low temperatures. This facto1 was species specific, causing aclheslon cinlv ot cells from the same species. These rcslllts ww interpreted to indicate that the sponge cell acllesion was cor~~poset~ of three basic components: the cell surface, divalent cations, and an intt*rcellular material. Tlwse components had been scjlaratcd dnring chemical dissociation and were capable of spontaneously and species specifically reassembling themscl\w to rt‘;orm an apparently normal cell adhesion. The implications of these results for models of cell adhesion are rliscrissed. REFERENCES
CUKI-IS, A. S. C. ( 1960). ~Nnturalisf 94, 3-56.
DEHAAN,
R. L.
( 1958).
Cell
Cdl
contacts:
migration
Chem. Busis Decchp. Baltimow, lmf. Contrih. 234, 339-377.
bornt’
ph~~sical consitl~r~~tions.
and morphogenetic
~nown1cnts.
AII~.
S!/UI~~.
1958, Johns Hopkins Univ. McCoillm-Putt
DELAUBENFELS, 11. ( 1932 ) Physiology and nlorphology of Porifcra csr~~~plifictl by lotrochotcl biratdate Higgin. Papem Tortugus Lab. 28, 37-66. IIUSHANE, C. P. ( 1943). The cmhrvology of vcrtehmtc pign~nt ~~~11s.()nwt. Rel;. Bid. 18, 109-127. FAWCEI-r, D. TV, (1958). Specializations of the cell surface, In “Frontiers in Cytology” (S. L. I’ala~, cd.), pp. 19-41. Yale IJniv. Press, Kew Haven, Connecticut.
46
TOM
HUMPHREYS
GALTSOFF, P. S. ( 1923). The amoeboid movement of dissociated sponge cells. Biol. Bull. 45, 153-161. GALTSOFF, P. S. ( 1925a). Regeneration after dissociation (an experimental study on sponges). I. Behavior of dissociated cells of Microciona prolifera under normal and abnormal conditions. 1. Exptl. Zool. 42, 183-221. GALTSOFF, P. S. (I925b). Regeneration after dissociation (an experimental study on sponges). II. Histogenesis of Microciona prolifera Verr. J. Exptl. Zool. 42, 223-251. GERISCH, A. ( 1959). Ein Submerskulturverfahren fur entwicklungs-physiologischc Untersuchungen an Dictyostelium discoideum. Naturwissenschaften 46, 654. GEHISCH, G. ( 1960 ) . Zellfunktionen und Zellfunktionswechsel in der Entwicklung von Dictyostelium discoideum. 1. Zellagglutionation und Induction der Fruchtkorperpolaritit. Arch. Entwicklungsmech. Organ. 152, 632-654. GERSH, I., and CATCHPOLE, H. R. (1949). The organization of ground substance and basement membranes and its significance in tissue injury, disease, and growth. Am. J. Anat. 85, 457-522. GRAY, J. (1926). The properties of an intercellular matrix and its relation to electrolytes. Brit. J. Exptl. Biol. 3, 167-187. GREGG, J. H. (1956). Serological investigations of cell adhesion in the slime molds Dictyostelium discoideum, D. purpureum, and Polysphondylium violaceum. .l. Gen. Physiol. 39, 813-820. GREGG, J. H., and TRYSTAD, C. W. (1958). Surface antigen defects contributing to developmental failure in aggregateless variants of the slime mold, Dictyostelium discoideum. Exptl. Cell Res. 15, 358369. HERBST, C. ( 1900). Ueber das Auseinandergehen von Furchungs und &webzellen in kalkfreien Medium. Arch. Entwicklungsmech. Orgun. 9, 424463. HOLTFHETER, J. ( 1939 ). Gewebaffinitiit, ein Mittel der embryonal Formbildung. Arch. Exptl. Zellforsch. Gewebezucht. 23, 169-209. HUMPHREYS, T. (1962) The mechanism of sponge cell adhesions. Thesis, The University of Chicago. HUMPHREYS, T., HUMPHREYS, S., and MOSCONA, A. A. (1960a). A procedure for obtaining completely dissociated sponge cells. Biol. B&l. 119, 294. HIJMPHREYS, T., HUMPHREYS, S., and MOSCONA, A. A. (196Ob). Rotationmediated aggregation of dissociated sponge cells. Biol. Bull. 119, 295. HUXLEY, J. ( 1911). Regeneration of Sycon. Phil. Trans. Roy. Sot. London 262, 165-190. MOSCONA, A. ( 1952). Cell suspensions from organ rudiments of chick embryos. Erptl. Cell Res. 3, 535-539. MOSCONA, A. ( 1956). Development of heterotypic combinations of dissociated embryonic chick cells. Proc. Sot. Erptl. Biol. Med. 92, 410-416. MOSCONA, A. ( 1957). The development in vitro of chime+ aggregates of dissociated embryonic chick and mouse cells. Proc. Natl. Acad. Sci., U.S. 43, 184-194. MOSCONA, A. (1960). Patterns and mechanisms of tissue reconstruction from dissociated cells. Symp. Sot. Study Develop. Growth 18, 45-70. MOSCONA, A. (1961a). Rotation-mediated histogenetic aggregation of dissociated
C;OMPONEA-T’S
OF
SPOh’GE
CELL
ADHESIONS
47
cells. A quantifiable approach to cell interactions in vitro. Exptl. Cell Res. 22, 455-475. MOSCONA, A. (196lb). Effect of temperature on adhesion to glass and histogenetic cohesion of dissociated cells. Nature 1980, 408-409. AIOSCONA, A. ( 1962). Analysis of cell recombination in cxperimtmtal synthesis of tissues in vitro., J. Cellular Camp. Physiol. GO, Suppl. 1, 65-80. HAPEH, K. B., and Taohr, C. ( 1941). Interspecific mixtures in the Dictyosteliaccac.
Am. J. Botcrq 28, 69-78. L. RI. J. (1958). The isolation of living cells from animal tissues. Intern Rev. Cytol. 7, 587-647. RIKCER, S. (1890). Concerning experiments to test t!le influence of lime, sodium, and potassium salts on the development of ova and growth of tadpoles. 1. Physial. (London) 11, 79-84. ROBEMTSON, 1, D. ( 1960). The molecular structure and contact relationships of cell membkmes. Pragr-. Bb&s. Biophys. Chem. 10, 34.3-418. SCHMITT, F. 0. ( 1941). Some protein patterns in ~11s. S!/?n/j. SW. Stuck/ KINALIXNI,
Develop. Grad
3, 1-20.
M. (195421). The role of specific surface antigens in ccl1 adhesion. I. The rcaggregation of sponge cells. Biol. Bull. 107, 130-148. SIXECEL, hl. (1954b). The role of specific surface antigens in ccl1 adhesion. II. Studies on embryonic amphibian cells. Biol. Bull. 107, 149-h-155. STEIXBERG, hi. S. (1958). On the chemical bonds between animals cells-A mcchnnism for type-specific association. Am. iVaturali.st 92, 65-81. SWIFT, C. H. (1914). Origin and early history of the primordial germ cells in the chick. Am. J. And 15, 483-516. TOWNES, 1'. L., and HOLTFHETER, J. (1955). Directed movements and selective adhesion of embryonic amphibian cells. J. Exptl. Zool. 128, 53-120. TYLER, A. (1947). An auto-antibody concept of cell structure, growth and diff’rrentiation. St/ml’. Sac. Study Deuelap. Growth 6, 7-19. ~VEXSS, L. (1960). The adhesion of cells. Intern. Rev. Cytol. 9, 187-225. WEISS, P. (1947). The problem of specificity in growth and development. Yale
SPIEGEL,
j. Bid. Med. 19, 235-278. \I’EISS, P. (1958). Cell contact. Intern. Rev. Cytol. 7, 391-424. \VEISS, P. and AXDIIES, G. (1952). Experiments on the fate of embryonic chick cells disseminated by the vascular route. J. Exptl. Zool. 121, 449-88. U'EISS, P., and MOSCONA, A. ( 1958). Type-specific morphogenesis of cartilages developed from dissociated limb and scleral mesenchyme in uitro. J. ET~~T!$.
Exptl. 3forphol. 6, 238-246. \VII.LIS, R. A. (1952). “The Spread of Tumors in the Human Body.” Buttcrworth, London. \\'II.SON, II. V. (1907). On some phenomena of conlescencc and regoncr”tion in sponges. J. Exptl. Zool. 5, 245-58. LVILSON, II. V. (1910). Development of sponges from dissociated tissue cells. Bull. Bur. Fi.rlzeries 30, l-30.
BIOLOGY,
Chemical
8, 27-47
Dissolution
and
of Sponge I. Isolation
in Vitro
Reconstruction
Cell Adhesions
and Functional Components
TOM llepc~rtnmlt
(1963)
Demonstration Involved1
HUMPHREYS~.:~
of Zoolog!j, University of Chicngo, The Maine Biologicul Laboratory, Woods Hole, hfassachrisctfs Accepted
of the
May
and
1, 1963
INTRODUCTION
The mechanisms by which the individual cells of multicellular organisms are held in their various specific groupings and ordered arrays are not known (Weiss, 1958). Many aspects of this selective cell adhesion have been strikingly revealed by numerous studies dealing with developmental phenomena. Cell adhesion is species specific in aggregation of dissociated sponge cells (Wilson, 1910; Galtsoff, 1923) and individual slime mold amebas (Raper and Thorn, 1941; Bomier and Adams, 1958); and tissue specific in aggregation of dissociated cells from vertebrate embroys (Townes and Holtfreter, 1955; Moscona, 1957). Sorting of aggregating cells (Huxley, 1911; Galtsoff, 1925b; Moscona, 1952, 1956, 1957)) morphogenctic movements during normal development (Swift, 1914; DuShane, 1943; Weiss and Andres, 1952 ), tissue architecture ( Weiss and Moscona, 1959 ) , and perhaps ’ This work was submitted in partial fulfillment of the requirements for the tlegrw of Doctor of Philosophy in the Department of Zoology, The Universit) of Chicago, Dr. A. A. Moscuna, thc~is advisor. It was supported by grants C-4272 from the National Cancer Institute, U. S. P. H. S., and from the*Dr. Wallace C. and Clara A. Abbott Fund of the University of Chicago to Dr. A. A. Moscona. I’ During the course of this study the author held tenures of a National Science t;oundation Predoctoral Fellowship nncl The hlorris Miller Wells Fellowship of tlw Cencrd Biological Supply Housr. ” Present address: Department of Biology, hlnssachuwtts Institutes of Techuology, Cnmhridgc, Massachusetts. 27
28
TOM
HUMPHREYS
even tumor metastasis (Willis, 1952) may be directed by selective cell adhesion. Although selective cell adhesion has been well documented and has evoked much interest (Moscona, 1960; DeHaan, 1958; Weiss, 1958; Rinaldini, 1958; L. Weiss, 1960; and others), the fundamental question of the general morphological and chemical nature of the cell adhesion is itself unclear. Present views suggest five basically different models: (1) divalent cation stabilization of an intercellular cement (Ringer, 1890; Herbst, 1900; Gray, 1926; Chambers and Chambers, 1961) ; (2) bonding between sterically complementary surface groups (Tyler, 1947; Weiss, 1947); (3) calcium bridge bonding between cell surfaces (Coman, 1954; Steinberg, 1958); (4) longrange bonding between cell membranes (Curtis, 1960, 1962); and (5) function of specific cell products acting at the cell surface (Moscona, 1960, 1961b, 1962). These proposed models have unique features which might be distinguished experimentally by appropriate tests. An aggregating system involving simple, defined and controllable techniques was sought. Dissociated sponge cells, the classical material for the study of cell aggregation (Wilson, 1907, 1910), seemed highly suitable, since they aggregate well in simple, defined salt solutions ( Galtsoff, 1925a; delaubenfels, 1932). Previous studies on aggregation of dissociated sponge cells employed mechanical dissociation and self-aggregation techniques which are unsuitable for an analysis of the physical and chemical structure of a cell adhesion. The need to eliminate dependence on cell migration had already been recognized and met for embryonic vertebrate cells (Moscona, 1960, 1961a) and for slime mold amebas (Gerisch, 1959, 1960). The aggregating cells were maintained in suspension and brought randomly into contact by agitating the culture. Rotationmediated aggregation (Moscona, 1961a) was easily adaptable for the study of sponge adhesion (Humphreys et al., 1960b). Also a chemical procedure for dissociation which would break the intermolecular bonds involved in the cell adhesion, but which would not destroy macromolecules that might be involved, was sought. Dissociation by removal of divalent cations was the most promising (Steinberg, 1958), although previous reports discounted the role of divalent cations in sponge cell adhesion (Agrell, 1951; Spiegel, 1954a). Exploratory experiments showed that sponge tissue could be dissociated in calcium-
COMPONENTS
OF
SPOXGE
CELL
ADIIESIOSS
29
and magnesium-free sea water. Thus a method using cold calciumand magnesium-free sea water was developed which produced SLISpensions of single, viable cells capable of aggregation and development into sponges (Humplrrevsi ct (II., 196Oa). This method of dissociation, in fact, did preserve the functional integrity of the molecules involved and permitted the separation of the cell adhesion into three components: the cell surface (represented by whole cells), divalent cations, and an organic factor. When placed back together ill vitro these three components would spontaneously reassemble to reform apparently normal cell adhesions ( Humphreys, 1962). RIATERIALS
ASD
hlETIIODS
Sponges were collected near \\‘oods IIole during the summer and fall and maintained in running sea water. cbnly specimens which had been in the laboratory less than 4 days mere L~YI since ciiltrires of older material contained mrich cellular debris which interfered with aggregation. Four species of monaxonic sponges, Miclociona plifcw ( referred to as AI), Nnliclow~ occlhtrl ( I1 ), Halichondric~ panicc~r were utilized. The work was conducted and Climza cdutu (C), CD)> mainly on 3ficlocioncl, and the descriptions refer only to this species rmless stated otherwise. The compositions of the artificial sea water used in these experiments are listed in Table 1. For chemical dissociation of A/, 1 gm of blotted tissue free of foreign material was immersed in SO ml calcium- and magnesiumfree artificial sea water at pH 7.2 (C\J’F-SF\‘, see Table l), at O”C, cut into 3 mm,: fragments, and soaked for 30 minutes. These fragments were dissociated by pressing through no. 25 standard ctuality bolting cloth (Wilson, 1907) ) into a second 80 ml of cold CMF-SW. The concentration of the resulting suspension of cell clumps and single cells, estimated by hemacytometer counts, ranged between 10 and 40 x 10” cells per milliliter depending on the time of year. The suspension was sedimented for 2 minutes at 2000 rpm, and the cells were resuspended in fresh, cold CAIF-S’IT’ at a concentration of 20 X 10’; cells per milliliter. The suspension was agitated for 6-9 hours in order to complete dissociation and to maintain the cells in suspension. The loose cell clusters remaining after this treatment were easily disrupted by flushing the suspension through a Pasteur pipette with a l-mm orifice, The single cells were gently sedimented and resuspended
30
TOM
HUMPHREYS
TABLE COMPOSITION
OF
_~RTIF~xAL
1
AND
M~DIFIE:D
SEA
WATISR~
Salt (grams per liter glass-distilled water) Type
of seit water
MBL-SW CMF-SW Ca- and Mgfree MBL-SW C&substituted MBL-SW Mg-srtbstituted MBL-SW &-substituted MBL-SW Ca- and Mg-substituted M BL-SW Ca- and Sr-substituted MBL-SW Mg- and &-substituted MBL-SW
MgCln 6HzC)
Stal 6Hz0
1.0 -
4.7 ~ -
_ _ _
0.7
6.8
-
-
0.18
-
0.7
-
12.4
-
0.18
21.i
-
0.i
-
-
24.7
-
0.7
3
21.7
-
0.7
3.4
24.i
-
0.7
KC1
CaCh
0.7 0.8 0.7
-
21.7
NaCI
N&O4
24.f 27.0 24.7
1.0 -
24.7
_
‘I
6.2
IG.3
6 .:3
-
8.2
6.2
a.2
0.18 0.18
-
-
0.18 0.18 0.18
-
0.18 0.18
in about 1.5 ml cold Marine Biological Laboratory formula artificial sea water (MBL-SW, see Table 1) or in experimental medium by several more pipettings. The cell concentration was determined again and the suspension diluted as required and immediately placed in the aggregation vessels. Mechanically dissociated cells were obtained by pressing 1 gm sponge tissue through bolting cloth directly into 80 ml sea water (Wilson, 1907) at 0°C. The following variations were required for dissociation of the other species; use of 2 gm H, D, and C tissue, concentration of D and C cells to 40 X lo6 per milliliter for the 6- to g-hour treatment in cold CMF-SW and doubling sedimentation time for D and C cells. Standard conditions for rotation-mediated aggregation (Moscona, 1961a) were 30 X 10GM cells, 15 X 10GH cells, or 40 X 10” D or C cells suspended in 3 ml sea water in 25ml Erlenmeyer flasks rotated at 80 rpm on a gyratory shaker with a three-quarter inch diameter of rotation. The experiments on effects of cell concentration, frequency of rotation, and the other early tests of sponge aggregation were carried out at room temperature (usually 25°C); all other variables
besides the one being tested were maintained at the standard ~1~s. In all other experiments the shakers were maintained at 22” t 0.5’ or 5” 2 1’ as indicated. For interspecific mixtures, one-half the usual number of cells from each species was used. For preparation of the organic factor, 5 gm of blotted Al tissue or 20 gm of II tissue, were placed in 150 ml cold CMF-STY, cut into l-cm pieces, and soaked for 10 minutes each in four changes of 150 ml CPLIF-S\\’ at 0”. The tissue was collected and pressed through no. 14 standard clnality bolting clotll into 50 ml cold CMF-SW. The resulting suspension was shaken gently for 3 hours at 0°C. It was then centrifuged at 0°C in a Servall SS-1 angle-head centrifuge for 10 minutes at 7500 rpm. Thr supernatant, which was light red (Al) or blue (H) und slightly turbid, was decanted from the large celhllar pellet and recentrifuged at 11,000 rpm for 90 minutes at 0°C. The clear, \‘er> slightly colored supernatant was decanted, and its activity was stabilized by a d c1’mg one part 18.5 mill CaCl’ solution to 9 parts of the factor solution. Reconstruction of adhesions was accomplished by mixing the factor solution 1:l with a suspension of chemically dissociated cells in MBLS1V and subjecting the mixture to rotation-mctliatetl aggregation at
5 cc . E\‘AI,UA’lTOIi
Chemical
OF METHODS AND CENEKAI, OF SPONGE ACCHECATION
ASPECTS
Dissociation
Sponge tissue was chemically dissociated into single, viable cells (Fig. 1) by the described procedure for removal of divalent cations. Soaking the tissue in CMF-S\f’ for only 30 minutes before mechanically disrupting the tissue loosened the cells considerably but did not yet free the numerous spicules, which upon longer treatment would also come out of the collagenous matrix. Dissociating at S”, adjusting the cell concentration to 20 x 10” cells per milliliter, and shaking the suspension during dissociation minimized cell damage to the extent that no detectable loss of cells occurred between final and initial determinations and little cellular debris appeared in the cultures. In order to establish that dissociation was due to the removal of calcium and magnesium, various permutations of the variables (temperature, pH, medium changes, ionic composition, mechanical dis-
32
TOM
HUMPHREYS
persal, etc.) were tested. In all cases the cells dissociated only when calcium and magnesium were removed. The removal of these ions is therefore clearly the effective parameter of the dissociation procedure, The dissociation procedure was developed for Microciona, but it also proved effective and satisfactory for dissociation of Haliclona, Halichondria, and Cliom with the small changes indicated in the methods section. Chemically dissociated cells of all four of these species self-aggregated on glass at room temperature (Wilson, 1910) like mechanically dissociated cells of their species. Rotation-Mediated
Aggregation
Chemically dissociated cells placed in sea water in rotating flasks began to adhere immediately at 22”. Within 15 minutes many cells were in small irregular clusters (Fig. 2a). Examination of living aggregates and of histological sections showed that they consisted of randomly mixed, closely apposed, round cells (Fig. 3a). By 30 minutes the aggregates had become larger through further accretion of free cells and fusion of the small clusters (Fig. 2b). Subsequently, the clusters rounded up and the cells moved into closer association. After about 1 hour all cells capable of aggregating had been incorporated into cell masses (Fig. 2c), and between 3 and 6 hours the aggregates attained final size. At 12 hours the aggregates resembled s-hour aggregates (Fig. 3b) histologically except that a layer of flattened cells had appeared on the surfaces of the aggregates which were thus round and smooth. Thereafter the aggregates remained much the same for many hours. At this time there were about 2000 spherical, compact, smooth aggregates averaging about 0.14 mm in diameter in each flask (Fig. 2d). Occasionally, for unknown reasons, a ring of cells formed on the flask at the air-liquid interphase. Aggregates were maintained in rotating flasks, the sea water being
FIG. 1. Chemically dissociated cells. (a) Living preparation at low power. Line indicates 50 p. (b) Fixed and stained. Note wide range in cell size. Line indicates 10 p. FIG. 2. Time sequence of rotation-mediated aggregation of chemically disFociated sponge cells: (a) 15 minutes, (b) 30 minutes, (c) 1 hour, (d) 12 hours. Ragged aggregates quickly form, and these round up and become smooth. Line indicates 200 JJ.
COhfI’ONESTS
OF
Sl’ONClr
C:ELL
.\I~IIESIOiKS
:‘,:3
Histological sections of sponge aggregates in rotation-mediated aggreFIG. 3. gation. At (a) 15 minutes, (b) 3 hours. The cells at first adhere over small areas and then move closely together. Line indicates 10 p. Sample of single cells and small clumps from a culture at 5°C for FIG. 4. 24 haurs. Line indicates 50 G.
<:Oh11’0NENTS
OF
SI’OXGE
CELL
$5
ADHESIOKS
changed every 48 hours for over 2 months. After lo-14 days mlder these conditions translucent, canal-like areas could be seen in some aggregates. In histological sections some of the aggregates fixed on the 19th day showed canal-like structures, newly formed spicules, and spongin. These results indicated that the culture conditions were satisfactory for 111tissue. Cells or aggregates of all other species of sponges tested do not survive more than a few days under any conditions tested. Variables in Rotation-Mediated
Aggregation
The standard conditions for aggregation were established by tests to determine the effects of frequency of rotation, cell concentration, and temperature. Frequelmy of rotation. Suspended, compact sponge aggregates formed over a range of 70-110 rpm; at the higher frequencies there was much cellular damage. As with vertebrate cells, the size of aggregates varied with frequency (Moscona, 1961a). The most gentle frequency which maintained all cells in suspension (80 rpm) was chosen. Cell concentration. 1Vhen cell concentration was varied, the final size of aggregates increased with concentration asymptotically to a maximum over the range of 0.17 to 17 X 10’; cells per milliliter. The meaning of these cell concentration effects is unknown. The cell concentration of 10 X 10’; cells per milliliter was chosen since it was at a point where variation in cell concentration minimally affected aggregate size. Temperatz~e. Aggregation was studied at temperatures ranging from 5°C to 30°C. At 5°C the chemically dissociated cells did not adhere appreciably (Fig. 4) although they were brought together by the agitation of the medium. They adhered into many small aggregates at lO”C, and optimal aggregation occurred at 18-25°C. Cells which failed to adhere at 5°C would aggregate normally if the temperature was raised to the optimum within 6 hours. At 30°C the cells were -
FIG. 5. Aggregates formed when (a) calcium, (b) mngncsium, or (c) strontium is snbstitutcd for the divalent cations of sea water. Line indicates 200 ,p. Frc. 6. Histological section of aggregate from reconstruction procedure at 5°C. The cells are in the association typical of early stages of aggrrgation at 22°C. 1,inc indicates 10 p.
36
TOM
HUMPHREYS
adversely affected and loose and fuzzy cell masses resulted. Like vertebrate cells ( Moscona 1961a), the aggregation of sponge cells is inhibited by suboptimal temperature (Galtsoff, 1925a). The Role of Divalent Cations The divalent cation requirement for aggregation was determined by studying the effects of a number of artificial, modified sea waters (Table 1) substituted for MBL-SW in the standard aggregation procedure. As described before, round, compact aggregates form in MBL-SW at 25”. If CMF-SW or MBL-SW without its divalent cationic salts was substituted for MBL-SW, the cells remained almost completely separate and did not adhere although brought together by agitation of the medium. These results, along with the results of dissociation, indicate that sponge cells are unable to adhere in the absence of calcium and magnesium, or possibly, divalent cations in general. The specificity of this divalent cationic requirement was tested by using modifications of MBL-SW in which the divalent cationic salts were replaced by combinations of calcium, magnesium, or strontium chloride (Table 1). Normal aggregates formed when the divalent cations of MBL-SW were substituted with (1) CaCl, alone, (2) equimolar quantities of CaCl, and MgCl,, or (3) equimolar quantities of CaCl, and SrCl?. Figure 5a shows aggregates formed in MBL-SW with calcium only. When MgCl, or equimolar quantities of MgCl, and SrCl? were substituted, compact aggregates about one-half normal size were formed (Fig. 5b). Only very small, loose cell clusters formed with strontium alone (Fig. 5~). Other multivalent ions were not tested because normal aggregation can occur without them and because these results establish a preferential requirement for calcium. General
Aspects of Sponge
Cell Adhesion
In the light of these findings that divalent cations are necessary for sponge cell adhesion the observations of Spiegel (1954a,b) should be reinterpreted. Confirming previous observations (Galtsoff, 1925a), Spiegel found that dissociated 1M sponge cells failed to aggregate in isotonic NaCl or KC1 or in EDTA containing sea water. However, when cells maintained in these solutions for 24 hours were swirled, instantaneous clumping occurred. This clumping was considered to
COMPOXENTS
OF
SPONGE
CELL
ADHESIOXS
37
be the beginning of true aggregation and thus suggested that divalent cations were required only for sponge cell migration, not for cell adhesion. With the present evidence, however, this clumping can no longer be equated with normal aggregation, particularly since it is possible that many of these cells did not survive; according to Galtsoff (1925a) and in our experience, many do not participate in aggregation when returned to sea water. The divalent cationic requirement for cell adhesion appeared to be totally satisfied by calcium since normal aggregates formed when it was the only divalent cation present. The role of magnesium in producing smaller aggregates is not clear; it may play a unique role in cell adhesion or it may only partially substitute for calcium. Sponge cells aggregating on a stationary surface require both calcium and magnesium ( Galtsoff, 1925a; delaubenfels, 1932). When each was tested alone, magnesium allowed far more aggregation than calcium. Galtsoff therefore concluded that magnesium was probably specifically necessary for cell migration and both calcium and magnesium were important for cell adhesion. These results indicate clearly that both these divalent cations have functions in cell aggregation which cannot be fulfilled by strontium or by each other. It is possible that magnesium is required for cell migration and calcium for cell adhesion; but in our experiments the small aggregates formed in the presence of magnesium alone appeared to be as firmly cohesive as normal aggregates. A more complete understanding of the specific roles of these two ions must await further analysis. The chemically dissociated cells did not adhere at low temperatures even in the presence of divalent cations. Similar effects of low temperature on self-aggregation of mechanically dissociated cells was thought to be due to the failure of cells to meet because cell migration was inhibited ( Galtsoff, 1925a). Since in rotation-mediated aggregation the cells are brought together, the failure of the chemically dissociated cells to aggregate must evidently be due to their lack of adhesiveness. LOW temperatures prevent the aggregation of proteolytically dissociated embryonic vertebrate cells, This has been interpreted as being the inability of the cells to regenerate cell surface products because of inhibition of metabolic activities (Moscona, 1961b). The results with chemically dissociated sponge cells can also be SO interpreted. Although the chemical dissociation of the sponge cells did not involve treatments that would be expected to destrov
38
TOM
HUMPHREYS
macromolecules, the assumption that the surface of these nonadhering cells was indeed, functionally intact requires further proof before any conclusions concerning the nature of the cell adhesion can be supported. IN
VITRO
RECONSTRUCTION
Adhesion of Mechanically
OF SPONGE
Dissociated
CELL
ADHESIONS
Cells at Low Temperatures
To find out whether cells would adhere at low temperatures if all components of the cell adhesion were present, the aggregation of mechanically dissociated cells at 5” was studied, since it seemed likely that the mechanical dissociation would not destroy or remove any macromolecule involved in adhesion. It was found, indeed, that mechanically dissociated cells adhered very rapidly in the rotating flask at 5°C and formed compact O.l- to 0.2-mm aggregates. By their rough outlines and histological appearance they resembled early stages of aggregates produced at 22°C. At 5” they would not progress beyond this stage; but, if the temperature was raised, they quickly rounded up and continued to develop. This block to development was probably due to the low temperature inhibition of cell movement (Galtsoff, 1925a), which prevented the randomly adhering cells from proceeding with reorganization until the temperature was raised. It appeared that mechanically dissociated cells retained some factors which had been lost by the chemically dissociated cells. Reconstruction
of ‘Cell Adhesions after Ch.emical Dissociation
The inability of chemically dissociated cells to adhere at 5” even though calcium had been added back was strikingly abolished if the cell-free supernatants from chemical dissociation were also added; the cells adhered into compact aggregates of randomly mixed cells resembling closely those of mechanically dissociated cells at 5”. At 5°C they remained at this stage for days. If the temperature was raised within 3 days they rounded up and proceeded to develop. It appeared that the supernatants from chemical dissociation contained the missing factor which had been dissolved from the cells by the removal of divalent cations. Divalent
Cation Requirement
In the presence of the factor and divalent cations, chemically dissociated cells adhered into one large aggregate at 5OC. With only the
factor and no divalent cations, they did not adhere at all. The specificity of this cation requirement was examined, and calcium and magnesium were found equally effective, but strontium was completely ineffective. Just as divalent cations were necessary for aggregation, they were also required for the development of cell adhesions in the presence of the factor. It appears then that during dissociation the cell adhesion has been separated into three components; the cell surface, divalent cations, and an organic factor; which when placed back together in C&D spontaneously reassemble to form new cell adhesions. Qlrantitatice Assuy
of the Active Fuctor
In order to obtain supernatants with a higher concentration of the active factor, a modified dissociation procedure was adopted for extraction of the material (see Methods section). This yielded a very active solution. When it was tested in the reconstruction procedure at 5”, all the cells in the flask adhered into one large, rough, cylindrical aggregate about 1.5 mm in diameter and 5-6 mm long (Fig. 7a). This large aggregate consisted of smaller compact cell masses held loosely together by strands of cells. There were numerous unoccupied areas between (Fig. 6). F irm round clots and mucous strings of amorphous material with scattered cells also appeared at this high concentration. As the factor was diluted the aggregates became smaller (Fig. 7b) and the amorphous material did not develop. At the lowest effective concentration many rough, compact aggregates about 0.1 and 2.0 mm in diameter were formed (Fig, 7~). At greater dilutions the cells behaved as if they were in MBL-SW alone (Fig. 7d). This distinct end point provided a possibility of quantitating the activity of a preparation. The least effective concentration of factor was therefore defined as one unit activity per milliliter. The number of units per milliliter in a particular preparation was equal to the dilution required to reach the threshold minimal activity. lMost preparations of crude factor had an activity of 128 units/ml. The activit) of the solution was rapidly lost unless calcium was added to a concentration of 1.8 mM. Source of the Active Facto? Three experiments were done to determine the source of the material: (1) a mock extraction of the factor was conducted using 14BL-SW rather than CMF-S\fy; (2) sponge tissue was homogenized
40
TOM
HUMPHREYS
FIG. 7. Aggregates from reconstruction procedure at 5°C. (a) End of cylindrical aggregate formed at very high concentrations of factor. (b) Aggregates at intermediate concentration. (c) Aggregates at lowest effective concentration. (d) Cell adhesion which occurs below effective concentration. FIG. 8. Haliclonn aggregates. (a) At 22”C, (b) at 5°C.
CO1\lI’ONENTS
OF
SPOSGE
(:ELL
.\DHESIOXS
41
in MBL-SW; and (3) chemically dissociated cells were homogenized in CMF-SW. Examination of the homogenized cells showed that a majority were disrupted. None of the supernatants obtained from the extractions were active; none caused cell adhesion. Species
Specificity
Species specificity of aggregation in sponges has been repeatedly described (Wilson, 1910; Galtsoff, 1923; delaubenfels, 1932). The existence of this specificity provided a very useful and important possibility of demonstrating the specificity of the reactions involved in reassembly of the cell adhesion. Rotutiotz-),lediatetl aggregation of Halichu. Kotation-lnediated aggregation of a second species. Ifaliclonu occzrlutu (II), was studied. Chemically dissociated tl cells aggregated rapidly at 22°C to form compact, round O.l- to 0%mm aggregates (Fig. 8a). The aggregation process for N was very much like that for hl cells at 28” except that the aggregates were short lived. At 5” much smaller, 0.05 mm, round aggregates formed from chemically dissociated H cells (Fig. Bb). Mechanically dissociated cells at 5” formed aggregates with sizes intermediate between 5” and 23” H aggregates. Hulicrona factor. Supernatants from the extraction procedure applied to H tissue with the slight modifications specified in the Methods section caused chemically dissociated H cells to adhere into round aggregates about 0.3 mm in diameter at 5” (Fig. 9). This is about 200 times the cell mass of aggregates formed in MBL-SW at 5”. There was a tenuous halo of clear amorphous material around each aggregate, and numerous firm, round clots with scattered small aggregates in the culture. Serial dilution of the H crude factor resulted in smaller aggregates until at about L3, ’ o the concentration no effect could be detected. The H factor required divalent cations. Interspecific mixtuws in ~otatiofz-mFdi,utetl ugwgation. $1 is bright red-orange, and II is bluish purple. This color difference can be used to determine the species of small aggregates and even single cells of the larger cell types in living preparations. The specificitv of cell
42
TOM
HUMPHREYS
adhesion in mixtures of M and H cells was thus examined. Intermingled chemically dissociated M cells and H cells aggregated at 22” onIy with their own species. W’ithin 15 minutes any aggregate observed was distinctly colored like one or the other species. Cells of the one species were rarely found associated with aggregates of the opposite species. Thus in these mixed suspensions, aggregated by rotation, cells of each species aggregated independently of the other; the final size of aggregates was generally smaller than for each species separately. Aggregation of mechanically dissociated H and M cells at 5” was equally specific. Specificity of the active factor. When M factor was added to chemically dissociated H cells at 5” or H factor was added to chemically dissociated M cells at 5”, they had no effect; the cells of both species behaved as if they were in plain MBL-SW. M factor added to a mixture of M cells and H cells caused the M cells to adhere into a large aggregate, while the H cells behaved as if they were in plain MBL-SW at 5”. H factor tested on a mixture of cells from the two species caused the H cells to form large 0.3-mm aggregates while the M cells behaved as if they were in MBL-SW. Finally, if M factor and H factor were added to a mixture of M cells and H cells, the cells from each species adhered into completely separate aggregates. After 15 minutes the cells in such a mixture were in large, rather loose but separate aggregates. The cells continued to adhere separately and finally formed aggregates exactly as they would if each species had been in a separate flask (Fig. 10). The failure of any of these preparations to develop interspecific adhesions demonstrated that the spontaneous reassembly of the cell adhesions involved specific bonds. This specificity evidently lies in the reactions between the cell surface and the factor of the other species would have resulted in interspecific adhesions in the bispecific cell mixtures. DISCUSSION
The aggregation resulting upon recombination of cells, factors, and divalent cations could be due either to normal cell adhesions or to some nonfunctional agglutination of cells. Various evidence supports the conclusion that the separated components are involved normally in cell adhesion and that the reassembled adhesions are similar to normal, functional adhesions. The aggregates resemble early stages of normal aggregation and can proceed with development if the
COhl1’OSEIUTS
OF
SPONGE
CELL
ADHESIONS
43
temperature is raised to 22”. Divalent cations are required as in normal sponge cell aggregation. Removal of divalent cations is required to release the factor from the cells, as it is required to dissociate cells. The factor cannot be extracted from the cells by homogenization, indicating that it is not a product of cell destruction during the dissociation procedure. Calcium stabilization of the factor demonstrates that divalent cations may be closely associated with its function. The species specificity of the factor parallels that of normal sponge aggregation and rules out the possibility that its activity is clue to a nonfunctional agglutination of cells caused by some general physical or chemical characteristic of the substance such as charge ( Schmitt, 1941) . Th ese various considerations indicate that the facto] represents a dissolved intercellular matrix functioning in cell adhesion by binding the adjacent cell membranes together. This agrees with previously postulated role of cell surface materials in cell aggregation suggested by studies on embryonic cells (Moscona 1960, 1962). The factor demonstrated in the reconstruction of sponge cell adhesions fits historical expectations of an intercellular cement (Ringer, 1890; Herbst, 1900; Gray. 1926; and others); that is, it reacts with divalent cationic bridges bind the material to the cell surface. The adhesion similar to these early ideas but including certain elaborations necessitated by the present data is supported. Sponge cells appear to be held together by an intercellular material bound to each cell surface bt~ specij% ho?zds involving divalent cations. The divalent cations are thought to be involved in the bonds between the material and the cell surface because the factor comes off the cell only when these ions are removed. In this binding the ions apparently do not simply counteract the effects of the negative charge on the cell surface (Schmitt, 1941; Curtis, 1962), but must interact with the intercellular material since the ions are necessary for maintenance of the factor’s functional activity. A possible explanation for their flmction is that divalent cationic bridges bind the material to the cell surface, The cationic and species specificities demonstrate that the bonds in which these divalent cations might be involved could not be simple electrostatic attractions between charged groups, but rather specific complementary reactions between chemical groups on the cell surface, groups in the material, and the cations. Another possibility is that the divalent cations hold the molecules of the intercellular material in the functional configuration necessary for their specific interaction with
44
TOM
HUMPHREYS
the cell surface. The nature of the chemical linkages within the intercellular material itself cannot be deduced from the present data; past ideas that they are cross links of a precipated calcium salt are interpretations which are not critically supported by evidence. Various other morphological and experimental observations relating to cell adhesion agree with the above model. The importance of divalent cations in cell adhesion has been repeatedly confirmed (Steinberg, 1958). Many observations, such as morphological identification of intercellular materials (Gersh and Catchpole, 1949; Fawcett, 1958) or enzymatic dissociation of cells (Moscona, 1952), have indicated the presence of intercellular materials (Rinaldini, 1958; Moscona, 1960). Cell adhesion is specific in many instances (Wilson, 1910; Galtsoff, 1925a; Holtfreter, 1939; Raper and Thorn, 1941; Townes and Holtfreter, 1955; Moscona, 1957). Also, antigenically specific cell surface substances, which may be equivalent to the specific intercellular material of the present studies, have been demonstrated to be important in cell adhesion (Spiegel, 1954a,b; Gregg, 1956; Gregg and Trystad, 1958). The demonstration of a component other than the cell surface and divalent cations in cell adhesions does not lend support to the models of cell adhesion based on calcium bridges (Coman, 1954; Steinberg, 1958) and long-range forces (Curtis, 1960). The 100-200 A electronlucid separation commonly observed between membranes of adjacent cells viewed with the electron microscope is the main evidence for these theories. However, the interpretation of this electron-lucid area as an empty space (Coman, 1954; Curtis, 1960, 1962) has been questioned (Robertson, 1960) and must be reconsidered with the present experimental demonstration of an intercellular material involved in cell adhesion. SUMMARY
The purpose of this work was to study the mechanism of cell adhesion using aggregation of dissociated sponge cells as the experimental system. A method of chemical dissociation was developed for marine sponges using cold calcium- and magnesium-free sea water. The viable, single cells resulting from this dissociation were able to aggregate and develop into functional sponges. Rotation-mediated aggregation was used to study various aspects of the aggregation of sponge cells.
C;Olrfl'OXENTS
Ok- SPONGE CXLL
i\DHESIOSS
-45
Using these techniques, a specific requirement for the divalent cations calcium and magnesium was established for sponge cell adhesion. Low temperature was found to inhibit adhesion of chemically dissociated cells even when the divalent cations were added back to the cells. hIcchanicullv dissociated cells aggregated rapidly at low temperatures. This difference was sho\\*n to be due to a factor released into the supernatant during chemical dissociation of sponge tissue. \Vhen this factor was added back to the chemically dissociated cells along with tlivalent cations, they adhered rapidly at low temperatures. This facto1 was species specific, causing aclheslon cinlv ot cells from the same species. These rcslllts ww interpreted to indicate that the sponge cell acllesion was cor~~poset~ of three basic components: the cell surface, divalent cations, and an intt*rcellular material. Tlwse components had been scjlaratcd dnring chemical dissociation and were capable of spontaneously and species specifically reassembling themscl\w to rt‘;orm an apparently normal cell adhesion. The implications of these results for models of cell adhesion are rliscrissed. REFERENCES
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bornt’
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AII~.
S!/UI~~.
1958, Johns Hopkins Univ. McCoillm-Putt
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