Cellular Adhesiveness and Extracellular Substrata

Cellular Adhesiveness and Extracellular Substrata FREDERICK GRINNELL Department of Cell Biology The University of Texas Heulth Science Center at Dallas Dallos. Texas

I . Introduction . . . . . . . . A . Adhesion and Separation . . . . . B. Endogenous Alterations of the Experimental Conditions . . . . . . . . C . Measurement of Cell Adhesion . . . I1. The Multistep Paradigm . . . . . . A. The Nature of the Substratum and Adsorption of Proteins . . . . . . . . B. Cell Contact with the Substratum . . . C . Formation of the Bonds of Attachment . . D . Cell Spreading onto the Substratum . . . . . . I11. The Role ofthe Substratum . A . Physical Structure of the Substratum . . B. Anchorage Dependence of Cell Growth . . C . Chemistry ofArtificia1 Substrata . . . D . Activation of Substrata for Tissue Culture Use E . Wettability and Protein Adsorption . . . IV. The Influence of Proteins on Cell Adhesion . A. PassiveandActiveAdhesion . . . . B. Observation of Proteins on the Substratum . . . . . . . . Surface C . Effects of Various Serum Factors . . . D . Model of Passive and Active Cell Adhesion . E . Cell Adhesion to Model Physiological Substrata V. The Role of Microexudates in Cell Adhesion . A. Observation of Microexudates . . . . B. Chemistry of Microexudates . . . . C . Functions of Microexudates . . . . . . . VI . Cell Contact with the Substratum . A. Role of Microvilli and Filopodia . . . B. BiochemistryofCell Contact . . . . VII . Cell Spreading onto the Substratum . . . A. Sequence of Events . . . . . . B. Role of Filopodia . . . . . . C . Substratum Requirements . . . . D. Role of Microfilaments and Microtubules . VIII . Location of Bonds of Cell Attachment and Their Ultrastructural Appearance . . . . . A . Marginal Location of Cell-Substratum Attachments . . . . . . . B. Cell-Substratum Attachments beneath the Cell . . . . . . . . Body . 65

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C . Ultrastructure of Cell Attachments . . . D . Adhesiveness of the Upper Cell Surface . IX . Biochemistry o f c e l l Adhesion . . . . A . Kinetics . . . . . . . . B. TemperatureDependence . . . . C . Energy Dependence . . . . . D . Cation Dependence . . . . . . E . Requirements for Free Sulfhydryl Groups . F . Inhibition by Trypsinization . . . . G . Other Inhibition Studies . . . . . H . Modulation of Cell Adhesion by Hormones and CAMP . . . . . . . . . I . Cell Adhesion Mutants . . . . . J . Adhesion of Enucleated Cells . . . . K . Summary of Biochemical Analyses . . . X . Cell Separation . . . . . . . A . Strength of Cell Adhesion . . . . B . Separation of Actively and Passively Attached Cells . . . . . . . . . C . Mechanism of Cell Separation . . . . D . Other Techniques of Separation . . . XI . Cell Surface Receptors . . . . . . A . Ligand-Induced Cell-Substratum Adhesion and Spreading . . . . . . . . B . Bridgingby Divalent Cations . . . . C . Glycosyl Transferases . . . . . D . LETS Protein . . . . . . . E . ATPase Activity . . . . . . F. Asialoglycoprotein-Binding Protein . . XI1 . Platelet Adhesion . . . . . . . A . Substrata for Platelet A d h e s i o n 4 n Vitro . B . Substrata for Platelet Adhesion-in Vivo . C . Biochemistry of Platelet Adhesion . . . D . Requirement for Plasma Components and Cell Surface Receptors . . . . . . XI11 . Macrophage, Monocyte, and PJeutrophil Adhesion A . Phagocytosis and Spreading . . . . B. Opsinin Requirement for Adhesion . . C . Divalent Cation Requirement . . . . D . Energy Dependence . . . . . E . Requirement for Sulfhydryl Croups . . F . Pseudopodia, Microfilaments, and Microtubules C. Separation of Macrophages from the Substratum H . Surface Receptors . . . . . . I . Margination and Emigration of Leukocytes . XIV . Red Blood Cell and Lymphocyte Adhesion . . A . Adhesion of Red Blood Cells . . . . B . Adhesion of Lymphocytes . . . . XV . Cell Migration and Chemotaxis . . . . A . Events in Cell Migration . . . . . B. Migration in Response to Adhesive Gradients

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ADHESIVENESS AND EXTRACELLULAR SUBSTRATA C. Selective Fixation by Ligands . . . . . D. Leukocyte Chemotaxis . . . . . . XVI. Cell Adhesion in Embryogenesis, Morphogenesis, and Wound Healing . . . . . . . . A. Modulation of Cell Behavior by Adhesion . . B. Collagen as an Inductive or Permissive Substratum . C. T h e Substatumas aTemplate . . . . . D. Changes in Cell Adhesiveness during Embryogenesis . . . . . . . . E. T h e Substratum and Cellular Events during Wound . . . . . . . . . Healing XVII. Cell Adhesion in MaIignancy . . . . . . A. Loss of Anchorage Dependence . . . . B. Changes in Cell Shape and Adhesiveness . . C. Alteration of CAMP Levels, LETS Protein, and . . . . . . . Cell Proteases . D. Loss of Contact Inhibition of Cell Overlap . . E. Cell Invasiveness . . . . . . . F. Cell Surface Negative Charges. . . . . G. Blood-Borne Metastasis . . . . . . H. Foreign Body-Induced Sarcomas . . . . XVIII. Conclusions . . . . . . . . . References . . . . . . . . .

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I. Introduction Cell adhesiveness is a fundamental cell property. It plays a role in developmental processes such as cell migration during embryogenesis and morphogenesis in response to particular extracellular matrices; it plays a role in homeostatic processes such as tissue and organ stability, thrombosis, inflammation, and wound healing; and it plays a role in the pathology of various disease states, for instance, in the invasive and metastatic behavior of malignant cells, in disorders of platelet function, and in disorders of leukocyte function. Two approaches to studying the problem of cell adhesion have been characterized: the adhesion of cells to each other, and the adhesion of cells to extracellular substrata (Fig. 1).This article is primarily concerned with the latter phenomenon. Studies have been carried out on the adhesive properties of fibroblasts, epithelial cells, macrophages , leukocytes, platelets, and red blood cells, and information is available on a variety of cell types both i n vitro and in vivo. Our goal is to bring together many of these studies and to emphasize those aspects of cell adhesiveness that indicate there are common underlying mechanisms in all systems. To carry out this project and consider all the literature that relates in one way or another to cellular adhesiveness is beyond the scope of

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ADHEWN

- SEPARATION

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CELL-SUBSTR

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FIG. 1. Models for the study of cellular adhesiveness. Details are in the text.

this article. Therefore we have chosen to include, perhaps sometimes arbitrarily, research we feel is most relevant to understanding &e mechanism of adhesiveness. Some important cell -extracellular substratum interactions, notably cell migration and phagocytosis, have been only partially touched on. Also, we have chosen to follow a biological and biochemical point of view and have largely omitted biophysical considerations. For additional information on cell -cell adhesion or the biophysics of cell adhesion, the reader is referred to the reviews by L. Weiss (1967a) and by Curtis (1967,1973); cell migration was the subject of a symposium several years ago (Porter and Fitzsimons, 1973); and Allison and Davies (1974) and Korn (1975)have reviewed phagocytosis and other endocytotic processes. This article is most complete in its review of fibroblast adhesion, and Sections I1 through XI are a summary of in vitro studies on fibroblasts along with some pertinent information on epithelium and nerve. Sections XI1 through XIV are less detailed and are concerned with both in vitro and in vivo studies on the adhesive properties of platelets, leukocytes, macrophages, and red blood cells. Finally, Sections XV through XVII are a general discussion of adhesiveness in cell migration and chemotaxis, embryogenesis, morphogenesis, wound healing, and malignancy.

A. ADHESIONAND SEPARATION Certain issues in understanding cellular adhesiveness require special attention. One of these is the distinction between cell adhesion

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and cell separation. Cell adhesion is a dynamic process (Fig. 1).The rate of cell adhesion depends on the rate of initial contact of cells with the substratum or with each other and the subsequent formation of attachment bonds. Ultimately, an equilibrium or pseudoequilibrium state is reached, and the cells attain a certain morphology a d strength of cell attachment depending on the number and organization of attachment bonds and cytoskeletal structures. However, cell separation involves bonds of attachment after they have already been formed and depends on their accessibility and sensitivity to being broken. Separation is thus independent of the contact interaction, whereas it is precisely the contact interaction that is rate-limiting for adhesion (Grinnell, 1976a). A second point is that there is no reason to expect cell adhesion and cell separation to occur at the same site. For instance, if the forces in the bond of attachment exceed the forces within the cell surface (i.e., the plasma membrane-glycocalyx continuum), separation may occur within the cell surface and not across the bond of attachment (L. Weiss, 1967a). This is in fact what happens (Section X). The distinction between adhesion and separation cannot be overemphasized and, in trying to understand cell behavior in vivo and in vitro, one must be critically aware that these processes are not the same.

B. ENDOGENOUS ALTERATIONS OF THE EXPERIMENTAL CONDITIONS

Another important problem involved in understanding cellular adhesiveness is defining the experimental conditions. Cells are metabolically active, and products of this activity (conditioning factors and microexudate) are continually secreted into the medium and onto the substratum surface. Thus the time period during which one carries out an experiment may have an influence on cell adhesion, because of the accumulation of synthesized substances that alter intrinsic cell adhesiveness. Moreover, if substances are absent that are required in the medium for long-term cell survival or growth, progressive cell death will interfere with cell adhesive properties. Cells may also undergo time-dependent repair processes. Therefore, if they have been reversibly perturbed prior to an adhesion experiment (e.g., by treatment with trypsin), whether or not the effects of this pertubation are observed will depend on the duration of the experiment relative to the time required for repair to occur.

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MEASUREMENTOF CELLADHESION The last point to be made concerns how cell adhesion and cell separation are determined. In general, whether or not a cell is attached to the substratum is arbitrarily and operationally defined according to the shear force it must resist to avoid being dislodged. The greater the shear force used, the greater the number of bonds of adhesion required before an individual cell is observed to be attached. Similarly, the greater the shear force used, the more readily cell separation occurs. Therefore, depending on methodology, substantial variation often exists from laboratory to laboratory in determining the rate or extent of cell adhesion and separation. C.

11. The Multistep Paradigm When cell adhesion is observed with light or scanning electron microscopy, many events are seen to occur, but overall there is a dramatic shape change from a rounded to a flattened morphology (Taylor, 1961; Witkowski and Brighton, 1971; Rajaraman et al., 1974). To understand this phenomenon, one should know what cell surface components are involved in adhesion, what constitutes the bond of adhesion, and what cell surface and cytoplasmic forces are responsible for the shape change. In the process of attempting to find the answers to these questions, it has become clear that cell adhesion occurs in a series of steps which can be distinguished biochemically and measured separately (Grinnell, 1976a). These are diagramed in Fig. 2. Before discussing them in detail in subsequent sections, it is useful to summarize the various steps.

ADSORPTION

ATTACHMENT

CONTACT

SPREADING

FIG.2. Multistep paradigm of cell adhesion. Details are in the text. From Grinnell

(1Y76a; Fig. 1).

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A. THENATURE OF THE SUBSTRATUM AND ADSORPTION OF PROTEINS Whether or not cell adhesion occurs onto a particular Substratum, or how it occurs, depends on two factors. First, the chemistry and structure of the substratum have a profound effect on adhesion. Second, if proteins are added to the medium (e.g., by adding serum), or if the cells produce a proteinaceous microexudate, the substratum will be-

come coated with adsorbed proteins and cell adhesion will be determined by the chemistry of the adsorbed components and how the cells interact with them. Cell adhesion to protein-free substrata appears to be a nonspecific adsorption phenomenon independent of the metabolic state or viability of the cells, whereas adhesion to proteincoated substrata is dependent on cell physiology and involves only a limited group of adsorbed proteins.

B. CELLCONTACTWITH THE SUBSTRATUM The initial interaction of cells with the substratum is dependent to a large extent on the ability of cells to protrude cytoplasmic microexten-

sions actively. Since cells carry a net negative surface charge at physiological pH and the substratum surface is also negatively charged in most cases, there is usually an electrostatic barrier between the two surfaces. Cytoplasmic microextensions seem to be important in penetrating this barrier, thereby bringing the cell surface close enough to the substratum surface to permit adhesion. OF THE BONDSOF ATTACHMENT C. FORMATION

Following contact of the cells with the substratum, bonds of attachment form. The bonds between cells and protein-free substrata are nonspecific, whereas those between cells and protein-coated substrata appear to be mediated by specific ligand-receptor-like interactions. Cell adhesion may require the redistribution of cell surface adhesion receptors.

D. CELLSPREADING ONTO THE SUBSTRATUM After initial contact and adhesion of the cells, there is a general reorganization of the microtubules and microfilaments that comprise the cell’s cytoskeleton. Cytoplasmic extensions called filopodia are protruded from the cell and become attached to the substratum, resulting in a progressive flattening of the cell on the substratum and an increase in strength of cell attachment.

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111. The Role of the Substratum A. PHYSICAL STRUCTURE OF THE SUBSTRATUM From the earliest studies, it has been clear that cells are rounded in suspension but spread out after attachment to a solid substratum. Moreover, their morphology depends on and responds to the shape and texture of the substratum. The general observation is that cells spread into highly polarized shapes in the direction of least curvature of the substratum. This finding has been made for cells attached to spider webs or plasma clots (Harrison, 1914), mica plates or glass fibers (P. A. Weiss, 1945),and a variety of other surfaces with natural or artificial grooves (P. A. Weiss, 1961, 1962; Rosenberg, 1963; Curtis and Varde, 1964; Rovensky et al., 1971; Ivanova and Margolis, 1973). A similar observation also has been made for elongating nerve axons (P. A. Weiss, 1945; Dunn, 1973; Ebendal, 1976; Cooper et aZ., 1976). The mechanism of contact orientation appears to involve the cell cytoskeleton. As discussed in Section VII, arrays of cytoplasmic microtubules and microfilaments oriented parallel to the substratum play an important role in cell spreading and orientation. Apparently, these linear elements do not bend readily. Therefore the direction of least curvature of the substratum becomes the direction of least resistance to cell spreading or axon elongation (Dunn and Heath, 1976). OF CELL GROWTH B. ANCHORAGEDEPENDENCE

Normal cells, but not transformed cells, require adhesion to a substratum in order to grow (Stoker et ul., 1968). Moreover, the substratum has to be of a certain size. Maroudas (1972, 1973a,b) discovered that, if the size and shape of the substratum do not permit cell spreading, growth is prevented. H e found that for normal cells to grow, their normal length when they are spread must be less than the circumference of glass bead substrata or less than the length of glass fiber substrata. C. CHEMISTRY OF ARTIFICIAL SUBSTRATA An early observation indicating the importance of substratum chemistry in adhesion was the dependence of adhesion on the type of glass substratum used. A critical number of negative charges on the glass was thought to be required (Rappaport et al., 1960; Rappaport, 1971). Maroudas (1975a, 1977)has reached a similar conclusion using chemically etched substrata and reported that for the substratum to be active it must contain at least 5 negatively charged groups per 100 A'. L. Weiss (1960) was the first to point out the requirement for sub-

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stratum wettability in adhesion. This notion was not supported by Taylor’s studies on cell adhesion to various substrata ( 1961); however, he compared protein-coated with nonprotein-coated substrata. This is not a valid comparison, since these conditions lead to different kinds of adhesion, and a reevaluation of Taylor’s work (Baier et al., 1968) indicates that his findings support the dependence of adhesion on wettability. In addition, many comparative studies have demonstrated preferential cell adhesion to wettable substrata (Carter, 1967a; Weiss and Blumenson, 1967; Hochmuth et al., 1972; Gail and Boone, 1972; Grinnell et uZ., 1972, 1973a; Maroudas, 1973b; Harris, 1973a; Ratner et al., 1975). Nerve axons also attach preferentially to wettable substrata (Letourneau, 1975b; Cooper et aZ., 1976). It should be pointed out that hydrogels such as agar are unlike other wettable substrata and do not support cell adhesion (L. Weiss, 1959); however, the reason for this difference is unknown.

D. ACTIVATIONOF SUBSTRATA FOR TISSUE CULTURE USE Petri dish polystyrene is a substratum of low wettability and does not generally support cell adhesion, although several exceptions have been reported (Ballard and Tomkins, 1970; Kolodny, 1972; Pouyssegur et al., 1977).Therefore, since growth of normal cells is anchorage-dependent (Stoker et al., 1968), such nonwettable substrata are not useful for routine tissue culture applications. However, nonwettable plastic can be converted to a wettable substratum for tissue culture by chemical etching techniques (Rubin, 1966a; Munder et al., 1971; Martin and Rubin, 1974) or by physical techniques such as “electret” formation (Murphy et al., 1971) and “glow discharge” (Amstein and Hartman, 1975).According to the Biquest Corporation (personal communication), Falcon wettable plastic is obtained by producing “a glow discharge between two electrodes at high voltage.” This kind of technique has had general applications in the plastic industry and has been patented in some instances (e.g., Ambroski, 1966). In principle, bombardment of the plastic with high-energy electrons breaks the polymer structure, resulting in the formation of negatively charged carbonyl groups as well as unsaturated groups and possibly peroxides, depending on the atmosphere in which the bombardment occurs.

E. WETTABILITY AND PROTEIN ADSORPTION Baier et al. (1968)have reviewed the role of wettability in adhesion.

In biological systems, substratum wettability seems to have its major influence on the nature of protein adsorption, which occurs almost

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FREDERICK GRINNELL

instantaneously on exposure of the substratum to protein-containing solutions (Baier, 1972; Vroman, 1972; Olsen and Kletschka, 1973). The conformation and biological activity of the adsorbed protein depends on the chemical characteristics of the substratum. For instance, Hageman factor (factor XI1)-the initiation protease in the intrinsic blood-clotting sequence-is preferentially activated from the proenzyme form by adsorption onto a wettable substratum, particularly one with repeating negative charges such as collagen (Vroman, 1967; Wilner et al., 1968a; Mason et al., 1972). In general, the substratum takes on the chemical characteristics of the adsorbed protein, and following adsorption hydrophobic substrata become more wettable and hydrophilic substrata become less so (Hayry et al., 1966; Vroman,

1967). As seen in the subsequent Section, serum and plasma proteins have

profound effects on cellular adhesiveness. The influence of substratum wettability on biological adhesion may be in part a result of the adsorption of particular proteins in active or inactive conformations or states.

IV. The Influence of Proteins on Cell Adhesion A.

PASSIVE AND

ACTIVEADHESION

Taylor (1961) was the first to draw a clear distinction between passive and active adhesion. In the absence of serum in the culture medium, cell adhesion to an artificial substratum occurs completely and almost instantaneously. It does not appear to depend on the cells being metabolically active or viable. Some of the accumulated evidence in support of this contention is shown in Table I. It can be seen that, in the absence of serum, adhesion is independent of temperature, pH, cell integrity, and cell fixation. In short, cell adhesion in serum-free medium probably occurs through direct adsorption of cells onto the substratum at the same sites at which proteins adsorb. Taylor (1961) also observed that cells spread from the point of contact in serum-free medium, whereas they spread b y protrusion of microextensions in serum-containing medium. A similar finding was made by Witkowski and Brighton (1972). In both studies, there was irregularity in the shapes of cells spread in the absence of serum which was interpreted to indicate a stronger cell-substratum interaction resulting in distortion of the cell contours. A similar effect occurs with nerve axons in serum-free medium (Luduena, 1973).

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TABLE I CHARACTERISTICS OF CELLADHESION IN THE PRESENCE AND ABSENCE OF SERUM IN THE MEDIUM Property

Without serum

With serum

Temperature dependence

No

Yes

pH dependence

No

Yes

Adhesion of lysed cells or cell fragments

Yes

No

Adhesion of fixed cells

Yes

No

Reference Nordling (1967); Unhjem and Prydz (1973); Rabinowitz and DeStefano (1973a) Taylor (1961); Takeichi and Okada (1972) Nordling (1967); George et al. (1971); Grinnell (1974a) Taylor (1961); Nordling (1967); Grinnell (1974a)

These two mechanisms of spreading-from point of contact and by cell protrusions-basically correspond to the “passive” and “active” spreading models described by Carter (1967a) and Wolpert et al. (1969). It now appears that, for a variety of cell types, there is no passive spreading phenomenon and cells remain rounded in serum-free medium. These include HeLa cells (Fisher et al., 1958; Michl, 1964; Nordling, 1967),human appendix A 1 cells (Lieberman and Ove, 1957, 1958), monkey kidney cells (P. A. Weiss, 1962), baby hamster kidney (BHK) cells (Wolpert et al., 1969), L cells (Price, 1970), and Ehrlich ascites tumor cells (Baier and Weiss, 1975). It may be an unfortunate misnomer that cell spreading in the absence of serum was called “passive.” Spreading in serum-free medium may depend upon the active secretion of microexudates on the substratum surface (Taylor, 1962; Takeichi, 1971; Yaoi and Kaneseki, 1972) thereby conditioning the substratum so as to enable spreading to occur (see Sections V and X1,D.). The inability of some cells to spread in serum-free medium may be because they cannot secrete an appropriate microexudate.

B. OBSERVATION OF PROTEINSON THE SUBSTRATUM SURFACE The adsorption of serum proteins has been measured by ellipsometric techniques, and the thickness of the adsorbed protein layer has been found to be 20-50 8, (Rosenberg, 1960; Taylor, 1961). The rate of protein adsorption from plasma is 10-20 A per minute (Vroman, 1967).

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FREDERICK GFUNNELL

The protein layer has also been observed ultrastructurally. When the attached cells are peeled away from the substratum, the fracture takes place between the protein layer and the substratum, and the protein layer appears as an electron-dense precipitate on which the cells rest (Brunk et al., 1971; Revel and Wolken, 1973; Pegrum and Maroudas, 1975; see Fig. 5). When the attached cells and the substratum are sectioned together, the protein layer appears as a -50-A electron-dense line between the cells and the substratum (Taylor, 1970; Grinnell et al., 1976; see Figs. 4 and 6). Finally, the protein layer on the substratum can also b e detected by staining techniques (Dillman and Miller, 1973) or by the binding of plant lectins (Rowlatt and Wicker, 1972). C. EFFECTS OF VARIOUSSERUMFACTORS Serum proteins affect cell adhesion by adsorbing onto the substratum surface, thereby preventing the passive adsorption of cells. At the same time, the adsorbed proteins become the sites to which cells attach, as first discussed by Lieberman and Ove (1958) and Taylor (1961).It has now become clear that all the direct influences on adhesion of serum in the medium can be obtained by pretreatment of the substratum with serum (Taylor, 1961; Nordling, 1967; Grinnell, 1976b,c). The question of specificity in the types of serum proteins that promote active cell adhesion has been of great interest. It now appears that serum proteins play many different roles in cell behavior. 1. lndirect Effects of Serum

L. Weiss (1959) reported an indirect requirement for serum in the reversal of trypsin damage to cells. Also, the stimulatory effect of fe1958) apparently is tuin on cell adhesion and spreading (Fisher et d., due in part to its antitrypsin action (Hebb and Chu, 1960).A second indirect effect of serum proteins occurs because most cells require serum in the medium in order to survive and grow (Eagle, 1955; Clarke et al., 1970; Paul et al., 1971; Temin et al., 1972; Houck and Cheng, 1973; Holley, 1975). Therefore, unless the cells are capable of secreting their own growth factors during long-term adhesion experiments, they lose viability and physiological adhesiveness.

2. Nunspecific Serum Inhibitory Factors Direct effects of serum proteins on cell adhesion have been measured in two ways. First, the presence of senim in the incubation results in a decrease in the rate of cell adhesion. Second, cell spreading requires the presence of serum in the incubation for many cell types. The decreased rate of' cell adhesion in serum-containing medium ap-

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pears to result because the sites on the substratum to which the cells could have adsorbed are blocked by the prior adsorption of the proteins onto these sites. This effect can be elicited by most serum proteins and does not appear to be specific (Nordling et al., 1964; Nordling, 1967). Serum lipoproteins (Myllyla et al., 1966) seem to be the most potent inhibitors.

3 . Specific Serum Activators For many cell types, active cell adhesion leading to cell spreading onto a substratum requires the presence of serum components adsorbed to the substratum surface. That only certain serum components are involved is shown by the complete inhibition of cell adhesion when the substratum adsorption sites are occupied by bovine serum albumin (BSA) (Unhjem and Prydz, 1973; Moore, 1976; Grinnell et aZ., 1977) or heat-treated serum (Roseman et al., 1974; Grinnell, 197613). The first spreading factor isolated from serum was described by Lieberman and Ove (1957,1958), and it was 15fold purified and believed to be a 7 s glycoprotein. About the same time, fetuin was reported to be a spreading factor (Puck et al., 1956; Fisher et aZ., 1958);however, later studies indicated that the spreading factor activity could be separated from fetuin by DEAE-cellulose column chromatography (Lieberman et al., 1959). Specific serum fractions that promote cell spreading also have been reported for HeLa cells (Unhjem and Prydz, 1973; Michl, 1964),and Holmes (1967) has partially isolated a human a1 glycoprotein which functions with several different cell types. Most recently, an adhesion and spreading factor for BHK cells (designated ASF) was purified 150-fold from fetal calf serum (Grinnell, 1976a,b,c; Grinnell et al., 1977). There are two active components, both of which are globular glycoproteins. The larger is 12.5s and contains polypeptides of approximately 215,000 molecular weight. The smaller is 9 s and contains polypeptides of 94,000,80,000, and 71,000 molecular weight. The mixed ASF preparation contains about 10% carbohydrate, including 3% sialic acid. Amino acid analysis indicates no unusual characteristics except the high content (-22%) of glutamic and aspartic residues and the lack of methionine. Also, the isoelectric point has been found to be 4.0. ASF-like activity is found in fetal calf, calf, porcine, rabbit, human, and chicken serum; and immunoglobulin purified from antiserum directed against fetal calf ASF cross-reacts and inhibits the spreading activity in calf and porcine sera completely, and in human serum partially. The chromatographic properties on DEAE-cellulose of the fac-

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FREDERICK GRINNELL

tors from all the sera are similar. In addition to BHK cells, ASF functions with HeLa, L, and Chinese hamster ovary (CHO) cells. Recent studies using human plasma have shown that the well-characterized plasma glycoprotein, cold insoluble globulin (Mosesson and Unfleet, 1970; Mosesson et al., 1975), and ASF are very similar (Grinnell and Hays, 197th). This conclusion is based upon the observations that: (1)clotting of plasma under conditions that remove cold insoluble globulin also remove the adhesion and spreading factor; (2) the activity of the adhesion and spreading factor cochromatographs with cold insoluble globulin antigenicity on DEAE-cellulose and the mobilities of adhesion and spreading factor and cold insoluble globulin are the same in disc gel electrophoresis; and (3) antibody which is directed against cold insoluble globulin cross-reacts with a single component in the adhesion and spreading factor and inhibits its activity. The antibody directed against human cold insoluble globulin also inhibits the adhesion and spreading activity associated with both the 12.5s and 9s components of fetal calf ASF. The activity of ASF depends on its adsorption onto the substratum surface, which occurs within seconds after exposure. Its affinity for the substratum is 50- to 1000-fold higher than that of BSA or bovine gamma globulin. With the most highly purified ASF preparations about 5 pg/ml is required in the medium to ensure complete cell spreading. Of this, a little less than 1pg becomes adsorbed to the substratum surface. Preadsorption of the substratum with other proteins inhibits the expression of ASF activity. Moreover, in competition experiments between ASF and albumin for substratum sites, it is possible to show that both the extent of cell adhesion and of cell spreading are correlated with the density of ASF adsorbed to the substratum (Grinnell et al., 1977). Fetal calf ASF has been subjected to a variety of chemical modifications following its adsorption onto the substratum in an attempt to map the active site (Grinnell and Minter, 1978). These studies have shown that modification of the carbohydrate portions of ASF by glycosidic enzymes or by periodate oxidation does not alter its activity. Modification of some protein portions of the factor by proteolytic enzymes or by specific modification of -COOH groups, tyrosine residues, or tryptophan residues results in a marked inhibition of ASF activity. However, modification of -SH groups, -NH, groups, or methionine residues does not affect factor activity. These findings suggest that the activity of the adsorbed cell adhesion and spreading factor depends upon specific protein portions of the factor and is independent of carbohydrate portions.

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11. MODEL OF PASSIVE AND ACTIVE CELL ADHESION

A simple model explaining the activity of ASF suggests that there is a receptor on the cell surface, which binds to ASF adsorbed onto the substratum surface. At the present time, it seems likely that ASF is functional only after its adsorption. No binding of radioactive ASF to the cell surface has been detected in solution (Grinnell and Minter, 1978).Moreover, there is no difference in the rate or extent of cell adhesion and spreading in comparing experiments in which ASF is on the substratum with other experiments in which ASF is both on the substratum and in the medium. If both adsorbed and soluble ASF had the same birding affinity for the cell surface receptor, cell adhesion and spreading ought to have been competitively inhibited by soluble ASF in the latter experiments. There are two possible mechanisms by which ASF may become functional following its adsorption onto the substratum. One is by a conformational change whereby ASF goes from an inactive form to an active form analogous to the activation of adsorbed Hageman factor (see Section 111,E). Alternatively, there may be a cooperative effect. For instance, if the dissociation constant of ASF and the adhesion receptor is only for a single interaction, then no binding of ASF with the cell surface would be detectable. However, if the dissociafor n-multiple substratumtion constant increases exponentially ( adsorbed ASF molecules interacting with a single cell, then the binding between the solid state ASF and the cells would be very strong. The various possible effects of serum proteins on cell adhesion can be summarized in reference to the model shown in Fig. 3 . In the ab-

FIG.3. Model of cell adhesion to adsorbed ASF. Details are i n the text.

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sence of proteins on the substratum, direct cell adsorption occurs, and this is passive adhesion. In the presence of proteins on the substratum, cells bind to the adsorbed substances. No cell -substratum interactions occur with nonfunctional proteins such as BSA or heated serum (Fig. 3, squares). Cell adhesion occurs through a ligand-receptor-like interaction between a cell surface receptor and ASF following its adsorption onto the substratum surface (Fig. 3, circles). The extent of cell adhesion and spreading depends on the density of ASF on the substratum. Finally, ASF in solution (Fig. 3, triangles) does not interact measurably with the cell surface. Among other things, this model predicts that ligands adsorbed to the substratum which are directed against the cell surface may induce active cell adhesion and spreading (see Fig. 8).This in fact occurs and is discussed in Section XI.

E. CELLADHESIONTO MODEL PHYSIOLOGICAL SUBSTRATA The extracellular matrix in situ is composed of collagenous fibers and a variety of other macromolecules including glycoaminoglycans.

During thrombosis and wound healing, fibrin also may form part of

the natural substratum. Unfortunately, relatively few studies on cell adhesion have been carried out with such substrata in uitro. A system for examining the adhesion of individual cells to isolated basement membrane has recently been described (Overton, 1977) and should

prove very useful in future analyses. Plasma clots were used classically as substrata for cell culture; however, the biochemistry of cell adhesion to these substrata has never been investigated systematically. There have been several recent reports comparing the adhesion of CHO cells on plastic and fibrin coated substrata (Nozowa, 1977; Nozowa and Guerrant, 1977). They found an increase in the degree of cell spreading and strength of cell adhesion on the fibrin substrata and the divalent cation requirements for adhesion to the two substrata were somewhat different. The possible role of serum or plasma proteins in addition to fibrin was not investigated and all of the studies were carried out in the presence of serum. Adhesion of cells to various kinds of collagen substrata has been studied more completely than adhesion to other model physiological substrata. The use of collagen as a substratum for cell growth in place of artificial materials is a well-established technique (Ehrmann and Gey, 1956; Bornstein, 1958). Significantly, cell morphology differs dramatically in fibroblasts embedded in gels of native collagen as compared to those attached to artificial substrata. The fibroblasts be-

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come aligned with collagen fibers and assume a bipolar, spindleshaped form (Elsdale and Bard, 1972). The biochemistry of fibroblast adhesion to dried collagen substrata has been studied with BHK cells by Klebe (1974, 1975). Many of the properties of this interaction are similar to the adhesion of these cells to tissue culture substrata; however, there is no passive adhesion of cells, i.e., adhesion in the absence of serxm. Klebe purified40-fold the calf serum factor required for adhesion and spreading of BHK cells onto dried colIagen substrata. This has been found to be the same adhesion and spreading factor required for BHK cell spreading on tissue culture substrata (Grinnell et al., 1977; Grinnell and Hays, 1978a), namely, cold insoluble globulin (see Pearlstein, 1976; Jilek and Horman, 1977). Kleinman et al. (1976) have shown that the factor binds to specific regions of collagen. Also, cold insoluble globulin is thought to be synthesized by fibroblasts in the connective tissue (Linder et al., 1975). It seems reasonable to suppose that this substance is secreted and becomes adsorbed to collagen fibers where it functions as the substratum for fibroblast adhesion and migration. Moreover, cold insoluble globulin can be cross-linked to fibrinogen by factor XI11 during clot formation (Mosher, 1975, 1976) and might thereby form the substratum for fibroblast adhesion and migration during wound healing, e.g., in the formation of granulation tissue. It should be pointed out, however, that the dried collagen substrata with which adhesion studies have so far been carried out are denatured and lack the native structure of collagen. The same results can be obtained using substrata coated by soluble gelatin. Using native collagen gels (Elsdale and Bard, 1972), we have recently found that BHK cells attach and partially spread onto these substrata in the absence of serum. So far, analysis of the rat tail collagen preparations which we have used in these studies has indicated that there is no contamination by endogenous cold insoluble globulin (F. Grinnell and D. Minter, unpublished observations). Therefore, the role of cold insoluble globulin in fibroblast adhesion to native collagen is still in doubt.

V. The Role of Microexudates in Cell Adhesion In many instances, cultured cells secrete and shed a variety of macromolecular substances into the medium and onto the substratum, and these can substitute for serum components. Cells can also leave fragments behind on the substratum as they move around. The various substratum-adsorbed components have been collectively called the

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microexudate, and the adsorbed substances along with those in solution are loosely termed conditioning factors. A. OBSERVATION OF MICROEXUDATES

The influence of microexudates on cell adhesion was first described

by P. A. Weiss (1945) who reported a colloidal exudate surrounding

cultured embryonic spinal ganglia to which outgrowing nerve axons were attached. However, it was much later before various investigators attempted to define microexudates in physical and chemical terms. Microexudates appear to be deposited on the substratum by cells both in the presence and absence of serum in the medium; however, there is some controversy as to the metabolic dependence of deposition. Using ellipsometry, Rosenberg (1960) reported the deposition of a 40-wlayer during the first few hours of cell culture in serum-free medium, which occurred even at low temperatures. With a similar technique, also in the absence of serum, Poste and Greenham (1971)found the layer to be 35-60 A, depending on cell type; however, deposition was inhibited b y low temperatures and also by cychloheximide or actinomycin D (Poste et aZ., 1973). Microexudates have also been observed with transmission electron microscopy (Flaxman et al., 1968; Yaoi and Kanaseki, 1972).

B. CHEMISTRY OF MICROEXUDATES Many experiments on microexudate formation and medium conditioning have been carried out either with serum in the medium or on the substratum, and this had complicated determination of the chemical and functional properties of conditioning factors. Depending on the system, a variety of different proteins is found in conditioned medium, including collagen (Goldberg and Green, 1964). Mucopolysaccharides are also present (Kraemer, 197la,b). Microexudate deposition depends not only on the cell type but also on the underlying substratum (Dodson and Hay, 1971). The precise origin of the microexudate is difficult to determine. It may contain shed cell surface components or secreted substances (Kapeller et al., 1973), and is probably also derived from cell fragments (L. Weiss and Coombs, 1963; L. Weiss and Lachmann, 1964; L. Weiss and Mayhew, 1967; Payne et al., 1973) such as the bulbous ends of filopodia or other cell microextensions (Dalen and Scheie, 1969; Price, 1970; Witkowski and Brighton, 1971; Rosen and Culp, 1977; Whur et aZ., 1977; see Fig. 7). Metabolic labeling experiments indicate the presence of protein, carbohydrate, and RNA in the microexudate (Terry and Culp, 1974; L. Weiss et al., 1975).

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Culp and his collaborators (Culp, 1974,1975,1976; Terry and Culp, 1974; Culp et al., 1975; Culp and Buniel, 1976; Mapstone and Culp, 1976) systematically studied the nature of microexudate left behind following EGTA separation of normal or transformed 3T3 cells from the substratum. The polypeptide composition of the microexudate has been defined by sodium dodecyl sulfate gel electrophoresis. The distribution of the microexudate i s topographically related to the morphology of attached cells in sparse cultures. Also, by using metabolic labeling, the rate of synthesis and turnover of microexudate have been studied. Although deposition of microexudate is inhibited at low temperatures, it is not inhibited by cycloheximide. The microexudate contains various glycoproteins and glycosoaminoglycans. It also contains a myosin-like protein, an actin-like protein, and a. high-molecular-weight glycoprotein called LETS protein (Section XI).The presence of an actin-like component in the microexudate has also been reported (McNutt et al., 1971; Lazarides, 1975a).

c.

FUNCTIONS OF MICROEXUDATES The possible role of conditioning factors in altering cell growth has been emphasized by Rubin (196613).When cells are initially attached in the absence of serum, they may deposit microexudate as an adhesion and spreading factor and thereby achieve the properties of cells attached in serum-containing medium (Taylor, 1962; Daniel, 1967; Takeichi, 1971; Yaoi and Kanaseki, 1972). Microexudation occurs during cell adhesion and spreading (Maslow and Weiss, 1972), and components of conditioned medium can be shown to substitute for serum in promoting the spreading of various adult and embryonic primary and cultured fibroblasts (Daniel, 1967; Takeichi, 1973; Yasuda, 1974; Moore, 1976; Pearlstein, 1976). Conditioned medium also contains growth factors (Dulak and Temin, 1973).It seems likely that cells able to attach and grow normally in serum-free medium (Evans et at., 1956; Rappaport, 1956; Waymouth, 1956; Hayashi and Sato, 1976) can do so by virtue of their ability to condition the medium and the substratum (Hodges and Melcher, 1976).

VI. Cell Contact with the Substratum In previous sections, there was a discussion of the influence of the substratum and its modifications on cell adhesion. The emphasis of this article now changes to consider the cellular events involved in the formation of bonds of attachment. In order for bonds of attachment to form, there must be contacts between the cell and substratum surfaces. This occurs first when cells initially attach to the substratum,

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and second when secondary cell attachments occur during cell spreading and migration.

A. ROLEOF MICROVILLI AND FILOPODIA P. A. Weiss (1945) recognized that new contacts between elongating nerve axons and the substratum were made at active cell margins h e called filopodia (it is not clear who originally coined the term “filopo-

dia”; similar structures have subsequently been called microspikes, attachment microextensions, retraction fibers, thin pseudopodia, and a variety of other names). Porter et al. (1945) were among the first to observe filopodia at the ultrastructural level. Theoretical considerations (Bangham and Pethica, 1960; Pethica, 1961; L. Weiss, 1964a) led to the conclusion that cell microextensions of low radius of curvature may be important in initial cell contacts, because they would facilitate cell penetration through the electrostatic barrier between the cells and the substratum. This barrier develops because mammalian cells carry a net negative charge at physiological pH (Mehrishi, 1972), and so do most substrata unless they are specially modified. In addition, various studies at the light and electron microscope levels support the idea that cell microvilli (diameter -0.1 pm, length up to 5 pm) are involved in initial cell adhesion and that filopodia (diameter -0.2 pm, length from 2 to 30 pm) which appear following initial cell adhesion are involved in cell spreading and migration (Lesseps, 1963; Taylor and Robbins, 1963; Taylor, 1966; Fisher and Cooper, 1967; Price, 1970; Vasiliev and Gelfand, 1973; Rajaraman et al., 1974; Springer et al., 1976; Grinnell et ul., 1976).

B. BIOCHEMISTRY OF CELLCONTACT Biochemical evidence for a role of cell protrusions in the contact process has also been reported and is based on experiments measuring cell adhesion in a centrifugal field (Milam et al., 1973). Under these experimental conditions, the centrifugal force (-750 x g ) bringing the cells against the substratum is probably greater than the force of the electrostatic barrier tending to hold apart the cells and the substratum (L. Weiss, 1976). Thus contact can be brought about artificially by this method, even in the absence of cellular activity. Centrifugation of cells against the substratum results in a marked increase in the rate of adhesion; however, the properties of attached cells are similar following adhesion at rest (1 x g ) or in a centrifugal field (Milam et al., 1973). With use ofthe centrifugation technique, a series of studies has been carried out to determine whether or not reagents and conditions known to inhibit cell adhesion at rest also inhibit adhesion in a

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centrifugal field (Grinnell, 1974a,b). Two classes of inhibitors have been distinguished: those that inhibit adhesion either at rest or in a centrifugal field, and those that inhibit adhesion at rest but not in a centrifugal field. The latter have been taken to be specific inhibitors of the contact process. They include the addition of cytochalasin B to the incubation medium, and carrying out adhesion in the cold or in the absence of Mg2+or Caz+.These results have been interpreted to mean that the contact process involves cytochalasin-sensitive structures and that the activity of these structures requires divalent cations and is inhibited in the cold. The evidence that microfilaments are required for the protrusion of microextensions is shown by the observed withdrawal of microextensions in the presence of cytochalasins (Wessells et al., 1971; Yamada et al., 1971; Goldman, 1972; Miranda et al., 1974a,b; Godman et al., 1975). The role of divalent cations in the protrusion of microextensions has also been discussed (Gingell et aE., 1969). It is not known how cells control the regions of the cell surface from which microextensions are protruded, but L. Weiss (1965)has suggested that this is dependent on local membrane deformability. Local regions of high deformability may be generated by a local high density of anionic surface charge (Wolpert and Gingell, 1968); however, the presence of anionic charges is apparently not necessary for the maintenance of cell microvilli (Grinnell et al., 1975). VII. Cell Spreading onto the Substratum A. SEQUENCE OF EVENTS

As soon as cell contact with the substratum occurs, bonds of attachment are formed. Active cell spreading is initiated by the radial extension of filopodia from the base of the attached cells (Taylor, 1961; Witkowski and Brighton, 1971; Kolodny, 1972; Rajaraman et al., 1974; Rovensky and Slavnaya, 1974; Vasiliev and Gelfand, 1976; Bragina et d., 1976; Rosen and Culp, 1977). The experiments of Rajaraman et al. (1974) with WI-38 cells were carried out in serum-free medium; however, recent evidence indicates that cell adhesion under these conditions is inhibited by sulfhydryl binding reagents (R. Rajaraman, personal communication), which is characteristic of adhesion to serum- or microexudate-coated substrata (Section IX). Therefore, the spreading of these cells in serum-free medium may depend upon their elaboration of a microexudate. At their extremities, these filopodia form attachments to the substratum around the entire cell circumference.

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Subsequently, there is a redistribution of cytoplasm as it flows out, along, and in between the filopodia, resulting in lamellar regions of spread cytoplasm called lamellipodia (Witkowski and Brighton, 1971, 1972; Vasiliev and Gelfand, 1973, 1976; Rajaraman et al., 1974; Springer et al., 1976). During this process, one or several lamellipodia often become dominant, and the cell can become highly polarized in shape (Vasiliev and Gelfand, 1973; Springer et al., 1976). During cell spreading, there is a substantial increase in apparent cell surface area. Several investigators have proposed that cell microvilli act as a surface reserve for this process (Follett and Goldman, 1970; Erickson and Trinkaus, 1976).In general, a correlation between the decrease in cell microvilli and the extent of cell spreading has been observed (O’Neill and Follett, 1970; Porter et al., 1973a; Willingham and Pastan, 1975a; Knutton et d., 1976). However, not all investigators have found this to be the case (e.g., McNutt et at., 1973). Cell spreading varies during the cell cycle. It is maximal during the mid-G, and S periods (Porter et al., I973a; Everhart and Rubin, 1974), and during mitosis spread cells round up (Terasima and Tolmach, 1963). Cell spreading also increases under culture conditions in which there are increased cell-cell interactions (Trinkaus, 1963; Rubin and Everhart, 1973; Middleton, 1976). B. ROLEOF FILOPODIA

Cell filopodia can play a variety of roles. Since cell spreading occurs along the path established by the filopodia, these structures play a decisive role in determining cell orientation in response to structural and adhesive gradients in the substratum. Thus they have an exploratory function (P. A. Weiss, 1945; Taylor and Robbins, 1963; Dalen and Scheie, 1969; Witkowski and Brighton, 1971; Rovensky and Slavnaya, 1974; Albrect-Buehler, 1976). Filopodia can also modify the substratum. Bulbous enlargements at the tips of attached filopodia may be left behind on the substratum to become part of the microexudate (Dalen and Scheie, 1969; Witkowski and Brighton, 1971). Also, particulate matter that is free on the substratum can become attached to filopodia at their tips and then carried off the substratum back onto the main body of the cell (Albrecht-Buehler and Goldman, 1976; Albrecht-Buehler and Lancaster, 1976). Filopodia are also found on the growth cones of nerve axons (P. A. Weiss, 1945; Yamada et d.,1971)and seem to play an exploratory role in axon-substratum interactions (Letourneau, 1975a,b). It should be emphasized that, although the direction of axon elongation is probably under the control of axon-substratum adhesion, elongation per se

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is a much different phenomenon than cell spreading (Strassman et al.,

1973; Letourneau and Wessells, 1974).

C. SUBSTRATUMREQUIREMENTS A primary requirement for cell spreading to occur is that extending filopodia be able to make firm adhesions to the substratum, otherwise the filopodia retract back into the cell body (Vasiliev and Gelfand, 1976; Albrecht-Buehler and Goldman, 1976). Not only must the substratum be able to support cell adhesion, but also, if the substratum has an irregular surface (e.g., a Millipore filter), discontinuities in the substratum must be close enough together so that they can be bridged by the filopodia, otherwise spreading does not take place (Ambrose, 1967). However, enhancing adhesion between the cells and the substratum promotes cell spreading; this can be done by incorporating any of a variety of cell surface-directed ligands into the substratum matrix (see Section XI). In order to spread, cells generate considerable tension on the substratum via the filopodia, which can result in the deformation of elastic substrata such as the fibers of a plasma clot. Thus, if the substratum lacks ridigity, cell spreading will not occur. A novel technique has been to measure cell spreading onto protein films at oil-water interfaces (Rosenberg, 1964; Maroudas, 1973b). Harris (1973b) tested cell spreading onto silicon fluids of varying viscosities in an attempt to relate the tensile force generated by various cell types. He found that leukocytes and macrophages spread onto fluid substrata with viscosities -lo4 times lower than those required for the spreading of fibroblasts. An epithelial cell line was found to require a substratum of intermediate viscosity. These results indicate that the relative tensile strengths generated by the cells are: fibroblasts > epithelium > leukocytes and macrophages.

D. ROLE OF MICROFILAMENTSAND MICROTUBULES The cytoplasmic driving force for cell spreading appears to depend on microfilaments. In the presence of cytochalasins, cell spreading either occurs more slowly than normal or not at all (Carter, 1967b; L. Weiss, 1972; Goldman et al., 1973a; Goldman and Knipe, 1973; Rabinovitch and DeStefano, 1973a; Jones and Partridge, 1974; Grinnell, 1974b; Miranda et al., 1974; Nath and Srere, 1977). Cytochalasins do not cause already spread cells to round up, which shows that the cellsubstratum bonds per se are not sensitive to disruption by these compounds; however, spread cells undergo a retraction phenomenon in

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the presence of cytochalasin, resulting in an arborized, partially rounded shape (Goldman, 1972; Sanger and Holtzer, 1972; Seraydarian et al., 1973; Goldman and Knipe, 1973; Ukena et al., 1974; Miranda et al., 1974a,b; Everhart and Rubin, 1974; Godman et al., 1975). A similar observation has been made for nerve growth cones (Yamada and Wessells, 1973). In the presence of cytochalasins, microfilaments do not disappear (Goldman, 1972); rather they contract back into the cytoplasm (Miranda et al., 1974b) and, as postulated b y Spooner (1973), cease interacting with the plasma membrane. Similarly, in nerve growth cones, the lattice microfilaments along the periphery disappear, although the centrally located sheath microfilaments remain intact (Yamada and Wessells, 1973; Luduena and Wessells, 1973). Microfilaments are believed to be contractile elements (Wessells et al., 1971; Pollard and Weihing, 1974), and in fully spread cells the filaments are distributed in linear bundles (stress fibers and cables) along the longitudinal axis of the cell and insert into the plasma membrane at the cell extremities (McNutt et al., 1971, 1973; Goldman et al., 1973a; Perdue, 1973; Goldman and Knipe, 1973). These bundles appear to contain actin, myosin, and tropomyosin (McNutt et al., 1971, 1973; Perdue, 1973; Goldman et al., 1975; Lazarides, 1975a,b; Goldman, 1975; Sanger, 1975; Pollack et al., 1975) and have contractile properties (Isenberg et al., 1976). Nevertheless, it is not clear how the contractile force is generated. It may be that microfilaments extend from a juxtanuclear location during cell spreading (Goldman and Follett, 1970); however, the bundles of microfilaments are first organized at the cell periphery and can be observed only after cell spreading has already started (Goldman et al., 1974a; Lazarides, 1975a; Goldman, 1975; Clarke e t al., 1975). Microtubules also appear to play a role in cell spreading but seem to be mainly involved in regulating the final shape of the spread cells. They also appear to extend from a juxtanuclear position during spreading (Osborn and Weber, 1976). In the presence of colchicine or other reagents that interfere with microtubule assembly, cell adhesion and spreading still can occur; however, the cells do not spread into polarized forms (Goldman and Knipe, 1973; L. Weiss, 1972; Jones and Partridge, 1974; Goldman e t al., 1973a; Rabinovitch and DeStefano, 1973a; Grinnell, 1974b; Ivanova et al., 1976). Moreover, treatment of spread, polarized cells with these reagents results in a loss of polarity (Vasiliev et ul., 1970; Rovensky et al., 1971; Goldman, 1971; Gail and Boone, 1971; Vasiliev and Gelfand, 1973).

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VIII. Location of Bonds of Cell Attachment and Their Ultrastructural Appearance As indicated in Section VII, cell attachments appear to form initially at local regions of cell-substratum contact and subsequently at the tips of filopodia Zxtending around the entire cell periphery to the substratum during cell spreading. Mitotic cells and trypsinized cells appear quite similar to initially attached cells and are also surrounded by filopodia attached to the substratum more or less equally around the entire cell periphery (Dalen and Scheie, 1969; Dalen and Todd; 1971; Porter et al., 1973a; Revel, 1974; Revel et al., 1974; Goldman et al., 1974a). OF CELL-SUBSTRATUM ATTACHMENTS A. MARGINALLOCATION Following cell spreading, cell -substratum attachments appear to be predominantly localized at the margins of lamellapodia. Releasing lamellapodia at their edges results in their contraction back into the cell body (Goodrich, 1924; Chambers and Fell, 1931; Algard, 1953; Trinkaus et al., 1971; Harris, 1 9 7 3 ~ )Similar . conclusions about the location of cell-substratum attachments have been reached by viewing cells sideways (Ingram, 1969; Hlinka and Sanders, 1972), with interference reflection microscopy (Curtis, 1964; Abercrombie and Dunn, 1975; Izzard and Lochner, 1976), and with electron microscopy (Kruse et al., 1970; Abercrombie et al., 1971; Brunk et al., 1971; Revel and Wolken, 1973; Revel et al., 1974). Based on these and other studies (Section VII1,C) sites of cell-substratum attachments are generally taken to be regions of close approach (- 100 A) between the cell and substratum surfaces. Under normal conditions the axons of cultured neurons also appear to be attached to the substratum exclusively at the margins of the growth cone region. However, if the axon-substratum attachment is anomalously strong (e.g., on polycationic substrata or in serum-free medium), the entire axon appears to be attached to the substratum (Bray and Bunge, 1973; Luduena, 1973; Letourneau, 1975a,b). Finally, epithelial cell sheets appear to be attached predominantly to the cells on the edges of the sheets (Chambers and Fell, 1931; Curtis and Varde, 1964), and these cells have filopodial attachments to the substratum at their peripheries (DiPasquale, 1975a,b).

B. CELL-SUBSTRATUM ATTACHMENTS BENEATH THE CELL BODY Ambrose (1961) has reported the presence of discontinuous cellsubstratum attachments beneath the entire cell using surface contact microscopy, and many electron microscope studies also have revealed

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the presence of close cell -substratum approaches beneath the cell (Cornell, 1969; Taylor, 1970; Brunk et al., 1971; Revel et uZ., 1974; Revel, 1974). Broad, flat regions of cell surface, which may or may not be sites of attachment, have also been observed (Revel and Wolken, 1973; Abercrombie et al., 1971; Price, 1970; Liebrich and Pawelletz, 1976). The finding that areas of cell membrane beneath the cell are left behind when tanned cells are mechanically removed from the substratum is particularly suggestive of attachments beneath the cell body (Revel et al., 1974).

C. ULTRASTRUCTURE OF CELL ATTACHMENTS A successful approach in identifying morphological cell -substratum attachments has been to look for in vitro correlates of in vivo structures. Hemidesmosomes have long been thought to be basal attachment sites of epithelium to the underlying basal lamina (P. A. Weiss and Ferris, 1954; Stern, 1965; Kelly, 1966), and similar structures have been visualized for epidermis (Flaxman et al., 1968; Cristophers and Wolff, 1975) and gingival epithelium (Taylor, 1970) cultured on artificial substrata. Figure 4 is from Taylor’s (1970) work and shows rat gingival epithelium on an epoxy substratum. Fibroblast cell -substratum specializations have also been observed

FIG.4. Rat gingival epithelial cell attached to an epoxy substraturn. The adsorbed senim line on the substratum surface is evident (double arrowhead), and numerous hemidesmosomes (single arrowheads) can be seen. The upper part of the substratum is darkened Iiy osmium penetration. From Taylor (1970; Fig, 10). x 88,000.

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FIG.5. Attachment of a mouse embryo fibroblast. The cells were attached to an Araldite substratum and peeled away from it prior to sectioning. The serum line is evident (double arrowhead), and an adhesion plaque is observed (single arrowhead). From Pegrum and Maroudas (1976; Fig. 3B). x 75,000.

in viuo, which consists of dense bundles of microfilaments just subjacent to the plasma membrane in regions of close cell-substratum apposition (Overton and Mapp, 1974; Ebendal, 1977). Similar structures observed in uitro have been called adhesion plaques (Brunk et al., 1971; Abercrombie et al., 1971; Goldman et al., 1974a; Pegrum and Maroudas, 1975; Goldman, 1975; Bragina et al., 1976). Figure 5 is an example of an adhesion plaque in a mouse embryo fibroblast that had been attached to an Araldite substratum (Pegmm and Maroudas, 1975). However, plaques have not been seen in many other ultrastructural studies (Cornell, 1969; Price, 1970; Douglas and Elser, 1972; Grinnell et al., 1976). Figure 6 is an example of a close cell-substratum apposition between a BHK cell and an epoxy substratum not characterized b y an adhesion plaque (Grinnell et d.,1976). In a recent systematic study of the timing of adhesion plaque formation, it was found that plaques formed only after initial adhesion and substantial cell spreading had already taken place (Bragina et al., 1976). This observation has especially important implications, because plaques are thought to be the membrane insertion sites for cytoplasmic microfilament bundles (Abercrombie et al., 1971; Harris, 1973c; Pollack et al., 1975; Goldman, 1975; Lazarides, 1975a,b). As described in Section VII, microfilament bundles begin to be organized at peripheral cell regions where close cell-substratum interac-

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FIG.6. Attachment ofa BHK cell to an epoxy substratum. The specimen was fixed with tannic acid-glutaraldehyde, which improves visualization of *theglycocalyx and plasma membrane. The serum line (single arrowheads) is $bout 50 A thick. The closest approach of the plasma membrane to the serum line is 90 A, and the intervening space is filled with extra plasma membrane material. No adhesion plaques are observed. Details in Grinnell et al., 1976. ~343,000.

tions occur, but this happens only after some cell spreading has already taken place. Thus it may be that plaques are initiation sites for the organization of microfilament bundles in the peripheral regions of cell-substratum interaction. An additional type of ultrastructural correlate of cell-substratum attachments are the attenuated bridges between the cell and substratum observed at the edges of some attached cells (Cooper and Fisher, 1968). The relationship of these structures to cell orientation has never been established; however, they appear to be regions where the cells are pulling away from the substratum surface. An example of attenuated attachment bridges on BHK cells growing on Lux coverslips is shown in Fig. 7.

D. ADHESIVENESSOF THE UPPER CELL SURFACE The observation that most cells appear to be attached only at their margins raises the question whether or not other parts of the cell are also adhesive. There is no evidence that prior to adhesion suspended cells have particular regions that are adhesive and others that are not. However, once cells are attached and spread, the upper surfaces of fi-

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FIG. 7. Attachment of a BHK cell to a Lux coverslip. Carbon-platinum replica. A filopodial attachment is evident (double arrowhead), and numerous attenuated attachment bridges are observed (single arrowheads).The granular deposit on the substratum may come from the attenuated attachment bridges when they break off. x 68,000. (F. Grinnell, J. Day, and C. R. Hackenbrock, unpublished observation.)

broblastic and epithelial cells have been reported to be nonadhesive; additional attachments to inert particles or to other cells can occur only at the cell margins (DiPasquale and Bell, 1974; Vasiliev and Gelfand, 1976). A similar observation has been made for different epithelial cell surfaces b y other investigators (Elsdale and Bard, 1974; DeRidder et al., 1975). There may also be a different distribution of cell-substratum glycoproteins on the lower and upper surfaces of attached cells (Noonan et al., 1976). Therefore the nonadhesiveness of the upper cell surface may indicate a lack of appropriate receptors. However, some sort of diffusion or other mechanical barrier may be present. For instance, the anionic surface sites of BHK cells are rapidly labeled in suspended cells by polycationic ferritin (Grinnell et al., 1975) but, by comparison, are very slowly labeled in attached and spread cells (Grinnell et aE., 1976).

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IX. Biochemistry of Cell Adhesion The various stages in cell adhesion have been described. It is now appropriate to consider some of the reagents and conditions that inhibit or promote cellular adhesiveness and try to relate their activity to the different stages of adhesion. In this section, the distinction made earlier between active and passive adhesion is further elaborated. Active adhesion is specific in the sense that it required added cofactors and depends on particular cell surface and cytoplasmic components. Passive adhesion is nonspecific in that it has no such apparent requirements. The emphasis of this article continues to be on active, specific adhesion.

A. KINETICS Most studies have confirmed Taylor’s (1961) analysis indicating that passive cell adhesion essentially occurs instantaneously, whereas active cell adhesion is a much slower process. The lag phase in active cell adhesion has been postulated to be the time required for local modification of the substratum by microexudation or for cells to make contact with the substratum through microvilli (L. Weiss and Harlos, 1972).Another possibility is that membrane receptors have to undergo some reorganization (perhaps patching) during adhesion (Shields and Pollock, 1974; Grinnell, 1974a; Maroudas, 1975b; Rees et al., 1977; Juliano and Gagalang, 1977). The observation that the rate of cell adhesion is dramatically increased b y the centrifugation of cells against the substratum suggests that a major part of the lag phase is the time required for contact to occur; however, even in a centrifugal field, adhesion is not instantaneous, and different cell types demonstrate different kinetics of adhesion (Milam et al., 1973; Grinnell, 1974b). Therefore the time required for local microexudation or receptor redistribution may also contribute to the lag phase.

B. TEMPERATURE DEPENDENCE The inhibition of cell-substratum adhesion b y a low (4°C) incubation temperature (Moscona, 1961) was taken as an indication that adhesion may be an energy-dependent process. This point was later emphasized by Wolpertet al. (1969). The temperature dependence of adhesion has been confirmed by many laboratories (Nordling, 1967; L. Weiss, 1964b; Michaelis and Dalgarno, 1971; Rabinowitz and DeStefano, 1973a; Wnhjem and Prydz, 1973; Grinnell, 1974a; Attramadal, 1975; Klebe, 1975; Ueda et al., 1976; Juliano and Gagalang, 1977; Nath and Srere, 1977).However, as mentioned earlier, it is characteristic only of active cell adhesion. Cell adhesion can be induced at 4°C

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if the cells are centrifuged against the substratum (Grinnell et al., 1973b), although not under all conditions (Nath and Srere, 1977). The cell contact process is probably more temperature-sensitive than the formation of bonds of attachment. Not only is filopodial activity known to be inhibited at 4°C (Albrecht-Buehler and Lancaster, 1976), but also, there may be a general increase in membrane rigidity resulting from a lipid-phase transition (Ueda et al., 1976).

C. ENERGYDEPENDENCE The energy dependence of cell adhesion is suggested by the inhibition of cell adhesion in the presence of various inhibitors of glyco-

lysis and oxidative phosphorylation (Michaelis and Dalgarno, 1971; Klebe, 1975; Juliano and Gagalang, 1977). Glycolysis alone is sufficient to support cell adhesion, as shown by the lack of inhibition of adhesion in the presence of inhibitors of oxidative phosphorylation or electron transport when there is glucose in the medium (Grinnell et al., 1972; Klebe, 1975).However, in a recent study with a variety of metabolic inhibitors, no clear correlation was found between cellular ATP concentration, respiration rates, and inhibition of adhesion by metabolic inhibitors (Nath and Srere, 1977). Filopodial activity also is energy-dependent (Albrecht-Buehler and Lancaster, 1976).

D. CATIONDEPENDENCE Divalent cations appear to be required for active cell adhesion, but not for passive adhesion (summarized in Table 11). Both Mg2+ and Ca2+seem to be active at physiological concentrations in many of the systems tested; however, some specific requirements have been reported. For instance, both Ca2+ and Mg2+ are required for optimal spreading of BHK cells onto serum-coated substrata, whereas either cation alone functions with substrata coated with ASF (Grinnell, 1976b,c). Several recent reports have shown that Mn2+may enhance adhesion even better than Mg2+or Ca2+(although concentrations of Mn2+much higher than physiological are required). Of particular interest is the observed stimulation of cell spreading by Mn2+ under serum-free conditions (Rabinovitch and DeStefano, 1973a; Pegrum and Maroudas, 1975; Allen and Minnikin, 1976). In the presence of Mn2+ cells rapidly elaborate a microexudate onto the substratum (Maroudas, 1977) and this may be an adhesion and spreading factor. In addition to divalent cations, monovalent cations (especially K+) may be important in cell adhesion (Rappaport et al., 1960; Rappaport, 1971); however, this point has not been studied further. A clear biochemical understanding of the divalent cation require-

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TABLE I1 DEPENDENCE OF CELL ADHESION

STUDIES ON THE CATION

Protein iii the inediutn or on the substratum

Requirement for divaleiit cations

Serum

Yes

Serum factors

Yes

Microexudate

Yes

None

NO

Reference Taylor (1961, 1962); Takeichi and Okada (1972); Grinriell (1974b); Nordling (1967); Rabinovitch and DeStefanct (1973a, 1975a) Licberman and Ove (1958); Fisher et u Z . (1958); Grinnell (1976a,h) Takeichi (1971); Yasuda (1974); Moore (1976) Taylor (1961, 1962); Berwick and Coman (1962); Rappaport (1971); Takeichi and Okada (1972); Takeichi (1971); Nordling (1967); Grinriell (1974b); Rabiiiovitch and DeStefano (1973a, 1975a)

ment in cell adhesion has yet to be obtained. Since partial cell adhesion occurs in a centrifugal field in the absence of divalent cations (Grinnell, 1974b) and divalent cations are required for filopodial activity (Albrect-Buehler and Lancaster, 1976), they probably have an important role in cell contact. Divalent cations may also play a part in the internal structure of the cell surface or as cofactors in the formation of the bond of attachment (Section X).

E. REQUIREMENT FOR FREESULFHYDRYL GROUPS Treatment of cells with reagents that block free sulfhydryl groups prevents subsequent active cell adhesion (Grinnell and Srere, 1971), although passive cell adhesion is unaffected by these reagents (Crinnell, 1974a). Also, treatment of initially attached cells prevents further attachments from occurring, which would otherwise result in an increase in the strength of cell adhesion and in cell spreading (Grinnell et al., 1973b). Many different types of blocking reagents are inhibitory, including mercurials, arsenicals, and alkylating reagents, but oxidizing reagents are ineffective. Also, the effect is reversible under ap-

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propriate conditions (Grinnell et al., 1972). The best reagents are those that can penetrate the plasma membrane, which indicates that the sensitive sites are probably not cell surface components; however, the sensitive sites are protected after the cells are attached and spread (Grinnell et al., 1973b). The precise site of action of sulfhydryl-binding reagents is still unknown. Centrifugation of cells with blocked sulfhydryl groups against the substratum does not overcome inhibition of adhesion, indicating that adhesion bond formation itself is somehow directly interfered with; yet this is hard to understand, since the reagents appear to act within the cells. One possibility is that cell surface adhesion receptors must undergo redistribution in order for adhesion to occur (Shields and Pollock, 1974; Grinnell, 1974a; Maroudas, 1975; Juliano and Gagalang, 1977; Rees et ul., 1977), and that this redistribution is blocked by sulfhydryl reagents. It has been found that sulfhydryl-blocking reagents inhibit tubulin polymerization (Mellon and Rebhun, 1976) and increase cell rigidity (LaCelle et al., 1976).Moreover, under some circumstances, sulfhydryl-binding reagents inhibit myosin ATPase activity and prevent the polymerization of G actin to F actin (Jocelyn, 1972). Since many different blocking reagents react with sulfhydryl groups, the interpretation of some adhesion-perturbation studies is difficult. For instance, the inhibition of adhesion by 1-fluoro-2,4-dinitrobenzene may occur at sulfhydryl sites and not at amino groups. Also, the chloromethyl ketone inhibitors of proteases shown to block cell adhesion when used at high concentrations (Whur et al., 1974; Grinnell, 1975) may in fact be blocking sulfhydryl groups (Rossman et al., 1974) and not cell proteases.

F. INHIBITION BY TRYPSINIZATION Treatment of cells with trypsin is a standard technique for accomplishing cell separation from a substratum and is discussed in some detail in Section X. Whether or not trypsin treatment alters subsequent cell adhesion has not been consistently agreed on. Kolodny ( 1972) found that trypsin-subcultured cells were normally adhesive; however, several other laboratories found trypsin treatment to inhibit subsequent active cell adhesion (L. Weiss and Kapes, 1966; Grinnell et al., 1973b; Juliano and Gagalang, 1977),although passive cell adhesion was unaffected (Grinnell, 1974a).The concentration of trypsin required to obtain inhibition is quite high (1.0-5.0 mg/ml). Under these conditions, loss of adhesiveness is reversible and can be regained in 2 4 hours by a process that requires de no00 protein synthesis (Grin-

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nell et ul., 197317).In addition to trypsin, treatment with chymotrypsin (L. Weiss and Kapes, 1966) or pronase (F. Grinnell, unpublished observation) also inhibits subsequent cell adhesion. Inhibition by trypsin is not overcome by centrifugation of cells against the substratum, which suggests that there is a direct influence on attachment bond formation (Grinnell, 1974b). It may be that the specific adhesion receptors are being removed or modified (Juliano and Gagalang, 1977). G. OTHERINHIBITIONSTUDIES As discussed in Section VI, cytochalasins inhibit cell adhesion, presumably by preventing the activity of cell microextensions. Local anesthetics and tranquilizers also inhibit cell adhesion when added to the incubation medium (Rabinovitch and DeStefano, 1973a, 1975a), and the inhibition is not overcome by centrifuging cells against the substratum (Grinnell, 1974b). These reagents also inhibit cell capping (Ryan et ul., 1974) and may prevent adhesion both by disrupting the cell cytoskeleton (Nicolson et al., 1976a) and by preventing reorganization of cell surface receptors (Rabinovitch and DeStefano, 1975a). Cell adhesion is not inhibited by treatment of cells in short-term cultures with inhibitors of protein synthesis, RNA synthesis, or D N A synthesis (Kolodny, 1972; L. Weiss and Chang, 1973; Grinnell et al., 1973b). Also, it is not inhibited by blocking cell surface amino groups (L. Weiss, 1974). Treatment of cells with various glycolytic enzymes including neuraminidase has not been reported to alter cell-substraturn adhesion, and the addition of individual sugars or nucleotide sugars to the incubation medium has not been reported to be inhibitory. AND CAMP H. MODULATION OF CELL ADHESION BY HORMONES Addition of the glucocorticoid dexamethasone phosphate to the medium has been reported to induce increased adhesiveness of rat hepatoma (HTC) cells over several days in culture (Ballard and Tomkins, 1969, 1970). Induction was inhibited by preventing protein or RNA synthesis. The adhesion assays were quite long, and it is not clear whether or not the induction resulted in an increase in intrinsic cellular adhesiveness or an increased ability of cells to elaborate microexudate. Other cell lines tested were not sensitive to induction. cAMP has also been implicated in cell adhesion and spreading. This is particularly well demonstrated with transformed cells that contain decreased intercellular levels of CAMP (Sheppard, 1972). The addition of dibutyryl cAMP to the incubation medium, or reagents that

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increase the intercellular concentration of CAMP, results in an increase in cell spreading (Hsie and Puck, 1971; Johnson et al., 1971; Porter et al., 1974; Willingham and Pastan, 1975a,b) and an increase in the strength of cell adhesion (Johnson and Pastan, 1972; Grinnell et al., 1973c; Shields and Pollack, 1974; Nozawa et at., 1975). However, when suspended cells are pretreated with cAMP derivatives, there is no increase in the subsequent rate of cell adhesion (Grinnell et al., 1973c; L. Weiss, 1973a). This means that the major effect of CAMP is probably on the spreading process and not on initial cell contact or formation of attachment bonds. The cAMP effect is reversed by Colcemid (Puck et al., 1972; Shields and Pollock, 1974). It has been proposed that cAMP enhances tubulin assembly and microtubule formation (Puck et al., 1972; Porter et al., 1974; Sloboda et al., 1975; Brinkley et al., 1975). I. CELLADHESION MUTANTS Several mutants have been isolated that have altered adhesiveness. Cold-sensitive mutants of BALB 3T3 (Willingham et al., 1973) and CHO cells (Crane and Thomas, 1976) have been reported that spread less well at the nonpermissive temperature. The poorly spread cells apparently have low levels of CAMP.Another BALB 3T3 mutant has been isolated that has a defect in the acetylation of glucosamine-6phosphate and is unable to spread normally (Pouyssegur and Pastan, 1976; Pouyssegur et al., 1977). The defect is overcome and normal spreading occurs if the mutant is fed N-acetylglucosamine. This mutant has a rate of cell adhesion similar to that of the wild type, but is more sensitive to inhibition by trypsin. The defect in adhesiveness may be in altered cell surface adhesion components. Mutants of BHK and CHO cells have been isolated that are insensitive to the lectin, ricin. The CHO mutant spreads less well than normal (Gottlieb et al., 1974), and some of the BHK mutants have decreased rates of cell adhesion (Edwards et d., 1976). Finally, Klebe et d., (1977) have isolated a CHO mutant requiring increased levels of serum and divalent cations in the medium in order to attach and spread.

J. ADHESIONOF ENUCLEATED CELLS The adhesiveness of cells resides in the plasma membrane and the cytoplasma as shown by studies with enucleated cells. Physically enucleated cell fragments remain attached and spread and continue migration on the substratum, at least for a short period of time (Goldstein et al., 1960). Neuronal processes severed from the neuron cell body also are capable of continued movement and extension (Shaw and

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Bray, 1977). Finally, several studies carried out with cells enucleated by cytochalasin B treatment have demonstrated the normal attachment and spreading of cytoplasts (Prescott et al., 1972; Goldman et ul., 1973b; Shay et al., 1974, 1977).

K. SUMMARY OF BIOCHEMICAL ANALYSES The experiments described in this section are helpful in sorting out

the different stages of active cell adhesion. The centrifugation technique has been particularly useful in distinguishing inhibition of cell

contact from inhibition of attachment bond formation. Based on this technique, it appears that low temperature, the lack of divalent cations, or the presence of cytochalasins inhibits cell adhesion primarily at the level of the contact process. However, sulfhydryl-binding reagents, trypsin treatment, or the presence of local anesthetics or tranquilizers inhibits formation of bonds of attachment, either by blocking cell surface adhesion receptors directly or by preventing receptor redistribution. These reagents may inhibit the contact process as well, but this cannot be determined from the centrifugation analysis. The CAMPdependence of cell adhesion clearly separates the rate of cell adhesion from the extent of cell spreading. This helps to emphasize the point that cell surface and cytoplasmic components contribute in multiple ways to the different stages of adhesion: Cell surface receptors form the bond of attachment; microfilaments control the rate of cell -substratum contact and spreading; microtubules control the polarity and extent of cell spreading. Adhesion mutants also demonstrate examples of both altered cytoplasmic and cell surface behavior, and these will very likely prove to be useful in the future, especially in determining the identity of the hypothetical cell surface adhesion receptor.

X. Cell Separation After cells become attached to the substratum, it is possible to study

cell separation. The methodology for carrying out cell separation is important for technical reasons relevant to the routine practice of tis-

sue and organ culture. Also, cell separation experiments measure different parameters than adhesion experiments. This is shown by the observation that several reagents that inhibit cell adhesion (e.g., sulfhydryl-binding reagents and cytochalasins) do not cause cell separation. However, some reagents (e.g., local anesthetics and trypsin treatment) both inhibit adhesion and cause separation.

ADHESIVENESS AND EXTRACELLULAR SUBSTRATA

A.

101

STRENGTH OF CELLADHESION

The susceptibility of attached cells to being separated from the substratum depends on the strength of cell adhesion. During the first 60 minutes of adhesion, over the same time period that spreading occurs, there is a progressive increase in the resistance of cells to separation by mechanical shear (Grinnell et al., 1973b). This increase in the strength of cell adhesion possibly is a result of increased numbers of attachment bonds; however, the shape of the cells may also be critical. Cells that have been previously spread but have then rounded up during mitosis remain attached to the substratum, but they are readily separated by mechanical shear. This is the basis of the mitotic selection technique (Terasima and Tolmach, 1963; Robbins and Marcus, 1964). The primary effect of reagents that cause cell separation is to induce the rounding-up of spread cells.

B. SEPARATION OF ACTIVELYAND PASSIVELY ATTACHED CELLS The most common technique for accomplishing cell separation is treatment of attached cells with hydrolytic enzymes or with divalent cation chelating reagents. These methods are also used for dissociating intact tissues (Rinaldini, 1958; Waymouth, 1974). As shown in Table 111, only cells that have been attached to the substratum in the presence of serum or attached to serum-coated or microexudatecoated substrata are separated from the substratum by these reagents. C. MECHANISMOF CELLSEPARATION During treatment wih EDTA or trypsin, cell rounding occurs just as in mitosis (Dornfield and Owczarzak, 1958; Dalen and Scheie, 1969; Dalen and Todd, 1971; Harris, 1973c; Revel, 1974; Revel et al., 1974; Rosen and Culp, 1977; Whur et al., 1977). Because rounding precedes separation, Revel (1974; Revel et al., 1974) has proposed that trypsin initially acts on the cell cytoskeleton and not on the bonds of attachments. In particular, cell microtubules may be involved in some way. Some cell rounding occurs in the cold (Carter, 1970; Revel et al., 1974) and, as discussed in Section VII, microtubules are probably important in controlling the extent of cell spreading. However, the. effects of trypsin are not mimicked by colchicine, therefore this explanation requires further clarification. ,The mechanism of cell rounding induced by EDTA in unclear. Surprisingly, the action of this compound is inhibited by low temperatures (Shields and Pollack, 1974; Attramadal, 1975). One effect of

102

FREDERICK GFUNNELL TABLE 111 STUDIESON THE SEPARATIONOF CELLSFROM BY TRYPSIN OR EDTA

Separation reagent Trypsin

EDTA

Protein in the medium or on the substratum during adhesion

THE

SUBSTRATUM

Separation

Reference

Serum

Yes

Microexudate None

Yes

Serum

Yes

Microexudate

Yes

None

NO

Wolpert et nl. (1969); Takeichi (1971); Unhjein and Prydz (1973); Culp and Buniel (1976) Takeichi (1971) Easty et nl. (1960); Takeichi (1971); Unhjem and Prydz (1973); Culp and Buniel (1976) Taylor (1961); Takeichi (1971); Unhjem and Prydz (1973); Culp and Buniel (1876) Taylor (1962); Takeichi (1971) Easty et (11. (1960); Taylor (1961, 1962); Takeichi (1971); Unhjem and Prydz (1973); Culp and Buniel (1976)

NO

EDTA may be to weaken the integrity of the cell surface (Dornfield and Owczarzak, 1958; L. Weiss, 1967b). Consistent with this notion is the finding that, in the presence of chelating reagents, pieces of the cell surface pinch off and are left behind on the substratum (Hynes et aZ., 1976; Rosen and Culp, 1977; Whur et al., 1977). L. Weiss and his collaborators have emphasized that the cleavage line in separation need not be at the bond of attachment and that separation probably occurs through rupture of the cell surface (L. Weiss, 1961a,b; L. Weiss and Coombs, 1963; L. Weiss and Mayhew, 1967). D. OTHERTECHNIQUES OF SEPARATION A relatively new method for cell separation which has important practical applications for the future is the treatment of attached cells with local anesthetics (Rabinovitch and DeStefano, 1975b).The initial effect of this treatment is also to induce cell rounding and local anesthetics probably also act at the level of the cell cytoskeleton (Nicolson et uZ., 1976a).

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Finally, in addition to the above techniques, a variety of other treatments has been shown under some circumstances and with some cell types to cause cell rounding. These include treatment of spread cells with antibody directed against the cell surface (O’Neill and Follett, 1970; Yamada et al., 1976a; Wylie et al., 1976) or with dextran sulfate (Bremerskov, 1973).Phenobarbital and strychnine also have been reported to induce cell rounding (Buchsbaum and Kuntz, 1954).

XI. Cell Surface Receptors In the preceding sections, many different aspects of active and passive cell adhesion were reviewed. It was pointed out that there is no evidence for specificity in passive cell adhesion. However, active cell adhesion is highly specific. It depends on the adsorption of certain serum components or microexudate onto the substratum (Sections IV and V), and it is altered by chemical modification of the cell surface and in mutants with modified cell surface components (Section IX). Moreover, there may be a rearrangement of specific cell surface components during adhesion (Sections VII and IX). These observations are consistent with the notion that there are specific adhesion receptors which are part of the cell surface (Martinez-Paloma, 1970; Hughes, 1973). The idea of highly specific adhesion receptors is well known in developmental biology (Tyler, 1946; P. A. Weiss, 1946). Cell surface ligand-receptor interactions are thought to mediate cell-cell recognition and adhesion (Moscona, 1968). Many systems have been characterized in which ligands of one sort or another are thought to be involved, although the details of the mechanisms have yet to be clarified (Roseman, 1970; Henkart et al., 1973; Balsam0 and Lilien, 1974; Rutishauser et al., 1976).

A. LIGAND-INDUCED CELL-SUBSTRATUM ADHESION AND SPREADING

The model of cell adhesion in Fig. 3 is one of specific cell surface receptors binding to ASF adsorbed onto the substratum surface. A prediction of this model is that, if ligands directed against the cell surface are adsorbed to the substratum, they may be able to substitute for ASF and induce adhesion and spreading by binding to cell surface components. This is illustrated diagrammatically in Fig. 8, where the ligands are shown on the substratum and the receptors (circles) are shown on the cell surface. The cationic proteins salamine and p o l y ( ~ lysine ) were found to

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FREDERICK GRINNELL

FIG.8. Model of cell adhesion to adsorbed ligands directed against the cell surface. Details are in the text.

substitute for serum on the substratum (Lieberman and Ove, 1958), and this finding was confirmed by many laboratories for a variety of cationic substances (Taylor, 1961; Nordling, 1967; Maciera-Coelho and Avrameas, 1972, 1974; Bases et al., 1973; Grinnell, 1975). The substratum-adsorbed polycationic substances are believed to act as ligands directed at negatively charged groups on the cell surface. Cell adhesion and spreading onto polycationic substrata are often even better than onto serum-coated substrata, and this technique has been used to improve cell growth (McKeehan and Ham, 1976) and to attach less adhesive cells in electron microscope studies (Mazia et al., 1975). When the substratum is coated with polyanionic ligands, instead of polycationic ligands, there is decreased cell adhesion (Nordling et al., 1964; Koike, 1964; Nordling, 1967; Macieira-Coelho and Avrameas, 1972; Macieira-Coelho et al., 1974; Clarke and Ryan, 1976). This may result from a nonspecific increase in electrostatic charge repulsion between the cells and the substratum. Cell adhesion and spreading are also promoted by other ligands directed against the cell surface which are adsorbed to the substratum, such as concanavalin A (Con A) and antiplasma membrane immunoglobulin (Grinnell, 1976b,c). A recent series of experiments compared cell adhesion and spreading activity onto substrata coated with polycationic ferritin (PCF), Con A, antibody directed against BHK plasma membranes (anti-BHK), and ASF (Grinnell and Hays, 1978b). Substrata coated with any of the ligands induces adhesion and spreading of BHK cells (Fig. 9). The morphology of spreading is very similar in every case, except that cells spread on PCF substrata (Fig. 9D) are often slightly more rounded. In the absence of the ligands or ASF on the substratum, passive adhesion occurs, and there is no cell spreading (Fig. 9A). Biochemical studies comparing spreading onto ligand-coated substrata with spreading onto ASF-coated substrata have shown that, for all the substrata, spreading can be inhibited by low incubation tem-

FIG.9. Spreading of BHK cells onto ligand-coated substrata. The substrata were treated for 5 minutes at room temperature with 1.0 ml of CaZf-and Mgz+-containing phosphate-buffered saline (PBS) containing: (A)no protein; (B)25 pg/ml of ASF; (C) 50 pgiml of Con A; (D) 460 pg/ml of PCF; (E) 142 pg/ml of the immunoglobulin fraction of anti-BHK cell surface antiserum. The treated substrata were then washed with deionizied water and incubated with BHK cells (10Tml) in Ca2+- and Mg2+-containing PBS for 30 minutes at 37°C. x 265.

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FREDERICK GRINNELL

peratures or b y the addition to the incubation medium of 2,4-dinitrophenol, N-ethylmaleimide, or cytochalasin B. Moreover, colchicine inhibits the polarity of cell spreading in every case. These findings support the notion that specific ligand-receptor interactions between the cell and substratum surfaces induce a cell spreading response similar, if not identical, to that observed with adsorbed ASF on the substratum. Although the cell surface ligands PCF, Con A, and anti-BHK induce cell spreading when they are adsorbed onto the substratum, they inhibit spreading when added to the medium. This is not surprising, since the ligands in solution would be expected to bind to the cell surface and compete with adsorbed ligand for cell surface sites (Grinnell, 1976c, 1977b; Grinnell and Hays, 197813).This finding is in marked contrast to the inactivity of ASF in solution (Section IV and Fig. 3) and further supports the notion that ASF is active only after its adsorption onto the substratum. B. BRIDGING BY DIVALENT CATIONS One of the earliest models of cell-substratum attachment involved electrostatic bridging by divalent cations (for reviews, see Weiss, 1967a; Curtis, 1973). This hypothesis is no longer attractive for a variety of reasons: (1)Cell adhesion can occur in the absence of divalent cations in a centrifugal field (Section VI); (2) EDTA causes cell rounding prior to cell separation and is inhibited in the cold (Section VII); and (3) EDTA-induced cell separation probably occurs through rupturing of the cell surface and not through breaking the cell attachment bond (Section VII). The requirement for divalent cations appears to involve the activity of microvilli and filopodia in cell contact and spreading, and maintenance of the integrity of the cell surface.

C. GLYCOSYL TRANSFERASES Roseman ( 1970) proposed that surface glycosyl transferases on one cell may react with appropriate receptors on a second cell surface to form a stable enzyme-substrate complex which would constitute the bond of attachment. This hypothesis has been recently reviewed by Shur and Roth (1975). Although several investigators have reported the presence of glycosyl transferases on the cell surface (e.g., Roth et al., 1971; Roth and White, 1972; Patt and Grimes, 1974; Bosmann, 1974; Sudo and Onodera, 1975), others believe that the evidence is insufficient and/or misinterpreted (Evans, 1974; Keenan and Moore, 1975; Deppert et al., 1974).

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The glycosyl transferase hypothesis has never been discussed for cell-substratum adhesion except in the work of Jamieson et al. (1971) on platelet-collagen adhesion (Section XII). It should be pointed out that the activity of glycosyl transferases requires divalent cations and is inhibited by sulfhydryl-binding reagents. However, the apparent unimportance of carbohydrate portions of the cell adhesion and spreading factor for its activity (Section IV,C) does not support a role of cell surface glycosyl transferase as the cell surface adhesion receptor. D. LETS PROTEIN Recently, evidence has been presented that a cell surface component called LETS protein plays a role in cell adhesion and spreading. This protein, whose name is derived from its Large size (ca. 250,000 polypeptides), External location on the cell surface, and Transformation Sensitivity (i.e., loss following malignant transformation), has been reviewed in detail (Hynes, 1976).Other laboratories have called this protein CSP, SFA, and, most recently, fibronectin. Since LETS protein is absent from transformed cells, as well as from some “untransformed” permanent cell lines (Yamada et al., 1977), which nevertheless attach to the substratum, it is questionable whether or not it is the cell surface receptor involved in the bond of attachment to the substratum. However, the presence or absence of LETS protein corresponds very well with the degree of cell spreading and the organization of cytoplasmic microfilaments involved in cell spreading. The evidence is: (1) Transformed cells, which lack LETS protein, are less well spread than normal cells (Section XVII); (2) LETS protein disappears from the cell surface during mitosis when cell rounding occurs (Hynes and Bye, 1974); (3) the removal of LETS protein from cells by urea treatment causes them to become less well spread on the substratum (Weston and Hendricks, 1972); (4)the addition of LETS protein to transformed cells increases their spreading (Yamada et al., 1976a,b) which is accompanied by a reappearance of microfilament bundles (Ali et al., 1977); (5)immunofluorescence studies indicate that LETS protein is distributed in fibrous networks on the surfaces of normal cells and perhaps at sites where microfilament bundles insert into the plasma membrane (Wartiovaara et al., 1974; Vaheri and Ruoslahti, 1975; Hynes et al., 1976); (6) the initial organization of LETS protein on the cell surface occurs after cell adhesion and spreading have already been initiated and is localized at the edges of lamellapodia in the regions where microfilament bundles first organize (Wartiovaara et al., 1974; see also Section VII).

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FREDERICK GRINNELL

This evidence is consistent with the notion that adhesion plaques, which are insertion sites of microfilament bundles and regions of close cell-substratum interactions (Section VIII), may be composed of local aggregates of LETS protein. Moreover, since these are the regions that are most firmly attached to the substratum, it is not surprising that LETS protein is partially left behind when cells are separated from the substratum under some conditions (Culp, 1976; Hynes et al., 1976). Although it seems unlikely that LETS protein is the cell surface adhesion receptor, LETS is closely related to the cell adhesion and spreading factor, i.e., cold insoluble globulin. A substance immunologically and structurally related to LETS protein was found in serum and believed to be cold insoluble globulin (Ruoslahti and Vaheri, 1975; Vaheri et al., 1976). Moreover, isolated LETS protein has cell adhesion and spreading activity for BHK cells on dried collagen substrata (Pearlstein, 1976) and on tissue culture substrata (F. Grinnell, unpublished observation). However, unlike cold insoluble globulin, LETS protein has lectin-like activity (Yamada et al., 1975).Also, there are differences in the structural and solubility properties of LETS and cold insoluble globulin. Another point of interest concerns the requirement by some cells for serum in the medium in order to spread. Primary cell types (e.g., human foreskin fibroblasts, chick embryo fibroblasts) and diploid cell types (e.g., WI-38, MRC-5) are capable of spreading in the absence of serum in the medium. In marked contrast, many “normal” cell lines (e.g., BHK-21-C13, BALB 3T3) and transformed cell lines (e.g., BHKpy, CHO, L) have a serum requirement for cell spreading. According to the recent quantitation of cellular LETS protein by Yamada et al. (1977), this pattern corresponds to cells with high and low levels of LETS protein, respectively. Finally, a LETS-like protein is known to be released into the culture medium by both normal and transformed cells although to much different extents (Ruoslahti et al., 1973; Vaheri and Ruoslahti, 19751, and, as mentioned earlier (Section V,C), adhesion and spreading factor is also secreted into the culture medium by some cell types. These considerations lead us to postulate the model shown in Fig. 10. We propose that cell surface LETS is either a marker for the capability of cells to secrete cold insoluble globulin (CIG) or its equivalent, or that cell surface LETS is a receptor, which when activated by contact between cells and a substratum, results in secretion of CIG. According to this hypothesis, cells with high levels of LETS can deposit their own CIG layer on the substratum and therefore spread in the absence of serum whereas cells with low surface LETS do not de-

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109

LETS

FIG.10. Model of cell spreading, the LETS protein, and insoluble globulin (CIG) synthesis. See text for details.

posit a CIG layer sufficient for spreading to occur, and therefore require the addition of serum.

E. ATPASEACTIVITY Other cell surface components that may play a role in cell adhesion and spreading are ecto-ATPases (Agren et al., 1971; Trams, 1974; Ronquist and Agren, 1975). Myosin may also be present at the cell surface (Willingham et al., 1974; Olden et al., 1976). The extracellular addition of ATP has been reported to influence the contractile properties of cells and their adhesion and spreading, however, not in a consistent fashion (H. Lettre’s work reviewed by Dornfield and Owczarzak, 1958; Ambrose, 1961; P. A. Weiss, 1962; Jones, 1966). The possibility that ecto-ATPases play a role in cell adhesion requires further study.

F. ASIALOGLYCOPROTEIN-BINDING PROTEIN The notion has been emphasized that cell adhesion and spreading can be mediated by any ligand-receptor interaction between the cell surface and adsorbed proteins on the substratum. Therefore, in concluding this section, it is worthwhile pointing out that a cell surface receptor on hepatocytes specifically binds asialoglycoproteins and is involved in their clearance from plasma (Ashwell and Morell, 1974). So far, this surface component has not been implicated in cell adhesion; however, it-and perhaps similar proteins on other cellswould have a potential for forming bonds of attachment between cells and appropriate glycoproteins adsorbed onto the substratum surface.

XII. Platelet Adhesion The adhesive properties of fibroblasts have been reviewed in detail in previous sections. The very brief review of platelet-substratum ad-

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hesion in this section is meant to illustrate several similarities between fibroblast and platelet adhesion. The adhesion of platelets to a substratum is the initiating event in the normal biological activity of platelets; however, it is only the first of a sequence of events that results in the release of platelet contents and platelet aggregation, which in turn leads to thrombus formation. Because of the importance of platelet interactions in vivo, sophisticated strategies for studying adhesion under in vivo conditions as well as in vitro have been developed. In addition, studies on clinical disorders of platelet function have contributed to the identification of the serum and platelet components required for adhesion. For the present purposes we are most interested in the initial platelet-substratum adhesion reaction regardless of whether or not it is followed b y release and aggregation.

A. SUBSTRATA FOR PLATELET ADHESION-in

Vitt-0

Two kinds of platelet-substratum adhesion have been described in vitro: adhesion to collagen, and adhesion to artificial. substrata. These are readily distinguished, since divalent cations are required for adhesion to an artificial substratum (Salzman, 1971; Nossel, 1975).

1 . Artificial Substrata The requirement for surface wettability in platelet adhesion to artificial substrata has been described (Salzman, 1971; Lyman and Kim, 1971; George, 1972; Olsen and Kletschka, 1973), and nonthrombic surfaces are usually either of low wettability or have a high negative charge (Gott and Furuse, 1971). Although there is some disagreement concerning the wettability dependence (Friedman et al., 1970; Mason et al., 1972), it may relate to the flow conditions of the experiments. Analagous to the case of fibroblast adhesion, proteins (from plasma) are adsorbed onto the substratum, and it is probably the composition of the adsorbed protein layer that determines platelet activity (Dutton et al., 1969; Baier, 1972; Vroman, 1972; Olsen and Kletschka, 1973). In particular, substratum-adsorbed fibrinogen may be involved (Zucker and Vroman, 1969; Packham et al., 1969), especially when it is partially polymerized (Niewiarowski, 1973). 2. Collagen Native collagen is a substratum for platelet adhesion, but thermally denatured collagen is not (Wilner et al., 196813).Apparently, the free amino groups of the collagen are critical in platelet adhesion and, when they are blocked by nonpolar groups, adhesion is inhibited

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(Nossel et al., 1969). When, however, the amino groups are blocked by succinylation, platelet adhesion still occurs, although at a decreased rate (Wilner et'aZ., 1971). The sugar residues of collagen may also be important in platelet adhesion, since adhesion is inhibited after treatment of collagen with periodate oxidation (Brass and Bensusan, 1976) or galactose oxidase (Nossel, 1975).

B. SUBSTRATA FOR PLATELET ADHESION-in Vivo The endothelial wall is not a substratum for platelet adhesion; however, following trauma, platelets adhere to the underlying connective tissue. The identity of the connective tissue component(s) that is the substratum for platelet adhesion is controversial. In suspensions of mesenteric tissue, platelets adhere to collagen fibers, and the process does not require divalent cations (Spaet and Zucker, 1964); however, following minimal trauma in situ, collagen fibers often might not be exposed. Under these conditions, adhesion takes place adjacent to endothelial junctions and may involve the deposition of fibrinogen or fibrin (Ashford and Freiman, 1968). Subendothelial basement membrane is also an in viuo substratum for platelet adhesion, and this reaction requires divalent cations (Stemerman et al., 1971; Baumgartner et al., 1971); noncollagenous microfibrils and amorphous collagen are probably the sites at which adhesion occurs (Stemerman et al., 1971; Baumgartner et al., 1971; Baumgartner and Haudenschild, 1972; Stemerman, 1974). Based the divalent cation requirement, the initial adhesion of platelets to subendothelium is more similar to adhesion to artificial substrata than adhesion to collagen fibers.

c.

BIOCHEMISTRYOF PLATELET ADHESION

The biochemical properties of platelet adhesion are similar to the properties of fibroblast adhesion, as shown by the following observations. (1) Platelet adhesion to artificial surfaces and to subendothelial basement membrane requires divalent cations (Ca2+or Mg2+);(2) platelet adhesion to glass is inhibited by cytochalasin B (Boyal Kay and Fudenberg, 1973) and, during adhesion, platelets extend microfilament- and microtubule-containing filopodia in an energy-requiring process (Hovig, 1974), which suggests that there is a contact process; ( 3 ) sulfhydryl binding reagents inhibit platelet adhesion to collagen (Al-Mondhiry and Spaet, 1970) and to glass (Mohammad et al., 1974), and reagents that penetrate the plasma membrane are the most effective; and (4) tranquilizers inhibit platelet adhesion to collagen (Jamieson et aZ., 1971) and to glass (Mohammad et al., 1974).

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D. REQUIREMENTFOR PLASMA COMPONENTS AND CELLSURFACERECEPTORS 1. Von Willebrand Factor

The possible role of adsorbed fibrinogen in platelet adhesion has been mentioned. Another plasma protein required for platelet adhesion to glass is von Willebrand factor (Salzman, 1963; Bouma et al., 1972), which is part of the factor VIII complex (H. J. Weiss et al., 1973). Patients lacking von Willebrand factor also have defective platelet-substratum adhesion (H. J. Weiss et al., 1975). The mechanism of action of von Willebrand factor is not understood; however, it probably becomes functional after binding to the platelet cell surface (Olson et al., 1975) and may be part of the thick surface coat on platelets observed by Behnke (1968).

2. Glycoprotein Defect in the Bernard -Soulier Syndrome Platelet-subendothelial adhesion is impaired in patients with the Bernard-Soulier syndrome (H. J. Weiss et al., 1975). In this case certain membrane glycopeptides present in normal platelets, which may be involved in adhesion, are apparently missing (Nurden and Caen, 1975; Jenkins et al., 1976; Tobelem et al., 1976). 3. Glucosyl Tran.sferase

The possible role of platelet surface glucosyl transferase in platelet adhesion to collagen has been emphasized by Jamieson and his collaborators (Jamieson et al., 1971; Jamieson, 1974). The evidence is based on coordinated inhibition of both glucose transfer to collagen and platelet adhesion to collagen by a variety of treatments. However, recent studies by Menashi et ul. (1976) indicate that, although denatured collagen is a substratum for platelet glucosyl transferase, native collagen is not. In any case, platelet membrane-binding sites for collagen fragments have recently been identified (Chiang and Kang, 1976). They may also be the complement component Clq-binding sites (Suba and Casko, 1976).The subsequent study of these sites may clarify whether or not glucosyl transferase activity is important. 4 . ATPase Activity Finally, a possible role for surface ATPases in platelet adhesion has been proposed (Robinson et al., 1965; Mason and Saba, 1969) but not demonstrated.

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XIII. Macrophage, Monocyte, and Neutrophil Adhesion A brief review of the adhesive properties of macrophages will suffice to show that many similarities in adhesive behavior are observed in macrophages and fibroblasts. Monocytes and neutrophils are discussed together with macrophages in this section. The classic study by Fenn (1923) demonstrated that rat peritoneal exudate cells adhere preferentially to wettable substrata, that adhesion and spreading occur either in the presence or absence of serum, and that, in the absence of serum, adhesion is neither temperaturenor divalent cation-dependent. It seems that for macrophages, as is the case for fibroblasts, there are active and passive modes of adhesion. The response of macrophages to the substratum is probably one manifestation of their phagocytic properties. A.

AND SPREADING PHAGOCYTOSIS

Phagocytosis occurs in two stages. The first involves attachment of the particle to be ingested to the cell surface, and the second involves uptake (Rabinovitch, 1967).Various kinds of particles can be phagocytosed; for some (e.g., living red blood cells) serum factors are required

to opsinize the particle surface, whereas others (e.g., latex beads) can be taken up directly (Cline and Lehrer, 1968). The attachment phase of phagocytosis is energy-independent; however, ingestion requires energy, and the energy obtained from glycolysis is sufficient (Cline and Lehrer, 1968; North, 1970; Karnovsky et al., 1970). North (1968,1970) has suggested that the spreading of macrophages onto a substratum is an attempt by the cells to phagocytose a particle of infinite diameter. This conclusion is based on microscopic observations and the demonstration that both uptake and spreading are inhibited by the sulfhydryl-binding reagent iodoacetate and stimulated by the exogenous addition of ATP to the incubation medium. Subsequently, Henson (1971) suggested that the neutrophils also try to phagocytose the substratum. He found that, when these cells adhere to Millipore filters coated with antibody-antigen complexes, they release their granules onto the filter.

B. OPSININREQUIREMENTFOR ADHESION Adhesion to glass bead columns has been shown to require serum in experiments with neutrophils and monocytes (Rabinowitz, 1964; Penny et al., 1966),and serum and plasma factors that promote macrophage spreading have been described (Leonard and Skeel, 1976; Bianco et al., 1976). Nevertheless, the addition of serum or opsiniza-

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tion of the substratum by serum factors is not an absolute requirement for cell spreading. Rabinovitch and DeStefano (1973b,c; 1974) found that, in addition to spreading onto substrata opsinized with antibodyantigen complexes, spreading of macrophages also occurs on uncoated substrata when the cells are treated with the proteolytic enzyme subtilisin, with dithiotreitol (DTT), or with buffer of low ionic strength or low pH. The addition of Mn2+(albeit at concentrations much higher than physiological) also induces cell spreading. Under all those conditions, spreading is retarded by whole serum. The induction of monocyte spreading in serum-free medium has also been observed (DeKaris et at., 1974; Rajaraman et at., 1977) and is stimulated by antibody-antigen complex opsinization of the substratum or b y treatment of the cells with DTT, Mn2+, or subtilisin (Douglas, 1976). Trypsin also has been reported to induce peritoneal macrophage spreading (Lutton, 1973), although another laboratory has found that it inhibits alveolar macrophage adhesion (McKeever and Gee, 1975).Whether or not any of these treatments promote release of microexudate onto the substratum has not been determined.

C. DIVALENT CATIONREQUIREMENT Divalent cations are also required for macrophage and leukocyte adhesion to plasma or serum-coated glass bead columns (Rabinowitz, 1964; Kvarstein, 1969b; Gamin, 1972), and for spreading under any conditions (Rabinovitch and DeStefano, 1973b,c, 1974). Mn" is best, and MgZ+is generally better than Ca2+.

D. ENERGYDEPENDENCE Inhibitors of glycolysis have been shown to prevent the spreading of peritoneal exudate macrophages (Hahinovitch and DeStefano, 1974); however, alveolar macrophage spreading is inhibited by an oxidative phosphorylation inhibitor alone (McKeever and Gee, 1975). A combination of glycolysis and oxidative phosphorylation inhibitors is required to prevent adhesion of leukocytes (Kvarstein, 1969a). In any event, energy appears to be required for adhesion and spreading.

E. REQUIREMENTFOR SULFHYDRYL GROUPS Reagents that block free sulfhydryl groups have been found to inhibit adhesion and spreading of peritoneal macrophages (North, 1968; Rabinovitch and DeStefano, 1974) and neutrophils (Giordano and Lichtman, 1973). These reagents also inhibit yhagocytosis b y neutrophils.

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F. PSEUDOPODIA, MICROFILAMENTS,AND MICROTUBULES Unlike fibroblasts, which are attached to the substratum only in peripheral regions, most of the macrophage undersurface is adherent (Chambers and Fell, 1931). The process of spreading probably requires microfilament activity, since cytochalasin B is inhibitory (Lutton, 1973; Rabinovitch and DeStefano, 1973b, 1974). As in the case of fibroblasts, cytochalasin B causes arborization and retraction of already attached macrophages (Axline and Reaven, 1974; Helentjaris et al., €976). Microtubules are also likely to be important, since colchicine has been shown to inhibit neutrophil adhesion to glass beads (Penny et al., 1966). However, the major effect of colchicine is apparently on the extent to which cell polarization can occur, which is particularly noticeable in moving cells (Bhisey and Freed, 1971; Crispe, 1976). Phagocytosis is also inhibited by cytochalasin B (Axline and Reaven, 1974). The mechanism of phagocytosis apparently involves a contact-initiated, energy-dependent extension of pseudopodia around the particle to be phagocytosed. However, the pseudopodia do not simply engulf the particle. Rather they spread (“zipper”) around it and must be able to make bonds of attachment along its entire circumference (Griffin et al., 1975, 1976). G. SEPARATION OF MACROPHAGESFROM THE SUBSTRATUM Unlike fibroblasts, macrophages are not very susceptible to separation from the substratum by proteolytic enzymes. As mentioned, subtilisin, and in some cases trypsin, may stimulate spreading. However, separation can be accomplished with EDTA. As is the case with fibroblasts, EDTA does not cause cell separation in the cold (Rabinovitch and DeStefano, 1976). Anesthetics and tranquilizers also cause macrophage separation and can be used to subculture these cells (Rabinovitch and DeStefano, 1974). With either EDTA or local anesthetics, the initial events in separation are the retraction of pseudopodia and cell rounding. Following cell rounding, filopodia are still observed to be attached to the substratum (Rabinovitch and DeStefano, 1976). H. SURFACERECEPTORS

The surface receptors for phagocytosis and for adhesion and spreading with antibody-antigen-opsinized surfaces have not been directly compared; however, it seems likely that the same ligand-receptor interactions between Fc receptors on the macrophage surface and im-

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mune complexes on the substratum surface are involved. However, the possible role of C3 receptors in adhesion and spreading has not been tested, and the nature of the receptors in opsinin-independent adhesion and spreading is entirely unknown. Michl et al. (1976a,b) recently reported that 2-deoxyglucose inhibits opsinin-dependent phagocytosis but not the opsinin-independent process; however, the mechanism of inhibition does not involve inhibition of glycolysis. This finding has not yet been extended to macrophage adhesion and spreading. I. MARGINATIONAND EMIGRATION OF LEUKOCYTES During the early phases of the acute inflammatory response neutrophils, and later monocytes, adhere to the endothelial cell wall and migrate in between endothelial cells into the connective tissue (Marchesi and Florey, 1960). Although soluble mediators are important in this process, it is not clear whether the change that results in adhesion occurs within the leukocytes or the glycocalyx of the endothelial cells (Luft, 1966; for a review, see Ryan and Majno, 1977). In any event, the change is highly localized (Cliff, 1966). By analogy to platelet adhesion, one might expect the initial change to be in the endothelium, and it has been suggested that adhesion occurs to local fibrin deposits (Allison et al., 1955).The presence of a fibrin network on macrophage surfaces but not on monocytes has been reported (Colvin and Dvorak, 1975). Similar to the in vitro situation, adhesion and migration of macrophages require divalent cations (Hersh and Bodey, 1970; Atherton and Born, 1972; Thompson et al., 1967). XIV. Red Blood Cell and Lymphocyte Adhesion Unlike the other cells discussed here, red blood cells and lymphocytes are relatively nonadhesive under normal in vitro conditions. A. ADHESION OF REDBLOODCELLS Red blood cells do not undergo active adhesion; however, the passive adhesion of red blood cells has been observed (Ponder, 1965; L. Weiss and Blumenson, 1967; George et nl., 1971; Hocmuth et al., 1972; Kowalczynska et al., 1973; Gingell and Fornes, 1975; Lewandowska et al., 1976). No metabolic activity of the cells is required for passive adhesion, and coating the substratum with serum proteins is inhibitory.

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B. ADHESION OF LYMPHOCYTES Although lymphocytes are relatively nonadhesive in vitro, they undergo adhesion to endothelial defects and migration is observed in vivo under certain circumstances (Schoefl, 1972). They may even selectively attach to specific organs (Woodruff and Gesner, 1968; Ford et d.,1976). When lymphocytes are stimulated in vitro with Con A, they become more adhesive to an artificial substratum (Mori et al., 1973; Wong et al., 1975). If ligands directed at the lymphocyte cell surface are incorporated into an extracellular matrix, lymphocytes adhere to the matrix (Rutishauser et nl., 1974). Moreover, lymphocytes can be induced to attach and spread onto a substratum coated with antibodyantigen complexes (Alexander and Henkart, 1976). In this case, spreading is quite similar to that occurring with fibroblasts and is inhibited by low temperature, azide, cytochalasin B, and the chelating reagents EDTA and EGTA. Moreover, the chelating reagents cause rounding of attached and spread cells. Thus lymphocytes have the potential to undergo contact-induced spreading given the appropriate cell surface-substratum interaction.

XV. Cell Migration and Chemotaxis Previous sections of this report dealt with the adhesive properties of specific cells. The remainder of the article concerns adhesiveness in terms of how it may influence cell behavior. The first consideration is the possible role of adhesion in cell migration and chemotaxis. A. EVENTSIN CELLMIGRATION

How cells are able to migrate and at the same time conserve their surface area is not clear. One hypothesis, which was proposed first for amoebae (Goldacre, 1961)and later for fibroblasts (Abercrombie et al., 1970; Harris, 1973b), is that new cell surface continually forms at the forward end of the cell and is resorbed at the rear. In any case, two mechanical events are required for migration to occur. One is the active extension and adhesion to the substratum of filopodia or lamellapodia (Harris and Dunn, 1972; Wessels et al., 1973; Takeuchi, 1976). Consistent with this are the many observations that cell migration is inhibited by cytochalasin B (e.g., Carter, 1967b; Spooner, 1973), colchicine, and Colcemid (Vasiliev et d., 1970; Goldman, 1971; Gail and Boone, 1971),and that migration requires divalent cations (Gail et al., 1973; Bore1 and Feurer, 1975). [Colchicine does not inhibit migration of monocytes and macrophages, but instead alters the movement from

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gliding (i.e., like fibroblasts) to ameboid (Bhisey and Freed, 1971; Crispe, 1976).] The other event is the separation of rearward attachments. How the cell accomplishes this is unknown. It may be that old attachments are left behind as part of the microexudate (L. Weiss, 1961a,b; note the possibility of broken-off attenuated attachment bridges in Fig. 7), but whether the cell secretes proteases or other substances to aid in shearing old bonds of attachment has not been established. The dependence of cell migration on cell separation implies that the strength of existing cell attachment bonds may be an important parameter in determining how much cells move. For instance, cell migration is increased when there is a decrease in strength of cell adhesion to the substratum (Gail and Boone, 1972). B. MIGRATIONIN RESPONSETO ADHESIVE GRADIENTS The strength of new cell adhesions may play a role in directing cell movements. If there is a gradient of potential adhesiveness within the substratum, cell movement will occur in the direction of increasing strength of adhesion (Carter, 1965, 1967b). This “haptotaxis” appears to be due to preferential stabilization of extended filopodia in the regions where they make firmer attachments to the substratum (Harris, 1973c; Letourneau, 1975a,b). This observation raises the possibility that directed cell movements in uiuo may be guided by potential adhesive gradients within the extracellular matrix. Cells would move so as to maximize their adhesive interactions or until they became so firmly attached that movement ceased (Gustafson and Wolpert, 1961, 1967). In uitro studies indicate that increased strength of cell attachment can result in decreased migration (Johnson and Pastan, 1972; Johnson et al., 1972; Rivkin et al., 1975). C. SELECTIVEFIXATIONBY LIGANDS An example of selective inhibition of migration due to increased strength of cell-substratum adhesion may be the effect of migration inhibition factor (MIF) on macrophages (Bennett and Bloom, 1967). This substance is apparently a cell surface ligand (Remold, 1975, 1976) and therefore has the potential to cross-link cells into the extracellular matrix. Moreover, MIF treatment has been found to result in increased strength of macrophage attachment (L. Weiss and Glaves, 1975). This kind of mechanism is supported by the observations that: (1)Con A has MIF-like activity for macrophages (Pelley and Schwartz, 1972); and (2) Con A and some other plant lectins increase the strength of fibroblast adhesion by cross-linking the cell and substratum surfaces (Gail and Boone, 1972; Grinnell, 1973; Sato and Taka-

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sawa-Nishizawa, 1974), and at the same time inhibiting cell migration (Gail and Boone, 1972; Friberg et al., 1972). D. LEUKOCYTE CHEMOTAXIS Although there is a possibility that leukocyte chemotaxis may be the result of a gradient of potential substratum adhesiveness (e.g., through a gradient of adsorbed ligands directed at the cell surface), there is no direct evidence to support this idea. It has been reported that chemotaxis depends on changes in cell surface components (Becker, 1975; Becker and Ward, 1967) and results from cytoplasmic flow in the direction of the chemotactic stimulus (Ramsey, 1974) or vectoral extension of pseudopodia (Gallin and Rosenthal, 1974). XVI. Cell Adhesion in Embryogenesis, Morphogenesis, and Wound Healing At the very beginning of this article it was mentioned that cell adhesion may play a role in development, in the directed migration of cells, and in cell-matrix interactions during morphogenesis. In this section, these and related topics are discussed. A. MODULATIONOF CELL BEHAVIORBY ADHESION Adhesion of cells to a substratum can have a marked influence on cell properties. For instance, normal cell growth is anchorage-dependent, as already discussed in Section 111. Adhesion also results in an increased capability of cells to transport metabolites (Foster and Pardee, 1969; Otsuka and Moskowitz, 1975; Pofit and Straws, 1977). Moreover, adhesion to artificial surfaces is necessary for the differentiation of neuroblastoma cells (Schubert et al., 1971, 1974) and teratocarcinoma embryoid bodies (Teresky et al., 1974; Gearhart and Mintz, 1974; Jack et al., 1975). B. COLLAGEN AS AN INDUCTIVE OR PERMISSIVE SUBSTRATUM Considerable evidence supports the notion that adhesion to collagen plays a special role in permitting cell morphogenesis to occur. For instance, embryonic epidermis requires dermis or collagen as a substratum in order to undergo morphogenesis i n vitro (Dodson, 1963, 1967; Wessells, 1964). Moreover, the type of specialized epidermis that develops is modulated by the underlying dermis (McLoughlin, 1963, 1968). Similarly, embryonic muscle differentiation occurs preferentially on a collagen substratum (Konigsberg and Hauschka, 1965; Hauschka and Konigsberg, 1966; Konigsberg, 1970). Collagen effects

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have also been reported in the morphogenesis of salivary, lung, ureteric bud, and corneal epithelium (Grobstein and Cohen, 1965; Wessells and Cohen, 1966, 1968; Bernfield and Wessells, 1970; Hay and Meier, 1976). Grobstein (1967) has discussed the role of collagen as a common extracellular inductive factor in epithelial morphogenesis; however, this role may be permissive of development rather than inductive in a directive sense (Wessells and Cohen, 1968). Collagen may also play a role in the modulation of cell phenotype. The implantation of demineralized bone matrix in test animals induces differentiation of osteoblasts and bone formation (Urist, 1970; Reddi and Anderson, 1976). The precise role of collagen in induction or modulation is not known. It may be that cells attach to collagen in particular conformations required for activity, or that cell adhesion to collagen is required for cells to secrete other molecules necessary for their activity (Bernfield and Wessells, 1970; Bernfield et d.,1972, 1973). For instance, the basal lamina of epithelium is composed at least in part of epithelial cell products (Kurt and Feldman, 1962; Pierce et d., 1963, 1964; Hay and Revel, 1963; Hay, 1964), and the type of basal lamina secreted may depend on the underlying demial components (McLoughlin, 1963, 1968; Dodson and Hay, 1971).

c.

THE SUBSTRATUM

AS A

TEMPLATE

A variety of shape changes and directed cell migrations occurs during embryogenesis (reviewed by Trinkaus, 1969), and cell -substratum adhesions may play an important role in these events. For instance, the possibility that the substratum can modulate selective conduction or fixation of cells (P. A. Weiss, 1946) has been demonstrated in vitro (Section XI), and the shape of fibroblasts in situ probably depends in part on the organization of the extracellular matrix (P. A. Weiss and Garber, 1952). The activity of filopodia in cell adhesion and cell movements during embryogenesis has been observed (Gustafson and Wolpert, 1961, 1967; DeHaan, 19f34), and Gustafson and Wolpert (1961, 1967) have developed a detailed model of directed migration under the influence of differential substratum adhesion. Moreover, they have proposed that relative cell-substratum and cell-cell interactions may control cell shape. It is now clear that acellular components, particularly the basal lamina and basement membrane, are the substrata for cell movements (Hay, 1968,1973; Trelstad and Coulombre, 1971) and may supply a physical scaffolding (Zwaan and Hendrix, 1973; Bard et al., 1975). Nevertheless, the hypothesis that differential adhesion gradients exist within the substratum is still unproved, and some recent ex-

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periments using the scanning electron microscope have failed to show a critical alignment of cells with components of the extracellular matrix (Nelson and Revel, 1975; Ebendal, 1977). CELL ADHESIVENESSDURING EMBRYOGENESIS The possible importance of the substratum should not overshadow the fact that the intrinsic adhesiveness of cells varies with their developmental age (Holtfreter, 1948,1968; Trinkaus, 1963,1969). There appear to b e changes in the ability of cells to protrude filopodia and other cytoplasmic extensions (Gustafson, 1973; Trinkaus, 1973), and changes in the degree to which cells can undergo spreading (Trinkaus, 1963, 1973).In chick embryo limb bud formation, differences in in vitro adhesive behavior of mutant and normal cells can be used to explain differences in in uivo histological shape of the developed structures (Ede and Flint, 1975). It has been proposed, but not demonstrated, that intrinsic differences in the embryonic cell surface may b e responsible for differences in adhesive behavior (Mestres and Hinrichsen, 1974). An alternative possibility is that changes in the organization and functioning of cell microfilaments and microtubules are critical. Alterations in cell shape can be explained by contraction or elongation of cytoskeletal components (Baker and Schroeder, 1967; Schroeder, 1970; Karfunkel, 1971; Burnside, 1973). D.

CHANGES IN

E. THESUBSTRATUM AND CELLULAR EVENTS DURING WOUND HEALING Altered cell adhesion and directed cell migration also occur during wound healing. In the epidermis, the basement membrane is the substratum for cell migration during this process (Khodadoust et al., 1968; Krawczyk, 1971). Hemidesmosomes, which are thought to be in situ epidermal cell-substratum adhesions, disappear before cell migration begins (P. A. Weiss, 1956; Croft and Tarin, 1970). Cell migration is then initiated by the protrusion of filopodia. Migration continues until closure, after which hemidesmosomes reform (Oldland and Ross, 1968; Croft and Tarin, 1970; Krawczyk, 1971). XVII. Cell Adhesion in Malignancy

The final topic to be discussed in this article is the question whether or not cell adhesiveness plays a role in the modified behavior of malignant cells. Malignant cells are characterized by a loss of growth control and the tendency to invade other tissues and, in some cases, to metastasize to sites distant from the primary tumor. Cowdry (1940)

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suggested that the invasiveness of malignant cells might b e encouraged by decreased adhesiveness, and several years later Coman (1944) reported squamous cell carcinomas to b e characterized by reduced strength of intercellular attachment. Comparative studies on the adhesive behavior of transformed and normal cells in vitro have revealed several differences. A.

LOSS O F ANCHORAGEDEPENDENCE

Unlike normal cells, transformed cells do not have to b e anchored to a substratum in order to grow (Stoker et al,, 1968).Thus a substratum such as agar, which is nonadhesive for cells in general, can be used for the selection of transformants (MacPherson and Montagnier, 1964). The loss of anchorage dependence may be related to a change in the ability of the cells to bind senim components and not directly related to cell adhesiveness. Normal BHK cells are released from anchorage dependence and can grow in suspension culture in the presence of 30%serum, a level much higher than that required for growth in a stationary culture (Paul et al., 1971). Transformed cells have a lower serum requirement than normal cells (Holley and Kiernan, 1968; Dulbecco, 1970) and bind serum components better than normal cells (Temnink and Spiele, 1974). Moreover, there is a thicker surface coat on transformed cells (Martinez-Palomo and Brailovsky, 1968; Martinez-Palomo et al., 1969; Poste, 1973), which may result from bound serum glycoproteins (Apffel and Peters, 1969). Thus cell adhesion may play a role in anchorage dependence insofar as normal cells are much better at binding the specific serum factors required for cell growth when these cells are anchored to a substratum compared to when they are in suspension; whereas malignant cells bind these components well regardless of whether the cells are attached or in suspension.

B. CHANGESIN CELLSHAPE AND ADHESIVENESS A second change in the adhesive properties of transformed cells compared to normal cells is that transformed cells are less well spread and less firmly attached to the substratum (Sanford et nl., 1967; Aaronson and Rowe, 1970; Barker and Sanford, 1970; Rovensky et al., 1971; 1974b; Domnina et al., 1972; Porter et al., 1973b; Goldman et d., Hale et ul., 1975; Erickson and Trinkaus, 1976). It should be pointed out that this does not necessarily mean that transformed cells attach more slowly to the substratum; in Gact, in some cases they may attach more rapidly (Coman, 1961).There are very few studies on the comparative rates of normal and transformed cell adhesion.

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The decreased spreading of transformed cells probably is a result of a less well-organized cytoskeleton (Ambrose et al., 1970). The lamellapodia are not well formed on transformed cells (Domnina et al., 1972), and there is less organization of the microfilaments (McNutt et al., 1971, 1973; Pollack et d., 1975) and the microtubules (Brinkley et al., 1975). Wang and Goldberg (1976) isolated a temperature-sensitive mutant of RSV-infected chick embryo fibroblasts that becomes less spread and loses its microfilament bundle organization at the permissive temperature. It should be pointed out that the morphology of cell spreading and strength of cell adhesion do not directly relate to anchorage dependence. Cell mutants have been described which are poorly spread but are nonetheless anchorage-dependent (Edwards et al., 1976; Pouyssegur et al., 1977; Willingham et al., 1977). C. ALTERATION OF CAMP LEVELS,LETS PROTEIN, AND CELLPROTEASES There are several possible causes for the decreased cytoskeletal organization of transformed cells. One is the decrease in CAMPlevels in transformed cells (Sheppard, 1972), which may result in defective microtubule assembly (Section VII). A second possibility is the absence on the transformed cell surface of LETS protein which probably plays a role in organizing the cell cytoskeleton during cell spreading (Section XV). Finally, the decreased spreading of transformed cells may result from the increased level of protease activity that transformed cells demonstrate (Bosmann, 1972; Ossowski et al., 1973; Unkeless et al., 1973; Pollack et al., 1974).This idea has been discussed by Hynes (1974). Inhibition of cellular protease activity results in an increase in cell spreading and strength of cell attachment (Goetz et al., 1972; Weber, 1975). Moreover, the addition of low concentrations of various proteases to the medium results in a slight decrease in cell spreading similar to that occurring following transformation (Zetter et al., 1976). It should be pointed out that the increased level of proteases, the disappearance of LETS protein from the cell surface, and the decreased level of internal CAMP may all be interrelated events (Sheppard, 1972; Hynes, 1976). D. Loss OF CONTACTINHIBITIONOF CELLOVERLAP The third change in the adhesive properties of transformed cells is the loss of contact inhibition of cell overlap. In sparse cultures, normal cells are not generally found on top of one another, and this phenomenon has been called contact inhibition of cell movement or contact in-

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hibition of overlap (Abercrombie and Heaysman, 1954).This should not be confused with density-dependent inhibition of cell growth. Several investigators have proposed that contact inhibition of cell overlap can be explained assuming different strengths of cell-cell and cell-substratum adhesion (Rubin, 1966b; Steinberg, 1973) by analogy to cells stopped at the border of a nonadhesive substratum (Carter, 1965,1967b). However, Abercrombie (1970)has emphasized that these explanations cannot account for the apparent paralysis and active retraction events in contact inhibition. Also, Heaysman and Pegrum (1973) have reported that specialized junctions form on contact of normal cells with each other but not on intercellular contact between transformants. This suggests that transformed cells may have lost the ability to recognize each other. Bell (1972) found that the presence of cells on top of one another results only from cell underlapping; therefore cells that are more spread and firmly attached to the substratum would be expected to resist underlapping more than cells that are less so. In this manner, the difference between overlapping of normal and transformed cells can be explained on the basis of their different degrees of spreading and strength of adhesion. Heaysman and Turin (1976) showed that contact inhibition of overlapping occurs in zinc-fixed and nonfixed cells under conditions in which there is no paralysis and retraction phenomenon. Moreover, Erickson (1976) has reported that both normal and transformed cells undergo paralysis and retraction when active cell margins collide. Thus the possibility has emerged that contact inhibition of overlap and contact-induced paralysis and retraction are two separate phenomena and that the loss of contact inhibition of overlap in transformed cells is a reflection of their reduced cell-substratum adhesion.

E. CELLINVASIVENESS The invasive tendency of malignant cells has variously been attributed to their loss of contact inhibition of movement and/or reduced adhesiveness (see Fidler, 1975, for a review). Cells that are invasive and eventually metastasize may be a less-adhesive subpopulation of the growing tumor (Fadei, 1975). As discussed earlier, decreased cellsubstratum adhesion may lead to an increase in migration (Section XV). A correlation between cell migration in vitro and tumorgenicity has been reported (Barski et al., 1976), and the evidence that cell migration is required for tumor invasiveness in v i v o has recently been reviewed (Stiuli and L. Weiss, 1977). The increased level of protease activity in malignant cells probably also encourages invasiveness by altering the connective tissue matrix (e.g., Sylven, 1973),and the pro-

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tease inhibitor trasylol can inhibit in vitro cell invasiveness (Latner et al., 1973). Several ultrastructural studies on invasive carcinomas have been carried out (Luibel et al., 1960; McNutt, 1976).There is a loss of desmosomes and hemidesmosomes, indicating decreased adhesiveness; the formation of breaks in the basal lamina, suggestive of protease activity; and the appearance of cytoplasmic protrusions into the surrounding extracellular space. It was pointed out that a loss of adhesiveness and the extension of filopodia are also initial events of cell migration during wound healing (Section XVI). F. CELLSURFACENEGATIVECHARGES One hypothesis formulated to account for increased invasiveness and decreased contact inhibition of malignant cells proposed that malignant cells have a higher negative surface charge than normal cells and therefore tend to repulse intercellular contacts (Ambrose, 1968). This notion is not widely accepted, because many different studies have failed to show consistent charge differences between normal and malignant cells (L. Weiss, 1973b). However, the average charge density on the cell surface and the specific distribution of charges are different properties (L. Weiss, 1968). Recent studies indicate that there may be specialized regions of negative charges on the cell surface, with the highest density of sites on cell microvilli (L. Weiss et al., 1972; L. Weiss and Subjeck, 1974; Grinnell et al., 1975).Therefore the possible role of the distribution of negative charges and malignant cell behavior requires further study. G. BLOOD-BORNE METASTASIS

Metastasis can occur through direct invasive extension of malignant cells into body cavities. It also takes place via the lymphatics or blood vessels following invasion of malignant cells into lymph vessels, venules, or small veins (Fidler, 1975).Only blood-borne metastasis is discussed in this article. The path of blood-borne metastasis was reported by Baserga and Saffiotti (1955), who emphasized that the tumor cells must pass from the circulation back into the connective tissue in order for a secondary tumor to form. The arrest of tumor cells in capillaries has not been found to be strictly a matter of physical lodgment (Wood, 1958; Zeidman, 1961) but may involve thrombus formation. The migration of tumor cells into the connective tissue occurs at endothelial defects (Wood, 1958), which explains why local trauma enhances metastasis (Fisher and Fisher, 1965). It has now been found that tumor cells form emboli with platelets

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(Warren, 1970) and with lymphocytes (Fidler et al., 1976).Adhesion of tumor cells to the endothelium probably occurs in conjunction with, and may depend on, the adhesion of platelets (Warren, 1970, 1976; Warren and Vales, 1972; Hilgard, 1973; Chew et al., 1976). It probably involves local deposition of fibrin or fibrinogen (Warren and Vales, 1972; Wood and Hilgard, 1973; L. Weiss, 1975; Chew et nl., 1976). The inhibition of metastasis by drugs that prevent platelet adhesion supports the notion that platelets are involved (Gastpar, 1974a,b). The relationship between the adhesive and metastatic properties of malignant cells is unclear. The treatment of tumor cells with sulfhydryl-binding reagents has been reported to inhibit their metastasis (Grasseti, 1970; Apffel and Walker, 1973),and the inhibition of metastasis by anticoagulants (Koike, 1964; Hoover and Ketchum, 1975) may even be a direct effect of these polyanionic compound on cell adhesion. The notion has also been discussed that tumor cells may become localized in different organs by attaching to specific endothelia through cell surface ligand-receptor interactions (Nicolson et al., 1976b). However, the localization may be due to survival factors rather than to preferential adhesion (Fidler, 1976).

H. FOREIGN BODY-INDUCED SARCOMAS The last topic to be discussed in this section is much different from those preceding and concerns the observation b y Turner (1941) that implantation of Bakelite disks results in the formation of sarcomas in rats. A variety of studies indicate that cell adhesion probably does not play a major role in this phenomenon. Oppenheimer et al. (1955, 1956, 1958) showed that tumor induction depends on the implant being a smooth plate or film. If the implant is perforated, induction does not occur. Induction is also more or less independent of the chemistry of the implant. Tumor formation occurs during a long latent period, within a dense connective tissue pocket formed around the implant; during this time, there are periods of both fibroblast proliferation and inactivity (Vasiliev et al., 1962). It should be pointed out that for at least part of the time the premalignant cells are attached to the implant (Brand et al., 1967). The disruption oftissue organization and the formation of an abnormal environment in the connective tissue pocket are generally considered to be the most important conditions leading to tumor formation. Although the possible importance of cellsubstratum adhesion is probably minimal in this phenomenon, it should not be discounted altogether. Boone (1975; Boone et al., 1976) has recently reported that normal BALB 3T3 cells are tumorigenic when attached to a substratum and then injected into test animals.

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XVIII. Conclusions The results presented in this article support the notion that adhesiveness is common to many different cell types under a variety of conditions, and that the underlying mechanisms are analogous for active cell-substratum adhesion occurring in vitro to artificial and to model physiological substrata and in vivo to fibrinogen or fibrin deposits, to the basement membrane, and to other acellular components of the connective tissue. We emphasize that, although a form of passive initial adhesion of cells to substrata can occur in vitro under protein-free conditions, this appears to be an nonphysiological interaction and is of doubtful biological relevance. Because adhesiveness has a common basis in many different systems, fibroblast adhesion in vitro has proven to be a good model system. The multistep paradigm of adhesion, which has evolved primarily from studies of fibroblasts, emphasizes that different events occur during the adhesion sequence: adsorption, contact, attachment, and spreading. This approach has been useful in trying to understand the role of adhesiveness in the many in vivo processes discussed here. There appear to be four main determinants of the cell-substratum interaction.

1. The substratum determines whether or not adhesion is possible. In vivo, there is a wide range of adhesiveness, from the nonadhesive surface on the luminal side of the vascular endothelium to the highly adhesive surfaces of collagen fibers and the basement membrane. In a sense, the functional roles of platelets and leukocytes in thrombosis and inflammation are controlled by this gross difference. The incorporation of ligands directed against cell surface components into the extracellular matrix can establish gradients of adhesiveness for particular cell types, which may be important in selective cell fixation, and cell migration can be shown to be directed by adhesive gradients in vitro. A question to be resolved is whether or not such gradients function in uivo. Since the extracellular matrix is the scaffolding to which adhesion occurs, its physical organization may also play a role in directing cell migration. Finally, the extracellular collagen matrix is inductive or permissive for morphogenetic events in a variety of systems. That this substratum induces cells to secrete other matrix materials necessary for morphogenesis is an attractive hypothesis. In general, there is a lack of knowledge of how the various protein and mucopolysaccharide components of the extracellular matrix interact with each other and influence cell-matrix interactions. Zn vitro stud-

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ies on the adhesion of fibroblasts to surfaces coated with various connective tissue components would b e useful.

2. Cold insoluble globulin adsorbed to the substratum is the site to which fibroblasts attach in vitro. The adhesion and spreading factor

for fibroblasts on tissue culture or dried collagen substrata is cold insoluble globulin. The glycoprotein must be adsorbed onto the substratum to be active. The protein portions of the molecule, in particular, carboxyl groups, tyrosine residues, and tryptophan residues, are most important in the interaction between cold insoluble globulin and the cell surface adhesion receptor. Cells able to spread in serum-free medium probably do so by secretion of cold insoluble globulin or its equivalent onto the substratum. Cells unable to spread in serum-free medium probably do not secrete sufficient cold insoluble globulin for spreading to occur and therefore require an exogenous source of the factor in the medium which is supplied by serum or plasma.

3. Microfilaments and microtubules control cell contact, spreading, and migration. Microfilaments are required for generating the motive force necessary for the activity of microvilli and filopodia. Microtubules provide the rigid structures necessary for obtaining polarity of cell shape. The ability of cells to attach (implant) to extracel-

lular substrata and to migrate depends on these activities. Adhesive changes in cells during embryogenesis, wound healing, and malignant invasion may be dependent in part on the alteration of these cytoskeletal elements.

4. Ligand -receptor interactions between the cell and extracellular matrix are probably the basis of the bond of attachment. This notion

had been developed predominantly from model system studies and indirect evidence. Thus the existence of a cell surface adhesion receptor is still hypothetical. Yet, this is perhaps the central problem in understanding adhesion. As presented in this article, various studies are being carried out in an attempt to resolve this question. Important areas include: (1)characterization of adhesion mutants, ( 2 )analysis of cell surface components left behind when cells are removed from the substrata, ( 3 )modulation of cell adhesion by altering cell surface components, for example, by proteolysis; and (4)analysis of the mechanism of action of specific serum and plasma factors required for adhesion and identification of their binding sites on the cell surface.

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ACKNOWLEDGMENTS

I am indebted to Dr. R. G. W. Anderson for his valuable criticism of the manuscript for this article, and to Ms. Mary Owens for her expert secretarial assistance. My research has been supported by a grant from the National Institutes of Health, CA 14609.

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