Cell. Vol. l&675-666,
November
Relationships and Actin
1976,
Copyright
0 1976 by MIT
between
Fibronectin
Richard 0. Hynes and Antonia T. Destree Department of Biology and Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139
Double label immunofluorescence was used to study the distribution of fibronectin (LETS protein), actin and intermediate filaments in cultured cells. No relationship was observed between fibronectin and intermediate filaments, but fibronectin and actin showed coincident staining in a large proportion of cells during spreading or when fully spread. The distributions of actin and fibronectin staining during the course of cell spreading progressed through a series of patterns. Certain actin patterns correlated with certain fibronectin patterns. When fibrillar patterns developed, there was correspondence between the two fibrillar arrays in 80-100% of the cells. These results suggest a transmembrane relationship between microfilament bundles and fibronectin. We propose that fibronectin may participate in the formation of attachment plaques and discuss the interrelationship between plaques, microfilament bundles and fibronectin in cell-substratum and cell-cell contacts. Introduction Interactions between externally exposed surface proteins and cytoskeletal proteins have frequently been proposed (Edelman, Yahara and Wang, 1973; Hynes, 1974, 1976; Berlin et al., 1974; Edelman, 1976; Nicolson, 1976a, 1976b). There is direct biochemical evidence for such transmembrane connections in erythrocytes (Ji and Nicolson, 1974; Yu and Branton, 1976; Liu, Fairbanks and Palek, 1977). A variety of more or less indirect experiments have suggested connections of the same sort in lymphocytes and fibroblasts (see reviews above). One set of results comes from analysis of redistribution of surface markers and concomitant analysis of the arrangement of cytoskeletal proteins (Albertini and Clark, 1975; Ash and Singer, 1976; Bourgignon and Singer, 1977; Ash, Louvard and Singer, 1977; Gabbiani et al., 1977; Schreiner et al., 1977). Most recently, associations between actin and H-2 antigens and immunoglobulins in lymphoid cells have been detected by biochemical co-fractionation (Flanagan and Koch, 1978; Koch and Smith 1978). We report here on the analysis, by double label immunofluorescence of fibroblastic cells, of the
,(LETS Protein)
distribution of actin and intermediate filaments inside the cells and of a major surface protein of fibroblasts outside the cells. Fibronectin is a large protease-sensitive external glycoprotein which we have previously called LETS protein (Hynes, 1973; Hynes and Bye, 1974; Mautner and Hynes, 1977; Ali et al., 1977); it has also been called CSP (Yamada, Yamada and Pastan, 1976). Fibronectin is a major surface protein with a number of interesting properties (reviewed in Hynes, 1974, 1976; Vaheri and Mosher, 1978). It is of particular relevance to the present work that high levels of fibronectin correlate with organized arrays of actin microfilaments. Normal growth-arrested cells have extensive submembranous microfilaments and actin microfilament bundles (Goldman and Knipe, 1972; McNutt, Culp and Black, 1971, 1973); they also have relatively large amounts of fibronectin (for reviews see Hynes, 1976; Vaheri and Mosher, 1978). Growing cells have less fibronectin (Critchley,l974; Hynes and Bye, 1974) and fewer submembranous microfilaments (McNutt et al., 1971, 1973). Transformed cells also have less fibronectin (see reviews) and fewer submembranous microfilaments than normal growth-arrested cells (Heaysman and Pegrum, 1973b), and have frequently lost actin microfilament bundles (McNutt et al., 1971,1973; Goldman, Chang and Williams, 1975; Pollack, Osborn and Weber, 1975; Weber et al., 1975; Edelman and Yahara, 1976; Wang and Goldberg, 1976; Ash, Vogt and Singer, 1976; Mautner and Hynes, 1977; Goldman, Yerna and Schloss, 1977; Tucker, Sanford and Frankel, 1978) as have protease-treated cells (Pollack and Rifkin, 1975). Addition of fibronectin to transformed cells causes reappearance of readily visible microfilament bundles (Ali et al., 1977; Willingham et al., 1977; Hynes et al., 1978), whereas cytochalasin B dissociates microfilament bundles and causes release of fibronectin (Ali and Hynes, 1977). These results suggest that there is some sort of relationship between actin and fibronectin; the possibility of a transmembrane linkage between them has been proposed (Hynes, 1974, 1976). In this paper we provide evidence for such a relationship by showing that the two proteins are frequently found in close proximity to one another. Results Double Label lmmunofluorescence Anti-fibronectin was conjugated with rhddamine and affinity-purified on fibronectin-Sepharose as described in Experimental Procedures. The conjugated antibody stained a network of extracellular fibrils on confluent NIL8 hamster cells as had been observed previously by indirect immunofluorescence; the cells themselves did not stain (Figure
Cell 876
Fibronectin 877
and Actin
Relationships
IA). This staining was eliminated if the coupled antibody was preabsorbed with fibronectin bound to Sepharose (Figure 16). Figure 1. also demonstrates the use of this conjugated antibody in a double label comparison of fibronectin and intermediate filament distributions in the same cells. Figures 1C and 1D show that, when staining was carried out as described in Experimental Procedures, the two stains showed no interference. The wavy pattern of intermediate filaments inside the cells was not coincident with the straighter extracellular fibrils stained with antifibronectin. When the intermediate filaments were induced to form a coil with colchicine (Hynes and Destree, 1978) and the cells were double-stained, the anti-fibronectin did not stain the coil (not shown). This discrimination was dependent upon the blocking step using normal rabbit serum. If this step was omitted, the anti-fibronectin did stain the intermediate filaments (Figures 1E and lF), presumably by way of free binding sites on the fluoresceinated goat anti-rabbit antibodies. On the basis of earlier results (Ali and Hynes, 1977; Mautner and Hynes, 1977)) we did not expect to observe any relationship between microtubules or intermediate filaments and fibronectin, and this is precisely the result we observed, as shown in Figure 1. These results indicate that the double labeling procedure stains unrelated structures without cross-reaction of the antibodies and suggest that the procedure can be used to investigate possible relationships between fibronectin and actin. Fibronectin
and Actin
in Spreading
Cells
NIL8 cells were trypsinized from near-confluent cultures and reseeded. Samples were fixed at intervals and stained for actin and fibronectin with the double label procedure. The results of this procedure are shown in Figure 2. Cells were rounded when initially attached and no clearly defined pattern of fluorescence was discernible. By 2 hr after seeding, virtually all cells showed patterned fluorescence with both stains. The patterns were classified as follows. The fibronectin patterns observed were: punctate staining generally in the central region of the cell, often in a circle (Figure 2A); punctate staining plus some short fibrils (Figure 2B); fibrillar staining with no clearly discernible ordered pattern (Figure 2C); and fibrillar staining
with a definite pattern-radial, spiral or chordal (Figures 2D-2F). We determined by careful focusing that all the fibronectin staining occurred at the bottom of the cell. Since the cells had been permeabilized, however, we cannot be certain that all these structures are external to the plasma membrane. The actin patterns observed were: staining of the ruffles around the edge of the cell and diffuse fluorescence over the rest of the cell but no fibrillar staining (Figure 2G); a similar pattern with, in addition, staining of circumferentially arranged fibrils (Figure 2H); the appearance of straight fibrils crossing the cell (Figures 2l-2K) [the circumferential fibrils were often present together with straight fibrils arranged radially, spirally or chordally (Figures 21 and 2J)]; and arrangements of actin fibrils in polygonal geodesic arrays usually found around the edge of the cell (Figure 2L). Figure 3 shows the results of scoring the occurrence of these different patterns during the course of spreading. The patterns were readily distinguishable (the counting was performed by one person on coverslips stained, coded and arranged in random order by another). The predominant early patterns were punctate fibronectin and actin in ruffles only. By 2-3 hr, punctate plus fibrillar staining of fibronectin and circumferential fibers of actin became the most prevalent patterns. These patterns were followed at later times by fibrillar fibronectin, initially having little order and later showing definite patterns and the appearance of straight actin fibers (microfilament bundles). Between 4 and 8 hr, a significant proportion of the cells also showed geodesic structures previously reported by Lazarides (1975, 1976) in spreading cells. These results suggest that the initial punctate fibronectin pattern gives rise to fibrillar patterns which develop increasing order with time. In a similar way, the pattern of actin staining progresses from “ruffles only,” through “ruffles plus circumferential bundles,” to straight microfilament bundles. The fibrillar patterns of fibronectin and the straight microfilament bundles increase with approximately the same kinetics, suggesting that they may be related. Scoring of individual cells for both their fibronectin and actin patterns showed that the patterns did correlate. Table 1 shows that the ruffles only actin pattern was accompanied by a complete absence of fibronectin pattern or, more often, by punctate -
Figure
1. Staining
of NIL8 Hamster
Cells
for Fibronectin
or Intermediate
Filaments
(A) Confluent cells stained with rhodamine-conjugated anti-fibronectin. (B) Parallel coverslip stained with rhodamine-conjugated antifibronectin which had bean preadsorbed with fibronectin-Sepharose. Absorption with Sepharose alone gave staining as in A. (C and D) Double label immunofluorescence for intermediate filaments (C) and fibronectin (D) using the protocol described in Experimental Procedures. Note the complete lack of correspondence between the two staining patterns. (E and F) As (C and D), except that the blocking step with normal rabbit serum was omitted. Note that the rhodamine anti-fibronectin stains the intermediate filaments. Photographed with 40x objective; bar represents 50 pm.
Cell 878
Figure
2. Staining
for Fibronectin
or Actin
in Spreading
NIL8 Cells
(A-F) Anti-fibronectin. (A) 2 hr after seeding, note punctate staining often arranged in fibrillar staining. (C) 4 hr, fibrillar stainingwithout a defined pattern. (D and E) 3 and 3.5 cell is beginning to develop definite asymmetry; fibronectin fibrillar pattern is related to (G-L) Anti-actin. (G) 1.5 hr, staining of ruffles and diffuse stain in body of cell, no fibrillar only; the other is better spread and shows strong staining of circumferential fibers. development of fine fibers crossing cell. (K) 3 hr, well-developed microfilament bundles. plus microfilament bundles across cells. All photographs taken with 83x objective. Bar in C represents 25 pm.
fibronectin. Appearance of the circumferential bundles of actin in cells was accompanied by a punctate or punctate plus fibrillar fibronectin pattern, and appearance of straight microfilament
a ring. (B) 2 hr. this cell shows both punctate and hr, fibrillar staining with a definite pattern. (F) 5 hr. the asymmetry. stain. (l-f) 1.5 hr, one cell shows staining of ruffles (I and J) 3 and 2.5 hr, circumferential stain plus (L) 5 hr. polygonal array of actin around cell edge
bundles in the actin pattern was almost invariably accompanied by fibrillar patterns of fibronectin. These results suggest that the patterns of the two fibrillar networks might be more directly related.
Fibronectin 879
and Actin
Relationships
Individual cells that showed rectilinear fibriHar patterns with both stains were scored for detectable correspondence between the patterns. We observed clear parallelism or colinearity of the actin microfilament bundles and the fibronectin fibrils in 84% (156) of the 186 cells scored in this way. We did not find convincing correspondences in the remaining cells. Figure 4 shows examples of individual cells stained with the two antisera. Although clear relationships can be seen between the two patterns, several points are worth noting. While the patterns are obviously related, they are not identical: not every fibronectin fibril corresponds with an actin microfilament bundle and not every microfilament bundle has a clear association with a fibronectin fibril. Furthermore, even when colinearity occurs
DEFINITE RUFFLES + CIRCUMFERENTIAL
%
80
5o 20
GEODESICS
cells were scored for pattern of staining. 100-200 cells were scored for each time point for each label. Results are expressed as the percentage of cells showing a given pattern. (Top panel) Fibronectin. (.-0-.) No pattern; (. . .O.. .) punctate; (--C -) punctate plus fibrillar; (--A-) fibrillar with no obvious pattern; (-•-) patterned fibrillar. (Bottom panel) Actin. (.-0-.) No pattern; (. . ‘0.. .) staining of ruffles; (--W-) staining of ruffles and circumferential fibrils; (-•-) definite straight microfilament bundles, whether or not circumferential staining also occurred; (--A--) geodesic polygonal networks. Examples of the different patterns are shown in Figure 2. The corresponding symbols in the two panels indicate corresponding patterns (see Figure 4, Table 1 and text).
-
HOURS Figure 3. Spreading and Actin (Bottom) Cells Table
were
stained
Time
Course;
at intervals
1. Correlations
between
Patterns
(hr) after Patterns
of Fibronectin
reseeding, of Fibronectin
(lop)
and individual and Actin Fibronectin
Actin
Pattern
Ruffles
Cell Spreading
Pattern
None Discernable
Punctate
1.5
9
26
2
3.0
4
11
2
Hours
Only
during
plus Circumferential
Fibrils
Bundles
Geodesic
Arrays
Patterned Fibrillar
1.5
76
37
6
27
56
6
2
3
1.5
3
17
11
13
3.0
2
20
22
45
1
21
58
4.0 Polygonal
Random Fibrillar
3.0 4.0 Microfilament
+
1
4.0 Ruffles
Punctate Fibrillar
1.5 3.0 4.0
2
5
4
14
Individual double-stained cells were scored for their patterns with both antibodies at three different times. 202 cells were counted for 1.5 hr, 204 for 3 hr. and 104 for the 4 hr time point. Note the time-dependent progression to more complex patterns and the predominance of cells on the diagonal of the table, indicating correlations between fibronectin and actin patterns. Cells in the last two lines of the last two columns (those showing fibrillar staining with both antibodies) were also scored for correspondences between the two stains. Correspondence was scored as: no obvious relationship (30 cells), parallel arrangement of fibronectin and actin but coincidence not clear (65 cells), clear coincidence (91 cells). Pooling those showing parallelism or coincidence of staining gave 156 out of 166 cells showing correspondence between the two patterns (64%).
Cell 860
Figure
4. Double
Label Staining
of NIL8 Cells during
Spreading
The figure shows pairs of photographs of actin and fibronectin staining of the same cells. (A, B, D and E) 1.5 hr. photographed with 63x objective, bar represents 50 pm. Note that the punctate fluorescence seen in D with antifibronectin is not observed in A with anti-actin. Conversely, anti-fibronectin does not stain the ruffles seen in A with anti-actin. (B and E) show cells in different stages. Two cells show no actin microfilament bundles and have punctate and short fibrillar fibronectin. One cell is better spread and shows fibrillar staining with both antibodies. All other panels were photographed with 40x objective. Bar in K represents 50 pm. (C and F) 3 hr. Note correspondence between actin and fibronectin fibrils. (G and J) 4 hr. Well-developed actin microfilament
Fibronectin 881
and Actin
Relationships
between actin and fibronectin, the two stains are not always co-extensive. This lack of complete correspondence indicates that the apparent codistribution does not arise merely from cross-reactions between the antibodies. The possible significance of the different arrangements seen is taken up in the Discussion.
Fibronectin and Actin in Well-Spread Cells By 8 hr of spreading, most cells were asymmetric and had well-defined actin and fibronectin fibrils (Figure 3). This situation persisted over the next 16-24 hr. If cells were allowed to continue growth in 5% serum, they became confluent and, as described previously (Mautner and Hynes, 1977), developed an extensive fibrillar network of fibronectin, most of which was on top of the cells. We could see no obvious correspondence between this pattern and the arrangement of actin microfilament bundles (MFB), which was not as clear as in sparse cultures. To investigate the relationships between fibronectin and actin in cells having large amounts of fibronectin, we turned to cultures arrested at subconfluence in low serum. We have previously shown that in such cultures most of the fibronectin is in fibrillar “footprints” beneath the cells and can be left behind on the substratum when the cells are detached (Hynes, Destree and Mautner, 1976; Mautner and Hynes, 1977; Hynes et al., 1978). In this condition we could observe well-displayed actin MFB and extensive extracellular networks of fibronectin. Figure 5 shows that there was very good correspondence between the two patterns. Virtually every cell in these cultures showed coincidence of some actin MFB and fibronectin. As in the spreading cells, the patterns were related but not identical. Careful focusing showed that actin MFB near the bottom of the cell corresponded to fibronectin fibrils beneath the cell, but that other arrays of actin higher up in the cell did not (see Figure 5). We observed several interesting patterns. The basal actin MFB frequently terminated before the periphery of the cell, whereas the colinear fibronectin often continued further toward the edge of the cell and even beyond it (compare Figures 5A5C with 5D-5F). In many instances we observed adjacent cells, each with arrays of basal MFB that appeared to be connected by fibronectin strands running from one cell to the other, each end of the fibronectin strand being colinear with an actin MFB (not shown). Another fairly common pattern consisted of arrays of actin MFB that coincided with
fibronectin along their
only near their ends lengths (Figure 5D).
or at intervals
Discussion This study has provided evidence that the double label immunofluorescence procedure does indeed work and that the apparent coincidences of staining do not arise from cross-reaction. Both antibodies (anti-fibronectin and anti-actin) are monospecific and do not cross-react by the Criteria of immunofluorescence (Mautner and Hyries, 1977) or by staining of gels (Burridge, 1976; our unpub’ lished data). Since both are rabbit antibodies, how-~ ever, the efficacy of the blocking step (see Experimental Procedures) must be established. Artifactual cross-reaction was not observed when unrelated structures (fibronectin and intermediate filaments) were stained using the procedure (Figure 1). Perhaps more convincingly, the actin-fibronectin double staining experiments (Figures 2, 4, and 5) contain internal evidence against cross-reaction. No fluorescein staining was seen beyond the boundaries of spread cells although rhodamine anti-fibronectin staining was apparent. In the early spreading cells, there was no fluorescein staining of the punctate pattern seen with the anti-fibronectin antibody. Thus the fluorescein goat anti-rabbit used to detect the anti-actin staining did not also detect the anti-fibronectin antibodies. Conversely, the rhodamine anti-fibronectin did not bind to the goat anti-rabbit or the anti-actin as shown by its failure to stain ruffles, circumferential microfilament bundles, geodesic arrays of actin, the periphery of well-spread cells or, indeed, many of the actin microfilament bundles in the cells. Coincident staining suggests coincident distribution of the two antigens. It is not clear, however, what coincidence means in this context since the resolution of the technique cannot be better than 0.2 pm. Thus the results indicate only that the two antigens are closer than the degree of resolution. A finer analysis of the degree of molecular proximity must await biochemical analysis. The apparent correspondence between actin and fibronectin becomes more convincing when complex patterns of one are replicated by the other, as shown in many of the Figures. We have investigated the correspondences in several ways: by preparing double exposures on color film, by tracing prints of the two stains and by projecting transparencies of one onto the other or onto tracings of the other. These exercises convince one of the correspondences, --____~
bundles coincide with fibronectin fibrils. Note that fibronectin fibrils are not continuous. (l-l and K) 4.5 hr. Well-developed microfilament bundles cross the cell and partially coincide with discontinuous fibronectin fibrils. Note that the polygonal array of actin (H) does not stain with anti-fibronectin (K). (I and J) 24 hr. Well-spread cell showing good microfilament bundles and corresponding fibronectin fibrils. Note that the two sets of fibrils are not co-extensive.
Cell 882
Figure
5. Double
Label lmmunofluorescence
of Well-Spread
Growth-Arrested
NILS Cells
Cells were seeded in 0.3% serum and fixed 4 days later. Photography was with 53x lens; bar represents 50 pm. (A-C) Anti-actin; (D-F) antifibronectin. In (A and D), note that in the upper right of the cell each microfilament bundle corresponds with a fibronectin fibril, and in the center of the cell many microfilament bundles lack corresponding fibronectin fibrils. Arrows in (D) indicate interrupted linear staining with antifibronectin. Also note that the cell boundary stains with anti-actin but not anti-fibronectin, whereas extracellular fibrils on the substratum and out-of-focus fibrils on top of the cell (top center, D) show the inverse staining pattern. The other four panels show good correspondence between anti-actin and anti-fibronectin. In (6 and C). arrows indicate microfilament bundles that terminate before the cell edge. Corresponding arrows in (E and F) indicate the fibronectin fibrils continuing beyond cell boundaries. In all panels, focus was at the bottom of the cell. Other arrays of actin microfilaments at higher planes of focus did not correspond with fibronectin staining, as seen clearly in (8).
but only indicate proximity. In the spreading cells, the development of the typical patterns of actin microfilament bundles proceeds from an initial concentration of actin in ruffles to circumferential fibrils and then to straight bundles running across the cell, sometimes passing through a stage characterized by polygonal or geodesic arrays of actin. Similar patterns have been described by Lazarides (1975,1976). We were concerned with the relationship of this pattern to the development of the fibronectin pattern. At early times, when cells contain only circumferential and no rectilinear actin fibrils, the predominant fibronectin pattern was punctate. .The specks of fibro-
nectin stain did not correspond to the circumferential actin fibers. When the punctate pattern was circular, it was usually central to the concentric actin fibers (see figures 4A and 4D). In almost all of the cells, rectilinear fibronectin fibrils appeared as soon as rectilinear actin fibrils did (Table l), and some correspondence between the two fibrillar arrays could also be observed in most cases (Figure 4). With time, both arrays increased in intensity of staining and in complexity, and the predominance of coincidences between the two patterns persisted. In well-spread, nonmotile cells (Figure 5), virtually 100% of the cells showed correspondence between the patterns.
Fibronectin 883
and Actin
Relationships
These results suggest a causal relationship between the two arrays of fibrils but the experiments do not allow us to determine the direction of causality. Earlier work has suggested causality in each direction: addition of fibronectin to cells lacking it promotes ordering of actin microfilament bundles (Ali et al., 1977), whereas addition of cytochalasin B, which disrupts actin microfilament bundles, causes increased release and decreased attachment of fibronectin (Ali and Hynes, 1977). One could postulate a relationship of reciprocal causality, but a final decision must await further work. In this context it is worth noting that we have examined two other cell types (rat embryo fibroblasts and human fibroblasts), both of which are extremely well-spread in sparse culture and have elaborate arrays of actin microfilament bundles. Under our experimental conditions these cells had rather little fibronectin, indicating that coincident fibronectin fibrils are not a necessary condition for the development of actin microfilament bundles (our unpublished data). The actin-containing structures described here have all been reported previously and their nature has also been analyzed by electron microscopy (Abercrombie, Heaysman and Pegrum, 1971; McNutt et al., 1971, 1973; Goldman and Knipe, 1972). It is worthwhile to consider the possible nature of the fibronectin-containing structures. All the structures reported in this paper that show relationships with actin lie at or near the bottom of the cell. The first to appear are the punctate patches seen in early spreading cells. These could be vesicles inside the cells (but near its base) or patches of fibronectin between the cell and the substratum. Punctate patterns of fibronectin have also been observed in well-spread epithelial cells (Chen et al., 1977; Mosher et al., 1977). These punctate patches of fibronectin might correspond to the attachment plaques seen with electron microscopy and interference microscopy (Abercrombie et al., 1971; lzzard and Lochner, 1976). These plaques are the points of close apposition and adhesion to the substratum (Abercrombie et al., 1971; Brunk et al.,1971), and similar plaques appear on contact with another cell (Heaysman and Pegrum, 1973a). Attachment plaques are frequently insertion points for actin microfilament bundles (Abercrombie et al., 1971; McNutt et al., 1971, 1973; Heaysman and Pegrum, 1973a; Revel and Wolken, 1973; Abercrombie and Dunn, 1975; lzzard and Lochner, 1976). They can be either punctate or elongate, as are the patterns of fibronectin staining reported here. Given the facts that fibronectin promotes cell adhesion and flattening (Yamada et al., 1976; Ali et al., 1977) and organization of microfilament bundles (Ali et al., 1977; Willingham et al., 1977; Hynes et al., 1978), and, in the present work, is frequently
coincident with actin microfilament bundles (Figures 4 and 5), it is reasonable to propose that fibronectin is a constituent of the attachment plSqUt?S. We are currently investigating this hypothesis. The observation that microfilament bundles are frequently coincident with fibronectin only at their ends or at intervals along their lengths (Figures 4 and 5) could be explained in a number of ways, One possibility is that these microfilament bundles, which are at or near the bottom of the cell, are closely apposed to the substratum only at some points, these being the positions where fibronectin staining is also observed. Also of interest is the observation that microfilament bundles which terminate at the cell periphery sometimes appear continuous with fibronectin strands that extend beyond the cell and even to adjacent cells where they may appear continuous with another microfilament bundle. This conformation suggests alignments of stress fibers with intercellular fibrillar connections containing fibronectin. Fibronectin fibrils have been reported in regions of cell-cell contact (Chen, Gallimore and McDougall, 1976; Mautner and Hynes, 1977), and actin microfilament bundles have been reported to arise from the point of cell-cell contact (Heaysman and Pegrum, 1973a; McNutt et al., 1973; Heggeness, Wang and Singer, 1977). We have more frequently observed that the actin structures terminate before the cell edge and the fibronectin fibril crosses the gap. The involvement of these structures in cell-cell contact areas merits further research Finally, we must consider how these results relate to recent reports that integral membrane proteins become aligned with microfilament bundles (Ash and Singer, 1976; Ash et al., 1977). In these experiments, various lectins and antibodies were used to stain living cells. The stains, which were evenly distributed at first, became (in some cells) concentrated with time over microfilament bundles, preddminantly over the top of the cell (Ash and Singer, 1976; Ash et al., 1977). In our experiments the cells were fixed prior to staining, making antibody-induced redistribution improbable. In any case, fibronectin on these cells is apparently not free to move laterally (Hynes et al., 1976; Mautner and Hynes, 1977), as others have also reported (Schlessinger et al., 1977). Fibronectin is a lectin receptor (Burridge, 1976; our unpublished results) and stains well with concanavalin A, one of the reagents used by Ash et al. (1976, 1977), but it is improbable that it would bind the specific antibodies tested (Ash et al ., 1977). Some time ago we suggested that fibronectin might be permanently connected with the cytoskeleton across the membrane (Hynes, 1974). This arrangement was offered as an explanation for the
Cell 884
restriction of lateral mobility of lectin binding sites ih normal cells that contain fibronectin and for the absence of restriction in transformed or proteasetreated cells that had been shown to lack fibronectin (Hynes, 1973). It was hypothesized that fibronectin might act as an anchor with which other lectin binding sites were associated either constitutively or after lectin-mediated lateral aggregation. The existence of extensive disulfide bonding among surface proteins (Hynes and Destree, 1977) could also contribute to this restriction of lateral mobility by promoting anchorage to fibronectin that is in turn anchored to the cytoskeleton. This hypothesis remains tenable, could explain some of the results of Ash, Singer et al., and is given some support by the data reported in this paper. Experimontrl Cells
Procedures
and Culture
Condltlons
The cells used were the NIL8 hamster fibroblastic cell line. Cells were cultured in Dulbecco’s modified Eagle’s medium with 5% fetal calf serum. They were seeded on coverslips (12 x 12 mm, Gold Seal) by trypsinization and plating in 5% fetal calf serum for analysis of spreading, or in 0.3% fetal calf serum for 2-4 days for analysis of well-spread, growth-arrested cultures.
Antlbodles The antisera used have all been described and characterized. They were anti-actin (Burridge, 1978; a gift from Keith Burridge), antiserum to the 58,000 dalton subunit of intermediate filaments (Hynes and Destree, 1978) and antifibronectin (anti-LETS protein; Mautner and Hynes, 1977). All three sera were raised using SDSgel-purified immunogens in rabbits, and for indirect staining were detected with fluorescein-conjugated goat anti-rabbit IgG (Miles). For double labeling, rhodamine-conjugated anti-fibronectin was prepared as follows. lmmunoglobulins were diluted to 10 mglml with 0.01 M Tris-HCI (pH 7.5). and tetramethyl rhodamine (Beckton, Dickinson and Co.) was added to give a final ratio of 15 fig/ mg protein: the pH was adjusted if necessary during the first hour. After incubation overnight at 4°C with stirring, unconjugated rhodamine was removed by passage over G25 Sephadex. The excluded peak was then passed over a column of Sepharose 28 coupled to hamster fibronectin (0.4 mglml of gel) purified as described (Mautner and Hynes, 1977; Hynes et al., 1978). After thorough washing with phosphate-buffered saline (PBS), bound antibody was eluted with 5 M Nal + 1 mM sodium thiosulfate, supplemented with 1 mg/ml ovalbumin, dialyzed extensively against PBS, checked by immunofluorescence (see Figure 1) and concentrated by vacuum dialysis or lyophilization if necessary. For absorption, rhodamine-conjugated anti-fibronectin was incubated for 30 min at 37°C with an equal volume of either fibronectin-Sepharose or Sepharose. The beads were removed by centrifugation and the supernatants were tested by immunofluorescence (Figure I).
Immunofluorescence lmmunofluorescence was performed on coverslips after fixation with formaldehyde and acetone treatment as described (Mautner and Hynes. 1977; Hynes and Destree, 1978). For double label immunofluorescence. the following protocol was used: stain with first antibody (anti-actin, 11100 dilution of IgG preparation, or anti-intermediate filament, l/25 dilution of whole serum), rinse; stain with fluoresceinated goat anti-rabbit IgG. l/IO dilution, rinse; incubate with normal rabbit serum, 1110 diltuion, rinse; stain with rhodamine-conjugated anti-fibronectin, rinse and mount. All staining steps were for 30 min at 37°C. The third step
with normal rabbit serum was essential to block free binding sites on the goat anti-rabbit IgG used to stain the anti-actin. If this step was omitted, the rabbit anti-fibronectin bound to actin-containing structures presumably via a ternary antibody complex (see Figure 1 and text). All photographs were taken on a Zeiss microscope equipped with epifluorescence optics, using a 63/l .4 or 40/I .O lens and exposure times of 30-60 set with Kodak Plus-X film, which was developed using Diafine.
Acknowledgments We are indebted and grateful to Keith Burridge for the anti-actin antibody. This work was supported by grants from the National Cancer Institute (to R.O.H. and S. E. Luria) and the American Cancer Society. R.O.H. is the recipient of an NIH Research Career Development Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
June
27,1978;
revised
August
2,1978
Refemnces Abercrombie. M. and Dunn, G. A. (1975). Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. Exp. Cell Res. 92, 57-62. Abercrombie, M., Heaysman, J. E. M. and Pegrum, S. M. (1971). The locomotion of fibroblasts. IV. Electron microscopy of the leading lamella. Exp. Cell Res. 67, 359-367. Albertini, D. F. and Clark, J. I. (1975). Membrane-microtubule interactions: concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins. Proc. Nat. Acad. Sci USA 72, 4976-4980. Ali, I. U. and Hynes, R. 0. (1977). Effects of cytochalasin B and colchicine on attachment of a major surface protein of fibroblasts. Biochim. Biophys. Acta 471, 16-24. Ali, I. U.. Mautner, V. Lanza, R. and Hynes, R. 0. (1977). tion of normal morphology: adhesion and cytoskeleton formed cells by addition of a transformation-sensitive protein. Cell 11, 115-126.
Restorain transsurface
Ash, J. F. and Singer, S. J. (1976). Concanavalin A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin-containing filaments. Proc. Nat. Acad. Sci. USA 73, 45754579. Ash, J. F., Vogt. P. K. and Singer, S. J. (1976). Reversion from transformed to normal phenotype by inhibition of protein synthesis in rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 73, 3603-3607. Ash, J. F.. Louvard, D. and Singer, S. J. (1977). Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. Proc. Nat. Acad. Sci. USA 74, 5584-5588. Berlin, Control
R. D., Oliver, J. M.. Ukena, T. E. and Yin, H. H. (1974). of cell surface topography. Nature 247, 45-48.
Bourguignon, L. Y. W. and Singer, S. J. (1977). Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc. Nat. Acad. Sci. USA 74, 50315035. Brunk, U., Ericsson, (1971). Specialization glia-like cells in vitro.
J. L. E., Ponten, J. and Westermark, B. of cell surfaces in contact-inhibitied human Exp. Cell Res. 67, 407-415.
Burridge. K. (1976). Changes in cellular glycoproteins after transformation: identification of specific glycoproteins and antigens in sodium dodecylsulfate gels. Proc. Nat. Acad. Sci. USA 73, 44574461. Chen.
L.
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and
McDougall,
J. K.
(1978).
Fibronectin 885
Correlation tein. Proc.
and Actin
Relationships
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LETS
pro-
Chen, L. B., Maitland, N., Gallimore, P. H. and McDougall,J. K. (1977). Detection of the large external transformation-sensitive protein on some epithelial cells. Exp. Cell Res. 106, 39-48. Critchiey. D. Ft. (1974). Cell surface proteins of NIL1 hamster fibroblasts labeled by a galactose oxidase, tritiated borohydride method. Cell3. 121-125. Edelman, G. M. (1978). and cell growth. Science
Surface modulation 792, 218-226.
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Edelman, G. M. and Yahara, I. (1976). Temperature-sensitive changes in surface modulating assemblies of fibroblasts transformed by mutants of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 73, 2047-2051. Edelman. G. M., Yahara, I. and Wang, J. L. (1973). Receptor mobility and receptor-cytoplasmic interactions in lymphocytes. Proc. Nat. Acad. Sci. 70. 1442-1448. Flanagan, J. and Koch, immunoglobulin attaches Gabbiani, G., Chaponnier, Actin and tubulin co-cap lymphocytes. Nature269,
G. L. E. (1978). Cross-linked to actin. Nature 273, 278-281. C.. Zumbe. A. and Vassalli. with surface immunoglobulins 697-898.
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L. R. (1978). Cell-to-substrate interference reflexion study J. Cell Sci. 21, 129-159.
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Ji, T. H. and Nicolson. G. L. (1974). Lectin binding and perturbation Of the outer surface of the cell membrane induces a transmembrane organizational alteration at the inner surface, Proc. Nat. Acad. Sci. USA 71, 2212-2216. Koch, G. L. E. and actin and the major 274-278.
Smith, M. J. (1978). An association between histocompatability antigen H-2. Nature 273,
Lazarides, E. (1975). lmmunofluorescence ture of actin filaments in tissue culture Cytochem. 23, 507-528.
studies on the struccells. J. Histochem.
Lazarides, E. (1976). Actin, a-actinin and tropomyosin interaction in the structural organization of actin filaments in non-muscle cells. J. Cell Biol. 68, 202-219. Liu, S. C., Fairbanks, G. and Palek, J. (1977). Spontaneous, reversible protein crosslinking in the human erythrocyte membrane, temperature and pH dependence. Biochemistry 16, 40664074.
Goldman, R. D. and Knipe. D. M. (1972). Functions of cytoplasmic fibers in nonmuscle cells. Cold Spring Harbor Symp. Quant. Viol. 37, 523-534.
McNutt. N. S., Gulp, L. A. and Black, P. H. (1971). inhibited revertant cell lines isolated from SV40-transformed II. Ultrastructural study. J. Cell Biol. 50. 691-708.
Goldman, R. D., Chang. C. and Williams, and behavior of hamster embryo cells adenovirus type 5. Cold Spring Harbor 801-814.
McNutt, N. S., Gulp, L. A. and Black, P. H. (1973). Contactinhibited revertant cell lines isolated from SV40-transformed cells. IV. Microfilament distribution and cell shape. J. Cell Viol. 56, 413428.
J. F. (1975). Properties transformed by human Symp. Quant. Biol. 39,
Goldman, R. D., Yerna. M. J. and Schloss, J. A. (1977). Localization and organization of microfilaments and related proteins in normal and virus-transformed cells. In Cell Shape and Surface Architecture, J. P. Revel, U. Henning and C. F. Fox, eds. (New York: Alan Ft. Liss), pp. 107-135. Heaysman, J. E. M. and Pegrum. S. M. (1973a). Early contacts between fibroblasts. An ultrastructural study. Exp. Cell Res. 78, 71-78. Heaysman. J. E. M. and Pegrum, S. M. (1973b). Methylcholanthrene-induced sarcoma and normal muscle fibroblasts of the mouse: a comparison of their ultrastructure. Differentiation 1, 191-198. Heggeness, M. H., Wang, K. and Singer, S. J. (1977). Intracellular distributions of mechanochemical proteins in cultured fibroblasts. Proc. Nat. Acad. Sci. USA 74, 3883-3887. Hynes. R. 0. (1973). Alteration of cell-surface proteins transformation and by proteolysis. Proc. Nat. Acad. Sci. 3170-3174. Hynes. R. 0. (1974). Role of surface alterations mation: the importance of proteases and surface 147-158. Hynes. mation.
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Hynes, R. 0. and Bye. J. M. (1974). Density and cell cycle dependence of cell surface proteins in hamster fibroblasts. Cell 3, 113-120. Hynes, R. 0. and Destree, A. T. (1977). bonding at the mammalian cell surface. Proc. 74, 2855-2859.
Extensive Nat. Acad.
Hynes. R. 0. and Destree, A. T. (1978). 10 nm filaments and transformed cells. Cell 73, 151-183.
disulfide Sci. USA in normal
Hynes, R. O., Destree, A. T. and Mautner, V. M. (1976). Spatial organization at the cell surface. In Membranes and Neoplasia: New Approaches and Strategies, V. T. Marchesi. ed. (New York: Alan R. Liss). pp. 189-201. Hynes. R. O., Ali, I. U., Destree, A. T.. Mautner, V., Perkins, M. E., Senger, D. R., Wagner, D. D. and Smith, K. K. (1978). A large
Mautner. V. and Hynes. R. 0. (1977). protein in relation to the cytoskeleton cells J. Cell Biol. 75, 743-758.
Contactcells.
Surface distribution of LETS of normal and transformed
Mosher, D. F., Saksela, O., Keski-Oja. J. and Vaheri, A. (1977). Distribution of a major surface-associated glycoprotein, fibronectin, in cultures of adherent cells. J. Supramol. Structure 6, 551557. Nicolson, G. L. (1976a). Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over cell surface components. Biochim. Biophys. Acta 457, 57-108. Nicolson, G. L. (1976b). Transmembrane control of the receptors on normal and tumor cells. II. Surface changes associated with transformation and malignancy. Biochim. Biophys. Acta 458, l72. Pollack. R. and Rifkin. D. (1975). Actin-containing cables within anchorage-dependent rat embryo cells are dissociated by plasmin and trypsin. Cell 6, 495-506. Pollack, R.. Osborn. M. and Weber, K. (1975). Patterns zation of actin and myosin in normal and transformed cells. Proc. Nat. Acad. Sci. USA 72, 994-998. Revel, gation 14.
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J. P. and Wolken, K. (1973). Electron microscope investiof the underside of cells in culture. Exp. Cell Res. 78, l-
Schlessinger, J.. Earak, L. S.. Hammes, G. G.. Yamada, K. M.. Pastan. I., Webb, W. W. and Elson, E. L. (1977). Mobility and distribution of a cell surface glycoprotein and its interaction with other membrane components. Proc. Nat. Acad. Sci. USA 74, 2909-2913. Schreiner, G. F., Fujiwara. K., Pollard, T. D. and Unanue. E. R. (1977). Redistribution of myosin accompanying capping of surface immunoglobulin. J. Exp. Med. 745, 1393-1398. Tucker, R. W., Sanford, K. K. and Frankel. F. R. (1978). Tubulin and actin in paired nonneoplastic and spontaneously transformed neoplastic cell lines in vitro: fluorescent antibody studies. Cell 13, 829-842. Vaheri, A. and Mosher, D. F. (1978). High molecular surface-associated glycoprotein (fibronectin) lost transformation. Biochim. Biophys. Acta. in press.
weight, cell in malignant
Cell 886
Wang, E. and Goldberg, A. R. (1976). organization and surface topography chick embryo fibroblasts with Rous Acad. Sci. USA 73, 40654069.
Changes in microfilament upon transformation of sarcoma virus. Proc. Nat.
Weber, K., Lazarides. E.. Goldman, R. D., Vogel, A. and Pollack. R. (1975). Localization and distribution of actin fibers in normal, transformed and revertant cells. Cold Spring Harbor Symp. Quant. Biol. 39, 363-369. Willingham, M. C., Yamada. K. M., Yamada, S. S., Pouyssegur, J. and Pastan. I. (1977). Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts. Cell 10, 375-360. Yamada. K. M.. Yamada, S. S. and Pastan, I. (1976). protein partially restores morphology, adhesiveness, inhibition of movement to transformed fibroblasts. Acad. Sci. USA 731217-1221.
Cell surface and contact Proc. Nat.
Yu. J. and Branton, D. (1976). Reconstitution of intramembrane particles in recombinants of erythrocyte protein band 3 and lipid: effects of spectrin actin association. Proc. Nat. Acad. Sci. USA 73.3891-3895. Nota
added
In Proof
M. Heggeness, J. Ash and S. Singer have recently reported (1976, Ann. NY Acad. Sci .312,414-417) some coincidence of actin and fibronectin in WI38 cells.
November
Relationships and Actin
1976,
Copyright
0 1976 by MIT
between
Fibronectin
Richard 0. Hynes and Antonia T. Destree Department of Biology and Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139
Double label immunofluorescence was used to study the distribution of fibronectin (LETS protein), actin and intermediate filaments in cultured cells. No relationship was observed between fibronectin and intermediate filaments, but fibronectin and actin showed coincident staining in a large proportion of cells during spreading or when fully spread. The distributions of actin and fibronectin staining during the course of cell spreading progressed through a series of patterns. Certain actin patterns correlated with certain fibronectin patterns. When fibrillar patterns developed, there was correspondence between the two fibrillar arrays in 80-100% of the cells. These results suggest a transmembrane relationship between microfilament bundles and fibronectin. We propose that fibronectin may participate in the formation of attachment plaques and discuss the interrelationship between plaques, microfilament bundles and fibronectin in cell-substratum and cell-cell contacts. Introduction Interactions between externally exposed surface proteins and cytoskeletal proteins have frequently been proposed (Edelman, Yahara and Wang, 1973; Hynes, 1974, 1976; Berlin et al., 1974; Edelman, 1976; Nicolson, 1976a, 1976b). There is direct biochemical evidence for such transmembrane connections in erythrocytes (Ji and Nicolson, 1974; Yu and Branton, 1976; Liu, Fairbanks and Palek, 1977). A variety of more or less indirect experiments have suggested connections of the same sort in lymphocytes and fibroblasts (see reviews above). One set of results comes from analysis of redistribution of surface markers and concomitant analysis of the arrangement of cytoskeletal proteins (Albertini and Clark, 1975; Ash and Singer, 1976; Bourgignon and Singer, 1977; Ash, Louvard and Singer, 1977; Gabbiani et al., 1977; Schreiner et al., 1977). Most recently, associations between actin and H-2 antigens and immunoglobulins in lymphoid cells have been detected by biochemical co-fractionation (Flanagan and Koch, 1978; Koch and Smith 1978). We report here on the analysis, by double label immunofluorescence of fibroblastic cells, of the
,(LETS Protein)
distribution of actin and intermediate filaments inside the cells and of a major surface protein of fibroblasts outside the cells. Fibronectin is a large protease-sensitive external glycoprotein which we have previously called LETS protein (Hynes, 1973; Hynes and Bye, 1974; Mautner and Hynes, 1977; Ali et al., 1977); it has also been called CSP (Yamada, Yamada and Pastan, 1976). Fibronectin is a major surface protein with a number of interesting properties (reviewed in Hynes, 1974, 1976; Vaheri and Mosher, 1978). It is of particular relevance to the present work that high levels of fibronectin correlate with organized arrays of actin microfilaments. Normal growth-arrested cells have extensive submembranous microfilaments and actin microfilament bundles (Goldman and Knipe, 1972; McNutt, Culp and Black, 1971, 1973); they also have relatively large amounts of fibronectin (for reviews see Hynes, 1976; Vaheri and Mosher, 1978). Growing cells have less fibronectin (Critchley,l974; Hynes and Bye, 1974) and fewer submembranous microfilaments (McNutt et al., 1971, 1973). Transformed cells also have less fibronectin (see reviews) and fewer submembranous microfilaments than normal growth-arrested cells (Heaysman and Pegrum, 1973b), and have frequently lost actin microfilament bundles (McNutt et al., 1971,1973; Goldman, Chang and Williams, 1975; Pollack, Osborn and Weber, 1975; Weber et al., 1975; Edelman and Yahara, 1976; Wang and Goldberg, 1976; Ash, Vogt and Singer, 1976; Mautner and Hynes, 1977; Goldman, Yerna and Schloss, 1977; Tucker, Sanford and Frankel, 1978) as have protease-treated cells (Pollack and Rifkin, 1975). Addition of fibronectin to transformed cells causes reappearance of readily visible microfilament bundles (Ali et al., 1977; Willingham et al., 1977; Hynes et al., 1978), whereas cytochalasin B dissociates microfilament bundles and causes release of fibronectin (Ali and Hynes, 1977). These results suggest that there is some sort of relationship between actin and fibronectin; the possibility of a transmembrane linkage between them has been proposed (Hynes, 1974, 1976). In this paper we provide evidence for such a relationship by showing that the two proteins are frequently found in close proximity to one another. Results Double Label lmmunofluorescence Anti-fibronectin was conjugated with rhddamine and affinity-purified on fibronectin-Sepharose as described in Experimental Procedures. The conjugated antibody stained a network of extracellular fibrils on confluent NIL8 hamster cells as had been observed previously by indirect immunofluorescence; the cells themselves did not stain (Figure
Cell 876
Fibronectin 877
and Actin
Relationships
IA). This staining was eliminated if the coupled antibody was preabsorbed with fibronectin bound to Sepharose (Figure 16). Figure 1. also demonstrates the use of this conjugated antibody in a double label comparison of fibronectin and intermediate filament distributions in the same cells. Figures 1C and 1D show that, when staining was carried out as described in Experimental Procedures, the two stains showed no interference. The wavy pattern of intermediate filaments inside the cells was not coincident with the straighter extracellular fibrils stained with antifibronectin. When the intermediate filaments were induced to form a coil with colchicine (Hynes and Destree, 1978) and the cells were double-stained, the anti-fibronectin did not stain the coil (not shown). This discrimination was dependent upon the blocking step using normal rabbit serum. If this step was omitted, the anti-fibronectin did stain the intermediate filaments (Figures 1E and lF), presumably by way of free binding sites on the fluoresceinated goat anti-rabbit antibodies. On the basis of earlier results (Ali and Hynes, 1977; Mautner and Hynes, 1977)) we did not expect to observe any relationship between microtubules or intermediate filaments and fibronectin, and this is precisely the result we observed, as shown in Figure 1. These results indicate that the double labeling procedure stains unrelated structures without cross-reaction of the antibodies and suggest that the procedure can be used to investigate possible relationships between fibronectin and actin. Fibronectin
and Actin
in Spreading
Cells
NIL8 cells were trypsinized from near-confluent cultures and reseeded. Samples were fixed at intervals and stained for actin and fibronectin with the double label procedure. The results of this procedure are shown in Figure 2. Cells were rounded when initially attached and no clearly defined pattern of fluorescence was discernible. By 2 hr after seeding, virtually all cells showed patterned fluorescence with both stains. The patterns were classified as follows. The fibronectin patterns observed were: punctate staining generally in the central region of the cell, often in a circle (Figure 2A); punctate staining plus some short fibrils (Figure 2B); fibrillar staining with no clearly discernible ordered pattern (Figure 2C); and fibrillar staining
with a definite pattern-radial, spiral or chordal (Figures 2D-2F). We determined by careful focusing that all the fibronectin staining occurred at the bottom of the cell. Since the cells had been permeabilized, however, we cannot be certain that all these structures are external to the plasma membrane. The actin patterns observed were: staining of the ruffles around the edge of the cell and diffuse fluorescence over the rest of the cell but no fibrillar staining (Figure 2G); a similar pattern with, in addition, staining of circumferentially arranged fibrils (Figure 2H); the appearance of straight fibrils crossing the cell (Figures 2l-2K) [the circumferential fibrils were often present together with straight fibrils arranged radially, spirally or chordally (Figures 21 and 2J)]; and arrangements of actin fibrils in polygonal geodesic arrays usually found around the edge of the cell (Figure 2L). Figure 3 shows the results of scoring the occurrence of these different patterns during the course of spreading. The patterns were readily distinguishable (the counting was performed by one person on coverslips stained, coded and arranged in random order by another). The predominant early patterns were punctate fibronectin and actin in ruffles only. By 2-3 hr, punctate plus fibrillar staining of fibronectin and circumferential fibers of actin became the most prevalent patterns. These patterns were followed at later times by fibrillar fibronectin, initially having little order and later showing definite patterns and the appearance of straight actin fibers (microfilament bundles). Between 4 and 8 hr, a significant proportion of the cells also showed geodesic structures previously reported by Lazarides (1975, 1976) in spreading cells. These results suggest that the initial punctate fibronectin pattern gives rise to fibrillar patterns which develop increasing order with time. In a similar way, the pattern of actin staining progresses from “ruffles only,” through “ruffles plus circumferential bundles,” to straight microfilament bundles. The fibrillar patterns of fibronectin and the straight microfilament bundles increase with approximately the same kinetics, suggesting that they may be related. Scoring of individual cells for both their fibronectin and actin patterns showed that the patterns did correlate. Table 1 shows that the ruffles only actin pattern was accompanied by a complete absence of fibronectin pattern or, more often, by punctate -
Figure
1. Staining
of NIL8 Hamster
Cells
for Fibronectin
or Intermediate
Filaments
(A) Confluent cells stained with rhodamine-conjugated anti-fibronectin. (B) Parallel coverslip stained with rhodamine-conjugated antifibronectin which had bean preadsorbed with fibronectin-Sepharose. Absorption with Sepharose alone gave staining as in A. (C and D) Double label immunofluorescence for intermediate filaments (C) and fibronectin (D) using the protocol described in Experimental Procedures. Note the complete lack of correspondence between the two staining patterns. (E and F) As (C and D), except that the blocking step with normal rabbit serum was omitted. Note that the rhodamine anti-fibronectin stains the intermediate filaments. Photographed with 40x objective; bar represents 50 pm.
Cell 878
Figure
2. Staining
for Fibronectin
or Actin
in Spreading
NIL8 Cells
(A-F) Anti-fibronectin. (A) 2 hr after seeding, note punctate staining often arranged in fibrillar staining. (C) 4 hr, fibrillar stainingwithout a defined pattern. (D and E) 3 and 3.5 cell is beginning to develop definite asymmetry; fibronectin fibrillar pattern is related to (G-L) Anti-actin. (G) 1.5 hr, staining of ruffles and diffuse stain in body of cell, no fibrillar only; the other is better spread and shows strong staining of circumferential fibers. development of fine fibers crossing cell. (K) 3 hr, well-developed microfilament bundles. plus microfilament bundles across cells. All photographs taken with 83x objective. Bar in C represents 25 pm.
fibronectin. Appearance of the circumferential bundles of actin in cells was accompanied by a punctate or punctate plus fibrillar fibronectin pattern, and appearance of straight microfilament
a ring. (B) 2 hr. this cell shows both punctate and hr, fibrillar staining with a definite pattern. (F) 5 hr. the asymmetry. stain. (l-f) 1.5 hr, one cell shows staining of ruffles (I and J) 3 and 2.5 hr, circumferential stain plus (L) 5 hr. polygonal array of actin around cell edge
bundles in the actin pattern was almost invariably accompanied by fibrillar patterns of fibronectin. These results suggest that the patterns of the two fibrillar networks might be more directly related.
Fibronectin 879
and Actin
Relationships
Individual cells that showed rectilinear fibriHar patterns with both stains were scored for detectable correspondence between the patterns. We observed clear parallelism or colinearity of the actin microfilament bundles and the fibronectin fibrils in 84% (156) of the 186 cells scored in this way. We did not find convincing correspondences in the remaining cells. Figure 4 shows examples of individual cells stained with the two antisera. Although clear relationships can be seen between the two patterns, several points are worth noting. While the patterns are obviously related, they are not identical: not every fibronectin fibril corresponds with an actin microfilament bundle and not every microfilament bundle has a clear association with a fibronectin fibril. Furthermore, even when colinearity occurs
DEFINITE RUFFLES + CIRCUMFERENTIAL
%
80
5o 20
GEODESICS
cells were scored for pattern of staining. 100-200 cells were scored for each time point for each label. Results are expressed as the percentage of cells showing a given pattern. (Top panel) Fibronectin. (.-0-.) No pattern; (. . .O.. .) punctate; (--C -) punctate plus fibrillar; (--A-) fibrillar with no obvious pattern; (-•-) patterned fibrillar. (Bottom panel) Actin. (.-0-.) No pattern; (. . ‘0.. .) staining of ruffles; (--W-) staining of ruffles and circumferential fibrils; (-•-) definite straight microfilament bundles, whether or not circumferential staining also occurred; (--A--) geodesic polygonal networks. Examples of the different patterns are shown in Figure 2. The corresponding symbols in the two panels indicate corresponding patterns (see Figure 4, Table 1 and text).
-
HOURS Figure 3. Spreading and Actin (Bottom) Cells Table
were
stained
Time
Course;
at intervals
1. Correlations
between
Patterns
(hr) after Patterns
of Fibronectin
reseeding, of Fibronectin
(lop)
and individual and Actin Fibronectin
Actin
Pattern
Ruffles
Cell Spreading
Pattern
None Discernable
Punctate
1.5
9
26
2
3.0
4
11
2
Hours
Only
during
plus Circumferential
Fibrils
Bundles
Geodesic
Arrays
Patterned Fibrillar
1.5
76
37
6
27
56
6
2
3
1.5
3
17
11
13
3.0
2
20
22
45
1
21
58
4.0 Polygonal
Random Fibrillar
3.0 4.0 Microfilament
+
1
4.0 Ruffles
Punctate Fibrillar
1.5 3.0 4.0
2
5
4
14
Individual double-stained cells were scored for their patterns with both antibodies at three different times. 202 cells were counted for 1.5 hr, 204 for 3 hr. and 104 for the 4 hr time point. Note the time-dependent progression to more complex patterns and the predominance of cells on the diagonal of the table, indicating correlations between fibronectin and actin patterns. Cells in the last two lines of the last two columns (those showing fibrillar staining with both antibodies) were also scored for correspondences between the two stains. Correspondence was scored as: no obvious relationship (30 cells), parallel arrangement of fibronectin and actin but coincidence not clear (65 cells), clear coincidence (91 cells). Pooling those showing parallelism or coincidence of staining gave 156 out of 166 cells showing correspondence between the two patterns (64%).
Cell 860
Figure
4. Double
Label Staining
of NIL8 Cells during
Spreading
The figure shows pairs of photographs of actin and fibronectin staining of the same cells. (A, B, D and E) 1.5 hr. photographed with 63x objective, bar represents 50 pm. Note that the punctate fluorescence seen in D with antifibronectin is not observed in A with anti-actin. Conversely, anti-fibronectin does not stain the ruffles seen in A with anti-actin. (B and E) show cells in different stages. Two cells show no actin microfilament bundles and have punctate and short fibrillar fibronectin. One cell is better spread and shows fibrillar staining with both antibodies. All other panels were photographed with 40x objective. Bar in K represents 50 pm. (C and F) 3 hr. Note correspondence between actin and fibronectin fibrils. (G and J) 4 hr. Well-developed actin microfilament
Fibronectin 881
and Actin
Relationships
between actin and fibronectin, the two stains are not always co-extensive. This lack of complete correspondence indicates that the apparent codistribution does not arise merely from cross-reactions between the antibodies. The possible significance of the different arrangements seen is taken up in the Discussion.
Fibronectin and Actin in Well-Spread Cells By 8 hr of spreading, most cells were asymmetric and had well-defined actin and fibronectin fibrils (Figure 3). This situation persisted over the next 16-24 hr. If cells were allowed to continue growth in 5% serum, they became confluent and, as described previously (Mautner and Hynes, 1977), developed an extensive fibrillar network of fibronectin, most of which was on top of the cells. We could see no obvious correspondence between this pattern and the arrangement of actin microfilament bundles (MFB), which was not as clear as in sparse cultures. To investigate the relationships between fibronectin and actin in cells having large amounts of fibronectin, we turned to cultures arrested at subconfluence in low serum. We have previously shown that in such cultures most of the fibronectin is in fibrillar “footprints” beneath the cells and can be left behind on the substratum when the cells are detached (Hynes, Destree and Mautner, 1976; Mautner and Hynes, 1977; Hynes et al., 1978). In this condition we could observe well-displayed actin MFB and extensive extracellular networks of fibronectin. Figure 5 shows that there was very good correspondence between the two patterns. Virtually every cell in these cultures showed coincidence of some actin MFB and fibronectin. As in the spreading cells, the patterns were related but not identical. Careful focusing showed that actin MFB near the bottom of the cell corresponded to fibronectin fibrils beneath the cell, but that other arrays of actin higher up in the cell did not (see Figure 5). We observed several interesting patterns. The basal actin MFB frequently terminated before the periphery of the cell, whereas the colinear fibronectin often continued further toward the edge of the cell and even beyond it (compare Figures 5A5C with 5D-5F). In many instances we observed adjacent cells, each with arrays of basal MFB that appeared to be connected by fibronectin strands running from one cell to the other, each end of the fibronectin strand being colinear with an actin MFB (not shown). Another fairly common pattern consisted of arrays of actin MFB that coincided with
fibronectin along their
only near their ends lengths (Figure 5D).
or at intervals
Discussion This study has provided evidence that the double label immunofluorescence procedure does indeed work and that the apparent coincidences of staining do not arise from cross-reaction. Both antibodies (anti-fibronectin and anti-actin) are monospecific and do not cross-react by the Criteria of immunofluorescence (Mautner and Hyries, 1977) or by staining of gels (Burridge, 1976; our unpub’ lished data). Since both are rabbit antibodies, how-~ ever, the efficacy of the blocking step (see Experimental Procedures) must be established. Artifactual cross-reaction was not observed when unrelated structures (fibronectin and intermediate filaments) were stained using the procedure (Figure 1). Perhaps more convincingly, the actin-fibronectin double staining experiments (Figures 2, 4, and 5) contain internal evidence against cross-reaction. No fluorescein staining was seen beyond the boundaries of spread cells although rhodamine anti-fibronectin staining was apparent. In the early spreading cells, there was no fluorescein staining of the punctate pattern seen with the anti-fibronectin antibody. Thus the fluorescein goat anti-rabbit used to detect the anti-actin staining did not also detect the anti-fibronectin antibodies. Conversely, the rhodamine anti-fibronectin did not bind to the goat anti-rabbit or the anti-actin as shown by its failure to stain ruffles, circumferential microfilament bundles, geodesic arrays of actin, the periphery of well-spread cells or, indeed, many of the actin microfilament bundles in the cells. Coincident staining suggests coincident distribution of the two antigens. It is not clear, however, what coincidence means in this context since the resolution of the technique cannot be better than 0.2 pm. Thus the results indicate only that the two antigens are closer than the degree of resolution. A finer analysis of the degree of molecular proximity must await biochemical analysis. The apparent correspondence between actin and fibronectin becomes more convincing when complex patterns of one are replicated by the other, as shown in many of the Figures. We have investigated the correspondences in several ways: by preparing double exposures on color film, by tracing prints of the two stains and by projecting transparencies of one onto the other or onto tracings of the other. These exercises convince one of the correspondences, --____~
bundles coincide with fibronectin fibrils. Note that fibronectin fibrils are not continuous. (l-l and K) 4.5 hr. Well-developed microfilament bundles cross the cell and partially coincide with discontinuous fibronectin fibrils. Note that the polygonal array of actin (H) does not stain with anti-fibronectin (K). (I and J) 24 hr. Well-spread cell showing good microfilament bundles and corresponding fibronectin fibrils. Note that the two sets of fibrils are not co-extensive.
Cell 882
Figure
5. Double
Label lmmunofluorescence
of Well-Spread
Growth-Arrested
NILS Cells
Cells were seeded in 0.3% serum and fixed 4 days later. Photography was with 53x lens; bar represents 50 pm. (A-C) Anti-actin; (D-F) antifibronectin. In (A and D), note that in the upper right of the cell each microfilament bundle corresponds with a fibronectin fibril, and in the center of the cell many microfilament bundles lack corresponding fibronectin fibrils. Arrows in (D) indicate interrupted linear staining with antifibronectin. Also note that the cell boundary stains with anti-actin but not anti-fibronectin, whereas extracellular fibrils on the substratum and out-of-focus fibrils on top of the cell (top center, D) show the inverse staining pattern. The other four panels show good correspondence between anti-actin and anti-fibronectin. In (6 and C). arrows indicate microfilament bundles that terminate before the cell edge. Corresponding arrows in (E and F) indicate the fibronectin fibrils continuing beyond cell boundaries. In all panels, focus was at the bottom of the cell. Other arrays of actin microfilaments at higher planes of focus did not correspond with fibronectin staining, as seen clearly in (8).
but only indicate proximity. In the spreading cells, the development of the typical patterns of actin microfilament bundles proceeds from an initial concentration of actin in ruffles to circumferential fibrils and then to straight bundles running across the cell, sometimes passing through a stage characterized by polygonal or geodesic arrays of actin. Similar patterns have been described by Lazarides (1975,1976). We were concerned with the relationship of this pattern to the development of the fibronectin pattern. At early times, when cells contain only circumferential and no rectilinear actin fibrils, the predominant fibronectin pattern was punctate. .The specks of fibro-
nectin stain did not correspond to the circumferential actin fibers. When the punctate pattern was circular, it was usually central to the concentric actin fibers (see figures 4A and 4D). In almost all of the cells, rectilinear fibronectin fibrils appeared as soon as rectilinear actin fibrils did (Table l), and some correspondence between the two fibrillar arrays could also be observed in most cases (Figure 4). With time, both arrays increased in intensity of staining and in complexity, and the predominance of coincidences between the two patterns persisted. In well-spread, nonmotile cells (Figure 5), virtually 100% of the cells showed correspondence between the patterns.
Fibronectin 883
and Actin
Relationships
These results suggest a causal relationship between the two arrays of fibrils but the experiments do not allow us to determine the direction of causality. Earlier work has suggested causality in each direction: addition of fibronectin to cells lacking it promotes ordering of actin microfilament bundles (Ali et al., 1977), whereas addition of cytochalasin B, which disrupts actin microfilament bundles, causes increased release and decreased attachment of fibronectin (Ali and Hynes, 1977). One could postulate a relationship of reciprocal causality, but a final decision must await further work. In this context it is worth noting that we have examined two other cell types (rat embryo fibroblasts and human fibroblasts), both of which are extremely well-spread in sparse culture and have elaborate arrays of actin microfilament bundles. Under our experimental conditions these cells had rather little fibronectin, indicating that coincident fibronectin fibrils are not a necessary condition for the development of actin microfilament bundles (our unpublished data). The actin-containing structures described here have all been reported previously and their nature has also been analyzed by electron microscopy (Abercrombie, Heaysman and Pegrum, 1971; McNutt et al., 1971, 1973; Goldman and Knipe, 1972). It is worthwhile to consider the possible nature of the fibronectin-containing structures. All the structures reported in this paper that show relationships with actin lie at or near the bottom of the cell. The first to appear are the punctate patches seen in early spreading cells. These could be vesicles inside the cells (but near its base) or patches of fibronectin between the cell and the substratum. Punctate patterns of fibronectin have also been observed in well-spread epithelial cells (Chen et al., 1977; Mosher et al., 1977). These punctate patches of fibronectin might correspond to the attachment plaques seen with electron microscopy and interference microscopy (Abercrombie et al., 1971; lzzard and Lochner, 1976). These plaques are the points of close apposition and adhesion to the substratum (Abercrombie et al., 1971; Brunk et al.,1971), and similar plaques appear on contact with another cell (Heaysman and Pegrum, 1973a). Attachment plaques are frequently insertion points for actin microfilament bundles (Abercrombie et al., 1971; McNutt et al., 1971, 1973; Heaysman and Pegrum, 1973a; Revel and Wolken, 1973; Abercrombie and Dunn, 1975; lzzard and Lochner, 1976). They can be either punctate or elongate, as are the patterns of fibronectin staining reported here. Given the facts that fibronectin promotes cell adhesion and flattening (Yamada et al., 1976; Ali et al., 1977) and organization of microfilament bundles (Ali et al., 1977; Willingham et al., 1977; Hynes et al., 1978), and, in the present work, is frequently
coincident with actin microfilament bundles (Figures 4 and 5), it is reasonable to propose that fibronectin is a constituent of the attachment plSqUt?S. We are currently investigating this hypothesis. The observation that microfilament bundles are frequently coincident with fibronectin only at their ends or at intervals along their lengths (Figures 4 and 5) could be explained in a number of ways, One possibility is that these microfilament bundles, which are at or near the bottom of the cell, are closely apposed to the substratum only at some points, these being the positions where fibronectin staining is also observed. Also of interest is the observation that microfilament bundles which terminate at the cell periphery sometimes appear continuous with fibronectin strands that extend beyond the cell and even to adjacent cells where they may appear continuous with another microfilament bundle. This conformation suggests alignments of stress fibers with intercellular fibrillar connections containing fibronectin. Fibronectin fibrils have been reported in regions of cell-cell contact (Chen, Gallimore and McDougall, 1976; Mautner and Hynes, 1977), and actin microfilament bundles have been reported to arise from the point of cell-cell contact (Heaysman and Pegrum, 1973a; McNutt et al., 1973; Heggeness, Wang and Singer, 1977). We have more frequently observed that the actin structures terminate before the cell edge and the fibronectin fibril crosses the gap. The involvement of these structures in cell-cell contact areas merits further research Finally, we must consider how these results relate to recent reports that integral membrane proteins become aligned with microfilament bundles (Ash and Singer, 1976; Ash et al., 1977). In these experiments, various lectins and antibodies were used to stain living cells. The stains, which were evenly distributed at first, became (in some cells) concentrated with time over microfilament bundles, preddminantly over the top of the cell (Ash and Singer, 1976; Ash et al., 1977). In our experiments the cells were fixed prior to staining, making antibody-induced redistribution improbable. In any case, fibronectin on these cells is apparently not free to move laterally (Hynes et al., 1976; Mautner and Hynes, 1977), as others have also reported (Schlessinger et al., 1977). Fibronectin is a lectin receptor (Burridge, 1976; our unpublished results) and stains well with concanavalin A, one of the reagents used by Ash et al. (1976, 1977), but it is improbable that it would bind the specific antibodies tested (Ash et al ., 1977). Some time ago we suggested that fibronectin might be permanently connected with the cytoskeleton across the membrane (Hynes, 1974). This arrangement was offered as an explanation for the
Cell 884
restriction of lateral mobility of lectin binding sites ih normal cells that contain fibronectin and for the absence of restriction in transformed or proteasetreated cells that had been shown to lack fibronectin (Hynes, 1973). It was hypothesized that fibronectin might act as an anchor with which other lectin binding sites were associated either constitutively or after lectin-mediated lateral aggregation. The existence of extensive disulfide bonding among surface proteins (Hynes and Destree, 1977) could also contribute to this restriction of lateral mobility by promoting anchorage to fibronectin that is in turn anchored to the cytoskeleton. This hypothesis remains tenable, could explain some of the results of Ash, Singer et al., and is given some support by the data reported in this paper. Experimontrl Cells
Procedures
and Culture
Condltlons
The cells used were the NIL8 hamster fibroblastic cell line. Cells were cultured in Dulbecco’s modified Eagle’s medium with 5% fetal calf serum. They were seeded on coverslips (12 x 12 mm, Gold Seal) by trypsinization and plating in 5% fetal calf serum for analysis of spreading, or in 0.3% fetal calf serum for 2-4 days for analysis of well-spread, growth-arrested cultures.
Antlbodles The antisera used have all been described and characterized. They were anti-actin (Burridge, 1978; a gift from Keith Burridge), antiserum to the 58,000 dalton subunit of intermediate filaments (Hynes and Destree, 1978) and antifibronectin (anti-LETS protein; Mautner and Hynes, 1977). All three sera were raised using SDSgel-purified immunogens in rabbits, and for indirect staining were detected with fluorescein-conjugated goat anti-rabbit IgG (Miles). For double labeling, rhodamine-conjugated anti-fibronectin was prepared as follows. lmmunoglobulins were diluted to 10 mglml with 0.01 M Tris-HCI (pH 7.5). and tetramethyl rhodamine (Beckton, Dickinson and Co.) was added to give a final ratio of 15 fig/ mg protein: the pH was adjusted if necessary during the first hour. After incubation overnight at 4°C with stirring, unconjugated rhodamine was removed by passage over G25 Sephadex. The excluded peak was then passed over a column of Sepharose 28 coupled to hamster fibronectin (0.4 mglml of gel) purified as described (Mautner and Hynes, 1977; Hynes et al., 1978). After thorough washing with phosphate-buffered saline (PBS), bound antibody was eluted with 5 M Nal + 1 mM sodium thiosulfate, supplemented with 1 mg/ml ovalbumin, dialyzed extensively against PBS, checked by immunofluorescence (see Figure 1) and concentrated by vacuum dialysis or lyophilization if necessary. For absorption, rhodamine-conjugated anti-fibronectin was incubated for 30 min at 37°C with an equal volume of either fibronectin-Sepharose or Sepharose. The beads were removed by centrifugation and the supernatants were tested by immunofluorescence (Figure I).
Immunofluorescence lmmunofluorescence was performed on coverslips after fixation with formaldehyde and acetone treatment as described (Mautner and Hynes. 1977; Hynes and Destree, 1978). For double label immunofluorescence. the following protocol was used: stain with first antibody (anti-actin, 11100 dilution of IgG preparation, or anti-intermediate filament, l/25 dilution of whole serum), rinse; stain with fluoresceinated goat anti-rabbit IgG. l/IO dilution, rinse; incubate with normal rabbit serum, 1110 diltuion, rinse; stain with rhodamine-conjugated anti-fibronectin, rinse and mount. All staining steps were for 30 min at 37°C. The third step
with normal rabbit serum was essential to block free binding sites on the goat anti-rabbit IgG used to stain the anti-actin. If this step was omitted, the rabbit anti-fibronectin bound to actin-containing structures presumably via a ternary antibody complex (see Figure 1 and text). All photographs were taken on a Zeiss microscope equipped with epifluorescence optics, using a 63/l .4 or 40/I .O lens and exposure times of 30-60 set with Kodak Plus-X film, which was developed using Diafine.
Acknowledgments We are indebted and grateful to Keith Burridge for the anti-actin antibody. This work was supported by grants from the National Cancer Institute (to R.O.H. and S. E. Luria) and the American Cancer Society. R.O.H. is the recipient of an NIH Research Career Development Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
June
27,1978;
revised
August
2,1978
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weight, cell in malignant
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Wang, E. and Goldberg, A. R. (1976). organization and surface topography chick embryo fibroblasts with Rous Acad. Sci. USA 73, 40654069.
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In Proof
M. Heggeness, J. Ash and S. Singer have recently reported (1976, Ann. NY Acad. Sci .312,414-417) some coincidence of actin and fibronectin in WI38 cells.
