The bases of the locomotory behaviour of fibroblasts

188

Experimental

THE

BASES

OF THE

Cell Research,

LOCOMOTORY

Suppl.

8, 188--158 (2961)

BEHAVIOUR

OF FIBROBLASTS M.ABERCROMBIE Department

of Anatomy

and Embryology,

University

College, London,

England

THE purpose of this paper is, firstly, to attempt to state a coherent framework of hypotheses about the stimulus-response mechanisms controlling the locomotory orientation of one kind of vertebrate cell, the tibroblast. Five propositions will be put forward: they cannot be considered well verified, but at least they seem to be hypotheses in immediate need of investigation. The derivation from these propositions of an account of the movements and patterns of whole populations of libroblasts will then be briefly considered, and finally some other cell types will be discussed. BEHAVIOUR A. The main

locomotory

OF INDIVIDUAL organ

FIBROBLASTS

of the fibroblast is its ruffled (undulating)

membrane. This membrane (henceforth called an RM) is seen most conspicuously at the front end of a hbroblast which is moving on glass or a similar structureless plane surface. In an embryonic chick heart tibroblast in these circumstances it is a membranous extension often wider than the rest of the cell, merging into a thicker process which itself merges into the perinuclear cytoplasm. cession

Its ruffling activity gives the impression

of waves beating from the edge inwards,

of a very irregular

predominantly

the visible waves are on the side of the membrane

suc-

backwards;

facing the liquid medium,

they progress fast enough to be seen by direct observation, and they are often detectably associated with pinocytosis. The size, shape and activity of RMs vary a good deal within one population

of tibroblasts,

and vary more

between populations of different sorts of libroblasts. They precede moving cells of other types, such as Schwann cells and nerve tibres. They seem to be always a product of the interaction of the cell and its solid substrate [25]: they do not seem to be produced free in the liquid medium, and the gross texture of the solid substrate, whether it is a broad plane surface or a system of tibres, has a profound influence on the size and form of the RM [12, 251. Several workers with microdissection have described the RM as the part of the cell most firmly attached to the substrate (in tibroblasts of birds [S], amphibia [6] and fish [13]). Experimental

Cell Research,

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8

Fibroblasf

behaviour

No consistent forward flow, relative to the centre of gravity of the cell, is observed in an RM, as it is in the pseudopodium of an amoeba. The assumption is that the RM moves forward on the substrate by some sort of multiple localised series of contractions and expansions co-ordinated with adhesions and de-adhesions, like a snail or earthworm. If the visible backwarddirected ruffling (which is on the side exposed to the liquid medium) represents waves of these events at the side in contact with the glass, then the movement of the membrane appears to he in principle that of an irregular earthworm, rather than an irregular snail. The backward direction of the waves implies that, as in the earthworm but not in the snail, the contracted parts of the RM must be adherent to the substratum, the expanded part free from it. Some of the rest of the cell behind the RM is also adherent to the substrate, or it would fall off in a hanging drop of liquid medium. A really long Schwann cell, which may be a thread ) mm in length, can usually be seen to be in contact with the glass over the terminal 10 p or so at each end (at one or both tips there is usually a small RM) and by its nuclear region, but to sag away from the glass between these attachments. In a fibroblast, the part of the cell behind the RM is presumably slid along the substrate, mainly by the work of the membrane, becoming drawn out into an elongated shape in the process. Its attachments to the substrate come loose (though major ones may distort the cell elastically before they do so) and new attachments form. It would, however, be rash to suppose that none of the locomotory work is done by the rest of the cell behind the RM; but, as will appear, any such work is, as a rule, subordinate to the activity of the membrane. B. An isolated fibroblast on a plane surface moves with changes of direction. A chick heart fibroblast isolated from other fibroblasts, on a glass surface which has no large-scale regularity of structure, usually moves about, though with stationary intervals. For at least 12 hours or so it does not need any contact with its fellows in order to move. It changes its direction at very irregular intervals, of the order of several hours. This occurs by the waning of an existing RM and the waxing of a new one. It is difficult to be sure that the former always precedes the latter, but it usually does. The new RM commonly appears as far away as possible from the place of the old, at the opposite end of the cell, thus reversing movement. But it may develop laterally; the cell has no fixed polarity, though it may be mentioned that in the absence of an RM the previous polarity still persists, since when an isolated bipolar cell goes into mitosis the telophase spindle usually has the same orientation as the original cell. Of course, it cannot be said that the waning or waxing of Experimental

Cell Research, Suppl. 8

190

M. Abercrombie

RMs results from an endogenous rhythm of change in the cell. Neither substrate nor liquid medium is likely to be perfectly homogeneous (in the plane of movement), so external stimuli may well be operative. Nevertheless, there is a background of waning and waxing of RMs in an environment as homogeneous as we can make it, independent of all relation with other cells. C. Ruffled membranes on an individual cell are competitive. A growing or a large active established AM seems to prevent the development of a second RM; a previously ruffled membrane that has ceased its ruffling or is regressing seems to permit it. An incompatibility of two RMs on one cell is suggested by the high frequency with which dells with a single RM are observed on a glass surface. That this is due to mutual interference, and not to the operation of some central controlling agent, can be shown experimentally by discouraging the appearance of any large RM,. when multiple small membranes result; and conversely by encouraging the emergence of a large RM, when the small ones do not appear. This is what Weiss and Garber [25] did by using plasma clots consisting of fibrin of various degrees of coarseness of tibre. Their result led them to formulate the hypothesis of competition. The existence of competition implies some communication system within the cell. Weiss and Garber [25] suggested that flow of cytoplasm into a large pseudopod drained available material away from any small incipient pseudopodia. It seems, however, that an RM can be dominant without expanding, and that only when it is expanding is there flow into it. Competition for cytoplasmic material in general seems a little unlikely, since an RM paralysed by contact inhibition, but still forming a rather massive connection to another cell, loses its competitive power. Curtis [lo] has suggested that the surface expansion caused by shear on a moving RM produces a corresponding surface contraction, viscosity increase and lowering of adhesiveness in the rest of the cell surface. Whatever the communication system is, it seems to work over distances of 50 p or more. But isolated Schwann cells, which may be much longer, not infrequently develop an RM at each end. D. Individual ruffled membranes can be inhibited or promoted by heterogeneities of the environment. Naturally, because of competitive interaction, selective inhibition of one membrane promotes development of another membrane, and vice versa, and the categorisation of agents as inhibitors or promotors is somewhat arbitrary. The main conclusions from existing knowledge may however be conveniently put by saying that contact inhibition selectively inhibits and contact guidance selectively promotes the life of an RM. Contact inhibition occurs in a high proportion of collisions between Experimental

Cell Research, Suppl. 8

Fibroblast behaviour fibroblasts [4]. The RM, according to the proposition A, must in at least one cell make the contact; it becomes paralysed soon after it has touched its fellow cell, in the sense that its ruffling ceases and if it was expanding in area, that expansion ceases too. In fact it seems to contract [2, 31. At about the same time the cell as a whole stops moving, its locomotor organ having ceased to work. It is to the cessation of locomotion that the term contact inhibition was first applied. The two cells are then adherent to each other [17, 191. A new RM (proposition B), released from dominance (proposition C), usually quickly starts to expand, and if it is successful the cell moves away in a new direction. Contact inhibition is perhaps partly a matter of relative advantage and disadvantage between competing membranes. When an RM in contact with another cell is in competition with an RM in contact with a glass surface, the latter evidently assumes dominance almost invariably: so that in a very sparse population of hbroblasts contact inhibition is substantially complete (Abercrombie and Gitlin, unpublished). When the competition lies between several contacts all with other tlbroblasts, as in a rather dense population, contact inhibition seems to be less effective [ 111. Nevertheless it is still highly effective in these circumstances, so it seems likely that in most contacts there is complete, rather than relative abrogation of a membrane’s effectiveness by contact inhibition, regardless of competition. What precisely this effect of contact may be is still largely a matter of speculation, and all that is necessary here is to list what appear to be the choices, with a few comments. (a) The unusually close adhesion of the RM to the cell it contacts directly or indirectly paralyses its activities. This is the only hypothesis that has been worked out in some physical detail [lo]. (b) As a result of the sudden change in chemical make-up of the environment to which the RM finds itself juxtaposed when contact occurs, the chemical make-up of the RM changes, in the way envisaged by Weiss [23] for mutual influence of two cells through their surfaces. This hypothesis depends on the controversial point as to whether cell surfaces can approach each other sufficiently closely to interact in this way [lo]. (c) There is complete inability of the RM to adhere to the exposed surface of the cell it contacts, except at its edges, so that the RM cannot continue its forward displacement. The exposed surface might resemble the coelomic surface of the peritoneum, well known to be non-adhesive. Sarcoma cells and macrophages succeed, however, in adhering to the free surfaces of fibroblasts, while being apparently no more adherent to their edges. Experimenlal

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192

Ad. Abercrombie

(d) Fibroblasts adhere to each other less than they do to the glass (or other) substrate, to such a degree that they are unable to pull themselves off the substrate by gripping the cell surface. This hypothesis provides no explanation for the paralysis of the RN’s ruffling movements and its other changes. (e) There will be a block to diffusion of substances out of or into the RM as a result of contact, and the change in the slope of the diffusion gradient near the Rhl surface may alter its behaviour. Such an effect should also be produced by any impermeable solid obstruction, but there seems no clear evidence yet as to whether contact inhibition follows in such circumstances. Opposite to the negative thigmotaxis evoked by contact with a homologous cell seems to he the effect of contact guidance [22, 231. This is a behaviour mechanism by which the movement of a cell is directed by an orientated structure of the substrate, and it may be regarded as the result of promotion by the substrate of the persistence of the RM that leads the cell along the orientated structure. In most situations it is, however, difficult to substantiate this view, since, because of the existence of RM competition, the behaviour can equally be interpreted as due to the discouragement of Rhls which attempt to extend at right angles to the direction of orientation. There is, however, an argument in favour of promotion. The orientated substrate may be regarded as a rail, specifying either of two directions. That the cell takes one has been asc.ribed by Weiss [24] to interaction by contact inhibition with other cells at one end of the cell and not at the other. While this may often be true, it is difficult to accept it for some cases, especially, we have found, for relatively isolated fibrosarcoma cells undergoing orientated movement on a substrate consisting of orientated fibroblasts. There is no contact inhibition localised to the rear end of these sarcoma cells since normal fibroblasts exert no contact inhibition on them. It is easy to see, however, that once they are on the substrate pointing in the right direction, conservation of the leading Rh4 will suppress any development of an RM at the rear, even though this is equally exposed ta the orientation of the substrate. Contact inhibition by homologous cells and contact guidance by orientated substrate are merely two instances that have been studied of the ways that RMs may be influenced by contact. We may expect to find inhibition or promotion of RM activity, of varying degrees, by many of the surfaces, both of extracellular substances and of heterologous cells, with which a fibroblast comes into contact. An instance of inhibition by a particular condition of the substrate has recently come to light. When fibroblasts with a low density of population, moving on a glass substrate, reach a part of the substrate from which other fibroblasts have beer) (for experimental purposes) Experimental

Cell Research, Suppl. 8

Fibroblast behaviour

193

scraped off, they stop, develop a new RM in another direction, and move away (Abercrombie and Gitlin, unpublished). This inhibition is evidently less severe than the usual contact inhibition between cells, because it can be competitively overridden. A densely packed group of tibroblasts will move over suc.h an inhibitory region of the substrate, presumably because cellular contact inhibition puts an RM at a greater disadvantage than does this inhibition by the substrate. Naturally such an inhibition by the substrate means a relative promotion of RM activity by an ordinary glass surface; and such relative degrees of inhibition or promotion give scope for the various subtle selective interactions that have been recognised such as cell recognition and [23]. It will be important to see whether the “selective contact guidance” different contact reactions can all be referred to different values on a single scale of adhesiveness [lo] or whether specific qualitative differences between the surfaces play a part. Something must now be said about influences not mediated by contacts, about the possibilities of chemotaxis, or movement orientated to a diffusion gradient. This is a kind of response which some vertebrate c.ells, the macrophages and polymorphs, are capable of performing [14]. -4 priori it would certainly seem that flbroblasts, with their elongation, and the competitive relations between their RMs. are well suited to chemotaxis. A gradient in a substance promoting, say, adhesion to substrate surely ought to give an RM at one end of the cell an advantage over an RM at the other. No such effect has, however, to my knowledge ever been fully reported, though Twitty and Niu [21] have mentioned that fibroblasts in a confined space disperse unusually, much as do the amphibian melanoblasts they primarily studied, which they attribute to a mutual negative chemotaxis. This needs further investigation; and perhaps too this important work on melanoblasts should be re-examined with contact inhibition in mind. E. The contacts that form between fibroblasts are adhesions which usually persist for a considerable time. When an RM is undergoing contact inhibition, it and its cell cease moving soon after contact has been made. The RM may then retract from the cell it has contacted, perhaps as a result of the light contraction of the RM that has already been mentioned. This is the contact retraction of Weiss ([24]; described also for Jensen rat sarcoma cells by Canti [7]). More commonly, however, it remains attached for a time as a rather firm adhesion [2, 17, 191. If there is cell-free space near, it is usually pulled away by the advent of a new RM and hence a new direction of movement. If it is in the midst of a dense population, and so is unable to move away, the adhesion may last very many hours. 13 - 60173256

Experimental

Cell Research, Suppl. 8

M. Abercrombie

194 BEHAVIOUR

OF FIBROBLAST

POPULATIONS

From the set of hypotheses just discussed about the orientation behaviour of individual fibroblasts one can derive much of the locomotory behaviour displayed by colonies of fibroblasts in vitro. Some aspects of this population behaviour will be mentioned here. TABLE

I.

Movements were recorded during equal time intervals on cells in growing cultures. The proportion of such movements which were to some extent directed away from the explant (“outward”) is recorded, classified according to the number of other cells with which each observed cell was in contact. Speed of movement does not differ significantly in different directions. (Data of Abercrombie and Heaysman (unpublished). Total number of readings

Proportion of “ outward” movements

0

457

1

616

2 3 4

692 542 316

596

127

0.62 0.72 0.71 0.76 0.78 0.87

Number of contacts

Dispersal occurs in a directional way from a populated to an unpopulated region. Dispersal, in other words, is not by anything approaching random movement, but shows a strong bias towards fibroblast-free areas of substrate. The strength of the bias in the movement of any individual cell depends on the density of population of the surrounding cells (Table I). The bias arises because, especially in a population linked by many adhesions (proposition E), it is difficult for most cells to move without running into some part of another, and undergoing contact inhibition. A cell at the periphery of the group, however, will sooner or later (proposition B) form an RM directed radially outwards into the cell-free space around the group; it will not now be inhibited by neighbours and it will be able to move outwards. Cells behind it can then follow. Lateral and. backward movement will always be less likely than outward, because neighbouring cells in those directions remain always near at hand, except for unsystematic oscillations. On a plane surface the cells will move as a monolayer; the more effective contact inhibition is, the more perfect the monolayer. When the substrate is a gel, it tends to become radially orientated [22] and this will reinforce the directional bias of movement. Experimental

Cell Research, Suppl. 8

Fibroblast behauiour

195

It follows from what has just been said that where no unpopulated space is available to a population of fibroblasts, the cells will immobilise each other. On a plane surface the monolayering tendency will persist in these circumstances. A population spreading from a centre will tend to assume a circular form. It is clear that if cells tend to move towards cell-free space, that is to say, at right angles to the periphery of the population, they will tend to smooth out an irregular periphery, filling indentations from both sides and broadening excrescences. A simple experiment shows, however, that this is not the only mechanism leading to circular form. When two explants are placed close together, each will become surrounded by a spreading circle of cells. When these circles meet, if the colony shape depends on cells always migrating ar right angles to the periphery, the total form of the pair of c.ultures should remain that of a pair of partly superimposed circles. it rapidly deviates from this, however, in that the indentations where the two circles meet quickly MI in. This is predictable from the basic propositions about frbroblast behaviour. Where the cells from the two explants meet in the indentation, they will become more closely packed, which will improve the efficiency with which they move towards cell-free space, by reducing the frequency of other movements (Table I). They will be directed predominantly outwards at right angles to a line joining the centres of the two explants. Consequently the indentation will be quickly filled. The cells of an excresence will conversely, by dispersing sideways, become less densely packed, more random in their movements, and less efficient in moving outwards than the rest of the cells of the culture. The equilibrium form is therefore circular, which preserves a similar packing all round the periphery, and this form will be quickly restored after disturbance. The cells of a fibroblast culture fail to scatter separately into the medium. The colony grows as a fairly coherent population .of cells. This is to be expected for the same reason, just discussed, that the cells of an excrescence become merged into the circular form of a culture. If a cell breaks free from the periphery, it becomes surrounded by cell-free space, and can form an effective RM freely in any direction. Unlike the cells behind it, it therefore ceases to move consistently outwards, and is soon caught again by the disciplined trend of the coherent cells. Small groups of two or three fibroblasts may be isolated at the periphery of a culture by scraping away the cells between them and the explant. The cells of these small groups move actively but in a relatively disorderly way, so that they are overtaken by the renewed outgrowth from the explant (Abercrombie and Gitlin, unpublished) Experimental

Cell Research, Suppl. 8

M. Abercrombie

196

BEHAVIOUR

OF OTHER

TYPES

OF CELLS

The chick heart tibroblast has a characteristic mode of locomotory behaviour, dominated in vitro by mutual contact inhibition of an absolutely effective nature and by contact guidance. Some libroblasts from other sources show similar behaviour [5] and it may be hazarded that this is the general tibroblast pattern. Each other cell type presumably has its own characteristic repertoire of behavioral responses, and it may perhaps be instructive briefly to mention some of the ways in which these differ from or resemble that of the tibroblast. The malignant tibroblast is a variant with reduced or absent contact inhibition, but no loss of contact guidance. The lack of inhibition by contact with normal fibroblasts permits sarcoma cells, as already mentioned, to exhibit contact guidance by a substrate of normal fibroblasts. This seems to be the explanation of their marked tendency to move in the same or the opposite direction as a sheet of moving tibroblasts on which they find themselves. It might be supposed that such a tendency could be explained by the persistence of a rudimentary degree of contact inhibition, which would make it easier to travel along tibroblasts orientated by movement than across them, since fewer contacts would be made. But this is negatived by the fact that rate of movement along the orientated libroblasts is just as good or rather better than along the plain substrate (glass) where there can be no contact inhibition at all. The lack of mutual contact inhibition amongst sarcoma cells, though it has not yet been shown to be total, should mean that they can produce contact guidance of each other, and in fact dense cords of similarly orientated librosarcoma cells can build up. This is evidently what Weiss [23], in the case of nerve fibres, called fasciculation. Schwann cells can perhaps show it too. It is not a form of behaviour that cells like normal fibroblasts with absolute mutual contact inhibition can show. But the manifestation in some circumstances of relative mutual contact inhibition is no bar to it. The wandering cells, macrophages and leucocytes, are, like sarcoma cells, immune to contact inhibition by fibroblasts. Other aspects of their behaviour are not yet worked out, apart from their chemotactic response, but this at present sharply distinguishes them from other cell types, in none of which, except perhaps melanoblasts [21], has chemdtaxis been unequivocally demonstrated. There are some points about the movement of epithelial sheets which may prove to be highly relevant to the further analysis of tibroblast behaviour. Experimental

Cell Research, Suppl. 8

Fibroblast behaviour It has been known for many years that an epithelium will advanc.e across a suitable substrate in so far as this is free of epithelium. As soon, however, as the advancing edge makes contact with the edge of another sheet of epithelium, it ceases to move, a phenomenon obviously related to the contact inhibition we have discussed in tlbroblasts. As early as 1915 Rand [20] succinctly will described this behaviour with the remark, “In general, an epithelium not tolerate a free edge”. The free edge in movement bears very conspicuous RMs projecting over the substrate in front of it. It is a crucial question whether these membranes of the peripheral cells are the locomotory organs, dragging the entire sheet behind them; or whether the under-surfaces in contact with the substrate of some or all of the cells behind the free edge are also in effective locomotor acitivity. The hypothesis that the edge alone is active in locomotion [16] may seem unlikely in view of the extent of the sheet that can be moved. It has, however, the merit that the cessation of movement on contact with another epithelial edge is immediately explicable. If the cells at the edge are inhibited in a way analogous to the contact inhibition of fibroblasts, the whole sheet must stop. The hypothesis has, on the other hand, a disadvantage, in that such cessation of movement does not always occur on contact, the epithelium heaping up into a multi-layered ridge at and near the original edge [9, 15, 181. This occurs also when it runs into some non-cellular obstruction on the substrate. Such behaviour is much more easily explained by the second hypothesis, that more than the edge of the sheet is taking part in the movement, and in these circumstances continues to push the edge against the obstruction. But this hypothesis has a problem in its turn in accounting for the stoppage of movement of the whole sheet by contact inhibition when this does oc.cur, since the information that there is no longer a free edge must somehow be transmitted through the sheet to stop the general movement: the stoppage incidentally occurs remarkably promptly [lS]. Similarly the general speed of movement must in some way be controlled by the edge, since when the peripheral part of the sheet and the region further back are on two different kinds of substrate, known to produce different speeds of movement, it is apparently the speed of the edge that conditions the speed of the whole [ 181. The epithelial problem is relevant to tlbroblast behaviour because fibroblasts, although they seldom form as coherent a sheet as do epithelial cells, do sometimes seem to move in such close array that it is doubtful whether RMs are present on all cells. They may therefore demand just the same choice of hypotheses as do epithelia, and it may be necessary to go beyond the bases of frbroblast behaviour suggested in the early part of this paper. Experimental

Cell Research, Suppl. 8

M. A bercrombie SUMMARY

A set of provisional hypotheses about the Iocomotory behaviour of individua1 fibroblasts is proposed, on which much of the pattern of movements of whole populations can be based. The hypotheses are as fohows. The locomotory organ of a fibroblast is a ruffled membrane. An isolated fihrobtast moves with changes of direction by waxing and waning of ruffled membranes at different parts of its margin. The ruffled membranes on an individual cell are competitive. They can either be inhibited or their life can be prolonged by the cell environment, notably by contact inhibition and contact guidance respectively. Mutual contacts form fairly persistent adhesions, Iinking fibroblasts into a meshwork. Some points in the behaviour of other cell types are considered in relation to that of fibrohlasts. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16.

17. 18. 19. 20.

21. 22. 23. 24. 25.

ABERCROMBIE. M.. Proc. Canad. Cancer Res. Conf. 4 (1961) (in press). ABERCRONIBIE~ M..and AMBROSE, E. J., Exptl. Cell R&ea~h~15~332~(1958). ABERCROMBIE, M. and HEAMMAN, J. E. M.. ibid. 5, 111 (1953). ibid. 6, 293 (1954). ABERCROMBIE, M. and KARTHAIJSER, H. M., ibid. 13,276 (1957). ALGARD, F. T., J. Exptl. 2001. 123, 499 (1953). CANTI, R. G., Arch. exptl. Zdlforsch. Gewebexucht. 6, 86 (1928). 199,380 (1931). CHAMBERS, R. and FELL, H. B., Proc. Roy. Sot. flondon) CHIAXUJ.AS, J. L., J. Exptl. Zool. 121, 383 (1952). CURTIS, A. S. G., Am. Naturalist 91, 37 (196). Proc. Natl. Cancer Inst. (in press). GARBER, B., Exptl. Cell Research 5, 132 (1953). GOODRICH, H. B., Biol. Bull. 46, 252 (1920). HARRIS, H., Physiol. Reu. 34, 529 (1954). HERRICK. E. H., Biol. Bull. 63. 271 (1932). HOLMES,‘S. J., .t. Exptl. Zool. i7, 28i (19i4). KREDEL, F., Bull. Johns Hopkins Hosp. 49, 216 (1927): LASH, J. W., J. Exptl. Zool. 128, 13 (1955). LEWIS. W.. Anal. Record 23. 387 11922). RAND,‘H. w., Wilhelm Rot& A& Entudcklungsmech. Organ. 41, 159 (1915). TWITTY. V. C. and Nru. M. C.. .I. Exptl. Zool. 125. 541 (1954). WEISS, P., ibid. 68, 39i(1934). Growth (Suppl.) 5, 163 (1941). Znfern. Reu. Cytol. 7, 391 (1958). WEISS, P. and GARBER, B., Proc. Natl. Acad. Sci. U.S. 38, 264 (1952).

Experimental

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