/. Embryol. exp. Morph. Vol. 32, I, pp. 57-68, 1974
Printed in Great Britain
57
The cellular basis of movement of the
migrating grex of the slime mould Dictyostelium
discoideum: chemotactic and reaggregation
behaviour of grex cells
By D. R. GARROD 1
From the Department of Biology, Southampton University
SUMMARY
The chemotactic behaviour of cells of the migrating grex of D. discoideum has been
studied by observing the responses of small groups of cells placed near to aggregation
streams, agar blocks containing cyclic AMP or migrating grexes. Grex cells placed near
aggregation streams were not attracted to the streams initially; after about 1 h, however,
some cells were attracted to the streams. Similarly, grex cells placed near agar blocks
containing cyclic AMP were not initially attracted towards the blocks, but some attraction
was observed after about 1 h; the most effective cyclic AMP concentration was 10~4 M.
Grex cells which were placed in heaps against the sides of aggregation streams, instead of
a short distance away, were often incorporated into the streams within | h. Grex cells were
not attracted towards migrating grexes.
Because of these results it is suggested that (1) migrating grex cells are not chemotactically
sensitive to cyclic AMP, but they acquire chemotactic sensitivity after separation from the
grex for about 1 h; (2) chemotaxis is probably not involved in the movement of the migrating
grex; (3) grex cells retain the adhesive property found in aggregating cells, which is involved
in the formation of end-to-end contacts necessary for the behaviour known as contact
following.
Reaggregation of dispersed grex cells was studied by time-lapse photography. Reaggregation
was found to be a two-stage process: first, an adhesive phase dependent on the development
of intercellular contacts at the expense of adhesions with the substratum; secondly, a
chemotactic phase which begins about 1 h after dispersal of the grex cells. It is suggested
that the first phase may be accounted for by assuming that the cells are more cohesive than
adhesive.
INTRODUCTION
In a previous paper (Garrod, 1969) a mechanism was proposed for the
polarized movement of the migrating grex of the slime mould Dictyostelium.
discoideum. The grex or pseudoplasmodium is an elongate mass of cells with
a distinct anterior tip and posterior end, which moves in a co-ordinated fashion
over the substratum. The cell mass is entirely surrounded by a slime sheath.
This sheath remains stationary relative to the substratum as the grex cells
1
Author's address: Department of Biology, Bassett Crescent East, The University,
Southampton SO9 3TTJ, U.K.
58
D. R. GARROD
advance through it, and collapses to form a slime trail at the posterior end
of the grex (Shaffer, 1965). It was suggested that the polarity of grex movement
is primarily controlled by the slime sheath.
This suggestion was based largely on observations of the motile behaviour
of grex cells. These observations showed that grex cell behaviour was similar
in certain important respects to that observed by other workers on cells at
earlier stages of the life-cycle (reviewed by Shaffer, 1962). In particular, their
pseudopodal activity was not inhibited merely by contact between the pseudopod
and the surface of another cell. (In contrast, many types of animal cells appear
to show inhibition of pseudopodal activity at points of mutual contact (Abercrombie & Ambrose, 1958; Trinkaus, Betchaku & Krulikowski, 1971).) Pseudopodal activity was inhibited, however, in situations where cell movement
seemed to be physically impeded, such as when a cell was totally surrounded
by other non-motile cells.
Because of this, it was suggested that the slime sheath might control the
polarity of grex movement by making it physically easier for the cells to move
forward rather than in any other direction. This could be the case, either if
the slime sheath were thinner at the grex tip than elsewhere on the grex
surface, or if the sheath were synthesized only at the tip. Evidence suggesting
a graded anterior-posterior change in slime sheath properties has been found
(Garrod, Palmer & Wolpert, 1970).
Recently, Loomis (1972) has described results which support this hypothesis.
Incorporation of [14C]acetylglucosamine into the slime sheath shows that sheath
synthesis takes place over the entire surface of the grex, indicating that the
sheath is indeed thinnest at the anterior tip and gradually increases in thickness
towards the rear. Also, microsurgical experiments seem to show that grex
cells move in the direction in which the slime sheath provides least resistance
to their movement. Firstly, if the tip of a grex was cut off and the slime sheath
scraped off from the cut surface of the posterior fragment, a new tip formed
at the cut surface and the posterior fragment recommenced migration. If the
slime sheath was not removed from the cut surface, the posterior fragment
did not migrate but developed directly into a fruiting body. Secondly, if both
the tip and the tail of a grex were cut off and the slime sheath removed from
both cut surfaces, the grex sometimes reversed its direction of movement.
This latter result is particularly important because it implies that there is no
underlying polarity in the cellular mass, and that the cells are perfectly capable
of moving backwards if the path of least resistance happens to lie in that
direction.
The evidence that the slime sheath controls polarity of grex movement seems
good but not conclusive, and in any case this need not necessarily be the
only mechanism involved in polarity control. In particular, the view has been
privately expressed that chemotactic movement of the grex cells may be
important in this respect. The cells at the tip of the grex might be a source of
Cellular basis of grex movement in slime mould
59
chemotactic attractant, which would guide the movement of the cells following
behind.
If such a chemotactic mechanism exists, the substance most likely to be
involved is cyclic AMP, which has been shown to be the chemotactic agent
during the aggregation stage of the life-cycle of D. discoideum (Konijn, Barkley,
Chang & Bonner, 1968). Cyclic AMP is produced at all stages of the life-cycle
of D. discoideum (Malkinson & Ashworth, 1973). Moreover, it has been suggested that there is a gradient of acrasin (cyclic AMP) emission along the
migrating grex of D. discoideum, 50% of all the cyclic AMP being produced
by the anterior one-tenth of the grex (Bonner, 1949). However, we have
reported two reasons for believing that chemotaxis is not important in grex
movement (Garrod & Malkinson, 1973). Firstly, even if such an emission
gradient exists in the intact grex, chemotactically sensitive cells from the
aggregation stage of the life-cycle did not seem able to detect it. Secondly, cells
of the migrating grex did not seem to be chemotactically sensitive.
This apparent lack of chemotactic sensitivity on the part of grex cells was
a surprising observation. On speaking to five other workers, all of whom
were engaged in studying some aspect of chemotaxis in the slime mould, I
found that all, in common with myself, had tacitly assumed that grex cells,
like aggregation stage cells, were chemotactically sensitive to cyclic AMP.
Because of this, and because of the importance of the observation in relation
to grex movement, this paper explores the chemotactic properties of grex cells
in a little more detail. It will be shown that grex cells are not chemotactically
sensitive when they are first removed from the grex, but that they reacquire
sensitivity when they have been separated from the grex for more than 1 h.
These results will be discussed in relation to grex movement and to the
reaggregation of dispersed grex cells.
MATERIALS AND METHODS
Dictyostelium discoideum strain NC-4 was cultured in association with
Aerobacter aerogenes on 2 % agar containing 0-1 % glucose and 0-1 % bacteriological peptone, and buffered to pH 6-3 with 0-01 M phosphate buffer. Plates
were incubated at 22 °C. Experimental manipulations were carried out with
fine glass needles or ball-tipped glass rods. Cyclic AMP (adenosine-3',5'-cyclic
phosphoric acid) was obtained from BDH Chemicals Ltd., Poole, England.
Time-lapse cinematography was carried out using a Wild phase-contrast
microscope fitted with a Vinten Scientific Camera. The film used was Kodak
Plus-X Reversal and a frame interval of 5 sec was chosen. Grex cells were
smeared on the surface of a thin layer of agar. A coverslip was mounted, using
a spacer, so that there was a small air gap between it and the agar surface.
The preparation was sealed with paraffin wax. To prevent the formation of
60
D. R. GARROD
condensation, a jet of air at a temperature just higher than that of the room
was directed onto the coverslip during filming.
RESULTS
Behaviour of grex cells in relation to aggregation streams
Aggregation streams of D. discoideum produce the chemotactic attractant,
cyclic AMP. Thus, if chemotactically sensitive cells are placed near to a stream,
they move directly towards it. It is therefore possible to test whether or not
cells are chemotactically sensitive by placing them near to an aggregation
stream and observing whether or not they are attracted to it. Experiments of
this type were carried out with cells from the migrating grex. These have been
briefly reported previously (Garrod & Malkinson, 1973).
Using a fine, ball-tipped glass rod, cells were picked up from a migrating
grex and placed in small heaps on one side of an aggregation stream, about
100-200 /mi distant from the edge of the stream. For comparison, heaps of
cells from another aggregation stream were placed on the opposite side of the
stream. The results of such an experiment are shown in Figs. 1-6. The heaped-up
stream cells began moving almost immediately towards the aggregation stream,
and after 25 min practically all of these cells had been absorbed by the stream.
The grex cells, however, did not move and after 25 min remained exactly
where they had been initially placed.
This result suggests that grex cells are not chemotactically sensitive to the
attractant produced by aggregation streams. A possible objection to this
conclusion is that the heaps of grex cells may be surrounded by a slime sheath,
which imprisons them and prevents their chemotactic sensitivity being expressed.
However, grex cells were not attracted towards aggregation streams, even
when they were smeared out as a monolayer over the agar surface (much as
in Fig. 14) near to streams.
When heaps of grex cells placed near aggregation streams were observed
for a longer time, results typified by those shown in Figs. 7-9 were obtained.
Grex cells placed near aggregation streams (Fig. 7) did not move after 30 min
(Fig. 8) but after a further 30 min some of the grex cells were being attracted
towards the streams (Fig. 9). In similar experiments, the time before attraction
of grex cells to streams was observed varied between 50 and 90 min, and was
most commonly between 60 and 70 min. This result suggests that grex cells
do not move initially towards aggregation streams, because they are not
chemotactically sensitive to stream attractant, but that after separation from
the grex for about 1 h they acquire sensitivity to this attractant, and the slime
sheath then does not prevent them from moving towards the stream.
If, instead of being placed a short distance away from a stream, heaps of
grex cells were placed in contact with the side of the stream, a different result
was obtained. Within about 30 min most groups of cells so placed moved
Cellular basis of grex movement in slime mould
61
•^6
Figs. 1-6. Photographs taken at 5 min intervals, showing behaviour of aggregation
stream cells (above) and migrating grex cells (below) placed in heaps near an
aggregation stream. The stream cells began moving towards the aggregation
stream almost immediately after being placed in position (Fig. 1), and after 25 min
almost all of them had moved to the side of the stream (Fig. 6). The grex cells
showed no sign of movement towards the stream during this 25 min period.
into the aggregation stream (Figs. 10, 11). Since these cells are not chemotactically sensitive, it is probable that their movement was guided by their
contacts with stream cells, a process known as 'contact following' (Shaffer,
1962).
62
D. R. GARROD
Figs. 7-9. Photographs taken at 30 min intervals, showing behaviour of migrating
grex cells placed in heaps near to aggregation streams and an aggregation centre.
Fig. 8 shows that after 30 min had elapsed the grex cells had not moved from their
initial positions shown in Fig. 7. Fig. 9 shows that after 60 min, movement of grex
cells towards the streams and centre was taking place. Of the four groups of grex
cells on the right-hand side of the photograph, three were moving almost entirely
towards a stream whereas one showed no sign of movement. Of the four groups of
grex cells on the left-hand side none was moving entirely, but small groups of
cells from two of the larger groups were being attracted (arrows).
Behaviour of grex cells in relation to agar blocks
containing cyclic AMP
It has been shown that aggregation-sensitive D. discoideum cells are attracted
to small blocks of agar containing cyclic AMP (Konijn et ah 1968); therefore,
experiments were designed to see if grex cells would also be attracted to such
blocks.
Preliminary experiments were carried out with cells from aggregation streams.
It was found that these cells were attracted towards blocks when the concentration of cyclic AMP in the block was between 10"4 and 10~6 M, which corresponds
to the range of sensitivity to cyclic AMP of aggregating cells found by Bonner
et al. (1969). In every case, the majority of stream cells placed near the agar
blocks were attracted.
Experiments with grex cells were carried out at cyclic AMP concentrations
between 10~3 and 10~7 M. Grex cells were either placed in small heaps near
to the blocks, or a grex was smeared out over the agar substratum immediately
surrounding the blocks. In most cases, no attraction of cells to the block was
Cellular basis ofgrex movement in slime mould
63
Figs. 10-11. Photographs taken at 30 min intervals, showing movement of migrating
grex cells placed in heaps touching the side of an aggregation stream. Four groups
of grex cells were placed against the stream (arrows in Fig. 10). After 30 min
three of these groups had been incorporated into the stream (Fig. 11).
found. When attraction was observed, it was incomplete, only a small proportion
of the cells being attracted. It was also delayed (compared with the response
shown by stream cells), being detectable between 1 and 2 h after setting up the
experiment. The most effective concentration of cyclic AMP was 10~4 M where,
in 6 cases out of 30, some of the cells were attracted. A single case of attraction
was recorded at 10~3 M, and another at 10~5 M cyclic AMP.
These results seem to suggest the same conclusion as those in which grex
cells were placed near to aggregation streams. When attraction to a cyclic
AMP-containing block took place, it was delayed in the case of grex cells,
compared with the rapid response shown by stream cells. It seems that grex
cells are not chemotactically sensitive to cyclic AMP, but that they acquire
sensitivity after having been separated from the grex for 1 h or more.
Behaviour ofgrex cells in relation to migrating grexes
Though cells taken from the migrating grex are not initially chemotactically
attracted either to blocks containing cyclic AMP or to aggregation streams, it
is nevertheless still possible that chemotaxis may be involved in grex movement.
It could be that another chemotactic mechanism, dependent on another
attracting substance, exists in the grex. In an attempt to check this possibility
so far as the present type of experiments would allow, the response of grex
cells to migrating grexes was tested. If the grex produced another chemotactic
agent to which its cells responded, grex cells might be attracted towards the
grex. (Assuming, of course, that the hypothetical second substance can diffuse
out of the grex through the slime sheath.) This was found not to be the case.
Small heaps of grex cells were lined up along one side of a migrating grex
and small heaps of stream cells along the other (Fig. 12). After 30 min (Fig. 13),
the stream cells, with the exception of a few stragglers, had been attracted
to the grex, but the grex cells had not moved. The migrating grex is a source
64
D. R. GARROD
r
o ->
.->
100/^m-
Figs. 12-13. Photographs taken at 30min intervals, showing the behaviour of
aggregation stream cells (below) and migrating grex cells (above) placed in heaps
near to a migrating grex. Thirty min after setting up the experiment (Fig. 13) the
majority of the stream cells had moved to the side of the grex, whereas the grex
cells had not moved.
of acrasin (cyclic AMP) which attracts aggregating cells (Bonner, 1949; Shaffer,
1957), but it does not chemotactically attract grex cells.
Reaggregation of grex cells dispersed on an agar surface
It is well known that if the migrating grex is stirred with a glass rod, it
will reform into a grex which may continue to migrate or, more commonly,
may develop directly into a fruiting body. If the grex is more widely dispersed
by smearing it out over the agar surface so that the majority of its cells are in
monolayer, the cells will reaggregate, usually forming a number of smaller
grexes, each of which may continue development. Preliminary observations
on grexes thus disturbed showed that reaggregation began almost immediately
after disaggregation had taken place, and was certainly well advanced 1 h
after disaggregation, which is the time when disaggregated grex cells seem
to acquire chemotactic sensitivity. These observations raise two questions.
Firstly, how does reaggregation take place if the cells are not chemotactically
sensitive? Secondly, what part, if any, does chemotaxis play in the reaggregation
process? In order to investigate these questions, more detailed observations
of grex cell reaggregation were made.
Figure 14 shows a preparation a few minutes after spreading the grex over
the surface of a thin layer of agar. It can be seen that the majority of cells are
in monolayer on the agar surface, though a few multilayered cell clumps
remain. The monolayer is irregular in shape. Time-lapse films show that the
cells are motile at this stage; they do not, however, show the directed movements
characteristic of chemotaxis. Figure 15 shows the same field of cells, 50 min
later. Considerable reaggregation has taken place by this time, so that the cells
are collected into groups rather than being widely dispersed over the surface.
The films show that this reaggregation is localized, in that the cells in a particular
area of the monolayer collect into an aggregate together with other cells with
Cellular basis of grex movement in slime mould
65
Figs. 14-17. Photographs showing stages in the reaggregation of migrating grex
cells dispersed on an agar surface. These are photographs (selected from a series
taken at 5 min intervals) of a preparation similar to those used in time-lapse
filming. Fig. 14 shows the initial appearance of the preparation: the majority of
the cells were spread as an irregular monolayer. Fig. 15 is the samefield50 min later
and shows the end of the first phase of reaggregation (see text). Some reaggregation
had taken place but was localized, in that cells which were close together in the
initial monolayer had collected together into aggregates. Note, for example, the
small groups of cells labelled a and b in Figs. 14 and 15. Fig. 16 shows the beginning
of the chemotactic phase of reaggregation (10 min after Fig. 15). The large
aggregate on the right-hand side of the photograph seemed to have become an
'aggregation centre' which attracted the other, smaller aggregates in the vicinity.
The cells in the latter aggregates took on the appearance of aggregation stream
cells. Fig. 17 shows a later stage (10 min after Fig. 16) in the chemotactic phase
of reaggregation.
5
EMB 32
66
D. R. GARROD
which they were either in contact, or nearly so, in the initial monolayer. This
phase of aggregation appears to be a 'zippering up' of intercellular contacts.
It can be seen from the films that cells within the aggregates move around
in circular pathways within the aggregates. The direction of this movement
can be either clockwise or anti-clockwise in different aggregates of the same
preparation. When the preparation is about 40 min old the situation becomes
entirely static with respect to reaggregation. The aggregates do not move and
there is no further reaggregation to form larger aggregates. The cells, however,
continue to rotate within the aggregates.
At a time which varied between 50 and 70 min in the seven preparations
studied, there was a dramatic change in this static situation - the onset of
a chemotactic phase of reaggregation. Some of the aggregates became aggregation centres, while other adjacent aggregates transformed into aggregation
streams which moved towards these centres (Fig. 16). In this way, some of the
initial aggregates became absorbed by others, so that a smaller number of
larger aggregates were formed.
These observations suggest that reaggregation of dispersed grex cells is a
two-stage process. Firstly, there is a phase in which localized reaggregation
occurs, probably by an increase in the extent of mutual adhesion by cells
which were initially either in contact or close enough to form contact by
random movement on extension of pseudopods. Secondly, there is a chemotactic phase in which directed movement, similar to and characteristic of the
aggregation stage of the life-cycle, takes place. The timing of the onset of this
chemotactic phase agrees with the suggested reacquisition of chemotactic
sensitivity, shown by grex cells placed near aggregation streams or agar blocks
containing cyclic AMP.
DISCUSSION
The suggestion that D. discoideum grex cells are not chemotactically sensitive
(Garrod & Malkinson, 1973) is supported by the more extensive results described
here. Cells isolated from the migrating grex did not respond chemotactically
either to aggregation streams, agar blocks containing cyclic AMP or other
migrating grexes. Thus, grex cells have lost the chemotactic sensitivity which
was acquired prior to aggregation. This implies that chemotaxis is of no
functional importance in grex migration.
That dispersed grex cells reacquire chemotactic sensitivity would seem to
be a fact important in relation to reaggregation. Although reaggregation can
take place to some extent before the cells become chemotactically sensitive,
the aggregates formed are generally small if the cells have been widely dispersed.
Also, many cells do not become incorporated into these initial aggregates probably because they have been too widely separated from other cells to
form mutual contacts. The onset of chemotaxis results in the formation of
larger aggregates because the small initial aggregates are attracted together.
Cellular basis of grex movement in slime mould
67
Previously isolated cells also become incorporated into these larger aggregates,
so that few cells become lost or wasted because of the disaggregation.
If grex cells seem to have lost the chemotactic behaviour of aggregating
cells, some of their adhesive properties would appear similar to those involved
in aggregation. It has been observed (Garrod, 1969) that grex cells show the
type of behaviour known as 'contact following', which has been shown to be
important in the aggregation process (Shaffer, 1962, 1964). Contact following
takes place because stable end-to-end intercellular contacts cause the cells to
follow each other. It has been demonstrated that 'aggregation-specific' adhesive
sites appear on the surfaces of slime mould cells prior to the onset of aggregation,
and that these sites appear to be involved in end-to-end contacts (Gerisch,
1968; Beug et al. 1970).
It must be assumed that movement of grex cells into aggregation streams
when they are placed close against the streams is directed by intercellular
contact (it cannot be directed by chemotaxis, because the cells are not chemotactically sensitive). Since grex cells can be guided by aggregating cells, it
seems probable that both possess identical surface adhesive sites, which are
involved in contact following at both stages. Gerisch has made a similar
suggestion, because he found that when a grex was placed in EDTA solution
the cells became partially dissociated, but remained attached to each other
by point contacts. The so-called 'aggregation-specific' adhesive sites of slime
mould cells are not disrupted by EDTA, so that the point contacts retained
by grex cells in EDTA may involve these sites (see Gerisch, 1968). The
observation that chemotactically insensitive cells can be guided into an aggregation stream, and presumably, from there into the aggregation centre, is a
positive demonstration of the view expressed by Shaffer (1962) that contact
and not chemotaxis guides the movement of cells in aggregation streams.
The process of reaggregation of dispersed grex cells seems to occur in two
distinct phases, only the second involving chemotaxis. It is necessary to seek
a mechanism for the initial phase of reaggregation. Initial reaggregation is
localized in the sense that cells which are near to each other or in contact on
the agar surface collect together into aggregates. The formation of aggregates
from a monolayer involves the development of intercellular adhesions at the
expense of cell to substratum adhesions. In principle, this type of behaviour
may be explained by assuming that the cells are more adhesive to each other
than to the agar substratum. Thermodynamically speaking, there would then
be a tendency for cells to maximize their area of mutual adhesion and minimize
their area of adhesion to the substratum; i.e. to form aggregates (see Martz &
Steinberg, 1973). Work on the adhesiveness of preaggregation cells of D. discoideum has suggested that at about the time of aggregation, the cells may
undergo a change such that their mutual adhesiveness exceeds their adhesiveness
to the substratum (Garrod, 1972). The present work suggests that similar
adhesive properties may persist into the migrating grex stage, and might
5-2
68
D. R. GARROD
account for the initial phase of grex reaggregation. These suggested changes
in the adhesiveness of slime mould cells are discussed in more detail in other
papers (Garrod & Ashworth, 1973; Garrod, 1974).
I thank Dr Patricia Cooke and Mr D. Forman for criticizing the manuscript, and Miss
Lynette Hand, Mrs Helen Creer and Mr J. L. Baynham for photographic and technical
assistance.
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GARROD,
(Received 4 October 1973)