FEMS Microbiology Letters 120 (1994) 1-8
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
FEMSLE 06039
MiniReview
The migration stage of Dictyostelium:
Behavior without muscles or nerves
J.T. Bonner
Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA
(Received 14 January 1994; revision received 23 April 1994; accepted 25 April 1994)
Abstract: The migrating slug of the cellular slime mould, Dictyostelium discoideum is capable of directed movement. A rather large
literature on how this simple group of amoebae move and orient is briefly summarized, All the amoebae in the slug move, and their
internal direction is controlled by chemotaxis, by relay waves of cAMP that start at the anterior tip. This moving mass of amoebae
can be oriented by directional light, as well as heat and chemical gradients. Such external factors could orient by shifting the
position of the cAMP pulse pacemaker in the tip, or by making the cells move faster or slower on one side of the anterior portion
of the slug, or a combination of the two.
Key words: Cellular slime mould; Dictyostelium; Slug movement; Taxes
Introduction
Dictyostelium discoideum's r e s p o n s e to starvation is for t h e s e p a r a t e a m o e b a e to s t r e a m tog e t h e r , to a g g r e g a t e , a n d f o r m a m u l t i c e l l u l a r
slug of h u n d r e d s o r t h o u s a n d s o f cells. T h e slug
n o t only m o v e s in a d i r e c t i o n a l fashion, b u t orie n t s with r e s p e c t to t h e i m m e d i a t e e n v i r o n m e n t
in t h e soil so t h a t it e n d s u p in a l o c a t i o n t h a t is
p r e s u m a b l y a d v a n t a g e o u s for fruiting a n d t h e disp e r s a l o f its spores.
In the recent literature there are a number of
s t u d i e s t h a t c o n c e r n t h e l o c o m o t i o n of m i g r a t i n g
slugs t h a t I w a n t to e x a m i n e , a l o n g with t h e m a n y
e a r l i e r ones, to see w h a t n e w insights w e m i g h t
have into t h e m e c h a n i s m s o f b o t h t h e m o v e m e n t
a n d the o r i e n t a t i o n o f t h e slugs. F r o m t h e e a r l i e s t
w o r k on c e l l u l a r slime m o l d s it was obvious t h a t
SSDI 0378-1097(94)00185-T
d u r i n g m i g r a t i o n t h e m u l t i c e l l u l a r m a s s m o v e d as
a unit; it m i g r a t e s as a b a g full o f cells, t h e bag
b e i n g t h e e x t e r n a l slime s h e a t h which t h e slug
s e c r e t e s as it moves. Since t h e r e is a c l e a r a n t e rior a n d p o s t e r i o r end, it leaves a c o l l a p s e d t u b e
o f slime s h e a t h b e h i n d (Fig. 1) [1].
How slugs move
Chemotaxis
O n e a s p e c t o f slug m o v e m e n t t h a t was e s t a b lished e a r l y is t h a t all t h e cells in t h e slug r e m a i n
u n d e r t h e t h r a l l of t h e c h e m o a t t r a c t a n t (or
acrasin, w h i c h in Dictyostelium is cyclic A M P ) ,
a n d as in a g g r e g a t i o n , pulses f r o m the tip p r o d u c e waves o f c h e m o t a x i s t h a t a t t r a c t s t h e cells
t o w a r d s t h e tip. T h e r e a r e m a n y w o r k e r s w h o
have contributed to our understanding of this
phenomenon, but it was first positively established in 1979 by Durston and Vork [2].
Role of the slime sheath
From the early work of Francis [3] it is clear
that the slime sheath is very thin at the anterior
tip, and is thicker further back. This was established in a detailed study by Farnsworth and
Loomis [4] using electron microscopy. However,
despite some attempts, there does not seem to be
any good evidence that the slime sheath contributes to the movement.
All the cells move
Chemotaxis within the slug could account for
the movement of individual cells, but how can
one account for the movement of the whole slug?
At first glance the problem seems to be suggesting that they can lift themselves by their own
bootstraps. One early idea which was put forward
by Shaffer [5] and later examined in more detail
by Odell and Bonner [6], was that one could get
forward movement if all the moving cells pushed
upon one another, provided the outlying cells got
some traction from the slime sheath. The model
also assumed that a substance increased the speed
Fig. 1, (,4,) Side view of a migrating slug of about 1 mm in length. (B) Top view of the migrating slug showing the slime tube
(photograph courtesy of D.F. Francis).
of the cells and diffused away from the surface of
the slug, so that it would be more concentrated
among the central cells which would then move
more rapidly than the peripheral cells. Various
predictions were made from this model. For instance, if the surface at the tip constrains the
forward movement there will be a reverse fountain movement of the internal cells; if the restraint on the tip is removed, the central cells will
move forward [6]. These predictions were supported by observations using vitally stained cells
as markers.
There have been a number of studies that
indicate that the cells can percolate through one
another. The first suggestion came from an early
experiment in which I grafted vitally stained anterior cells into the posterior end of slug, and they
moved forward within the slug to assume an
anterior position [1]. The really elegant studies
were those of Yamamoto [7] in which he persuaded vitally stained slugs (which normally have
a darkly stained anterior prestalk region) to enter
an agar tunnel. When they migrated to the dead
end of the tunnel, they turned around and went
out the open end; it was like turning a sock inside
out except that it occurred by the dark red cells
percolating through the posterior, colourless, prespore cells. This switching of the organizing tip
from the dead end of the tunnel to the open end
probably reflects the greater oxygen availability at
the open end, for it is known from the work of
Sternfeld and David [8[ that an oxygen gradient
will orient the tip.
In the 1950's I showed that some of the cells
move faster than the slug itself, while others
moved more slowly and fell back to the rear of
the slug. This was part of the evidence that there
was a sorting out of cells leading to an anterior
prestalk zone, and a posterior prespore zone.
These early observations have been greatly extended by numerous workers (see Nanjundiah
and Saran [9] for a review).
Recently, earlier observations of others have
been systematically investigated by Inagaki, Ishida
and Inouye (personal communication). They followed vitally stained cells with time lapse video,
and showed that individual cells from different
parts of the slug are often (but not always) highly
variable in their rate of movement, and even
more importantly, there is clearly no large scale,
or mass movement of cells within any part of the
slug.
Speed versus size
The speed of a slug is related to its size. In
some early work we noticed that larger slugs
moved faster than smaller ones, and suggested
the speed was proportional to the volume of the
slug. First Francis [3], and more recently Inouye
and Takeuchi [10] showed that the speed of a slug
does not correlate well with its volume, but perfectly with its length. If two slugs of roughly equal
volume (and age) are compared, but one is short
and fat and the other thin and long, the latter will
move faster. Furthermore, Inouye and Takeuchi
showed that for slugs of equal size, younger ones
move faster than older ones. Establishing the
cause of these size-speed-age relations will be a
matter of considerable interest.
Zone of motive force
One of the interesting aspects of locomotion is
the demonstration by Inouye and Takeuchi [10,11]
(and initially suggested by Francis [3]) that the
motive force for movement is greater in the anterior prestalk zone than in the posterior prestalk
zone. They showed this in two ways: (1) if anterior tips are removed from slugs and their speed
measured, they are faster than would be expected
of slugs their size [10]; and (2) by measuring the
motive force of slugs, or parts of slugs in agar
tunnels, they showed that anterior slug segments
had approximately three times the motive force
of posterior ones [11]. (They also reported that
the posterior portion can move on their own in
the agar tunnels. Normally, on an agar plate, a
posterior isolate will round up and only regain
locomotion after some hours).
The prestalk and prespore zones do not form
immediately after aggregation, and, unlike older
slugs, young slugs stain evenly with vital dyes over
their length. It has been shown that the prestalk
cells produce more cAMP, have more cAMP
binding sites, and have more cell-bound phosphodiesterase than the prespore ceils, and therefore
would be more responsive to gradients and pulses
of cyclic AMP (this is the work of a number of
authors; see [12] for some of the work and a list
of references). Presumably this is why the prestalk cells accumulate at the anterior end early in
migration. One obvious conclusion is that these
more active prestalk cells become the engine that
pull the slug forward.
Helical and linear cell movement
Adding to the earlier observations of Durston
and Vork [2], Siegert and Weijer [13] showed in
some elegant experiments that the forward movement of the slug can be accompanied by a helical
or scroll movement of the internal cells. These
two studies differ in the reported frequency of
scroll versus linear movement of the cells, but
there are many differences in the external conditions and the strains used. It is well known that
the movement of aggregating rings of cells can
often turn into spirals. Since one finds both spiral
and linear movement in aggregation patterns as
well as slugs, one must assume that the amoebae
can shift from one mode to the other. There is
considerable evidence that the cyclic A M P
chemotaxis, which is controlled by pulses emanating from the tip in both aggregates and slugs, is
responsible for these patterns. McNally (personal
communication) has some interesting new evidence of this at the mound stage, which is the
transition point between aggregation and migration. The switch is also predicted from mathematical models (see Cox [14] for a review; see also
Steinbock, et al. [15].)
Siegert and Weijer [13] note that the scroll
waves stop near the prestalk-prespore junction
and suggest that this is because the posterior
prespore region contains less excitable cells: their
cAMP oscillations are less rapid and prespore
cells are less responsive to cAMP chemotaxis.
From this they argue that because the ceils in the
scroll of the anterior region are not moving directly forward, they could not be responsible for
the motive force of the slug. This conclusion is
not consistent with the evidence I have just presented. They show that the cells in the anterior
scroll move about 40% faster than the prespore
cells; perhaps the scroll occurs because the ceils
move so rapidly they begin to spiral for they are
bound within the slime sheath and are held within
the confines of the slug by mutual adhesion between the cells.
How slugs orient
Two hypothetical possibilities
Migrating slugs are photo-, thermo- and
chemotactic. The question is, how do they orient
to these external stimuli? There are two hypotheses as to the basic mechanism. One is that the
cells are stimulated to move faster or slower on
one side of the tip, and this causes the tip to turn.
I will call this the differential speed hypothesis.
The other is that, since cells are being attracted
to the cAMP pacemaker at the very tip of the
slug, any external stimulus that shifts the location
of this apical pacemaker, or dominant cAMP
source, will cause a redirection of the ceils that
are attracted to it, leading to a shift in the orientation of the tip. Because this tip, with its special
properties, has often been called an 'organizer', I
will call this the shifting organizer hypothesis.
Note that these two hypotheses are not mutually
exclusive, and furthermore, since the organizer
entrains cells, it is not easy to tell them apart.
Phototaxis
There are many reasons to indicate that the
extremely sensitive orientation of slugs towards
light involves the lens effect. Francis [16] was the
first to put this on a firm footing by irradiating
one side of a slug near the tip, and showing that
the slug moved away from that side. The cylindrical curvature of the slug acts as a magnifying lens
and will focus light coming from one direction
onto the further side of the slug. If we are to
explain the turning towards the light by the differential speed hypothesis, the light must increase
the speed of cells. There is evidence that this is
so for unilateral light as Poff and Loomis, and
Kitami have shown (for references see [16]). On
the other hand it could be that the light focused
on the far side produces a repellent that pushes
the pacemaker source of cAMP to one side,
thereby complying with the shifting organizer hypothesis.
One approach to get at the mechanism of
phototaxis was to look at the effect of light on
separate amoebae. Contrary to various studies,
including my own, H~ider and his co-workers [18]
showed that if the experiment was done correctly,
individual vegetative amoebae were capable of
phototaxis. However, it is not clear that amoeba
phototaxis is necessarily related to slug phototaxis
because their action spectra are somewhat different, although a mutant incapable of slug phototaxis showed greatly reduced light responses for
its amoebae.
This brings up another way of seeking mechanisms of phototaxis: finding phototaxis mutants.
This approach, first explored by Loomis, has resulted in the discovery of a number of mutant
strains in different laboratories that potentially
could be of great interest, but so far it is not
known what the lesions are, that is, what is different about their internal chemistry so that they fail
to respond to light. In one study Fisher and
Williams [19] have interesting evidence that some
slug phototaxis mutants are also considerably impaired in their ability to respond to heat gradients.
Thermotaxis
In an early study [1] we demonstrated that
slugs were also extraordinarily sensitive to temperature gradients and this was confirmed by Poff
and Skokut [20]. A temperature gradient of
0.0004°C across a small slug is sufficient for it to
orient. A particularly interesting discovery was
that of Whitaker and Poff [21] who showed that
slugs were positively thermotactic above the temperature at which the slime mold was reared, and
negatively thermotactic below. As they pointed
out such a reversal would encourage the slugs to
go towards the surface of the soil, their natural
habitat, both in the warmth of the day, and the
cool of the night, presumably to come to a more
advantageous place to fruit.
Chemotaxis
The earliest evidence for slug chemotaxis was
that of Kenneth Raper who reported in 1939 that
slugs migrated towards drops of acid on an agar
plate. For many years, and many attempts, we
and others were unable to repeat this experiment.
Success finally arrived in 1985 when we were able
to get slug orientation in a p H gradient [1].
We became interested i.n the 1960's in the
possibility that cell masses could be oriented by
volatile substances. If cell masses of developing
fruiting bodies (which show the same taxes as
migrating slugs) are close to one another as they
rise they will lean away from one another [1]. The
best evidence that this was a volatile substance
came from placing a piece of activated charcoal
near the rising cell mass, and it would lean towards and into the charcoal; the repellant was
being adsorbed by the charcoal. Later we [1] and
Feit and Sollitto [22] independently showed that
this substance was N H 3. Kosugi and Inouye [23]
not only confirmed this result, but showed that
the effect was probably due to a change in the
internal p H of the cells, because besides NH3,
other small amines were also effective in turning
the slug tip.
The next logical step was to show that an
increase in external N H 3 increased the rate of
movement [1]. This turned out to be the case for
both slugs and for separate amoebae over a specified range of concentrations (although the latter
needed a lower concentration of N H 3 to increase
their speed).
We then examined the effect of N H 3 on photoand thermotaxis. If N H 3 is added to an atmosphere surrounding migrating slugs, they completely lose their ability to either orient in unidirectional light, or in a temperature gradient. From
this we made the obvious hypothesis that somehow light increases the production of N H 3 on
one side of the slug and that the slug turns
because of an increase in the rate of movement of
the amoebae. To test this we were able to show
that slugs in the light produce more N H 3 than
those in the dark. A similar argument was made
for thermotaxis, but because there is both positive and negative thermotaxis, as we have seen,
the case becomes dangerously hypothetical.
It occurred to me that since slugs produce
N H 3 through proteolysis, and since the change in
pH has to be internal within the cells, perhaps
there might be some way of affecting proteolysis
within the slug so that one could control the
turning of the tip [24]. By absorbing proteases on
polyacrylamide beads and placing the beads near
the tip of the slug, it turned away in the matter of
a few minutes. It is assumed that this is because
proteins, presumably on the slime sheath, are
degraded, and ultimately NH 3 is released in a
local patch which increases the speed of the cells
on that side. Conversely, if certain protease inhibitors are applied, using the same kind of bead,
the slug will move more slowly on that side, and
curve around the bead. I have interpreted all
these results, and the previously mentioned ones
on photo- and thermotaxis, in terms of the differential speed hypothesis, largely because NH 3 does
indeed directly affect the speed of cells. On the
other hand, there is nothing that rules out the
organizer shifting hypothesis, for among other
things, it is known that NH 3 inhibits cAMP production and it could be causing the cAMP pacemaker to shift its position.
There is another consideration that is relevant
here. If the tip's cells are undergoing a scroll
motion, then how would an increase in speed of
those cells effect a turning? According to Seigert
and Weijer [13] the prestalk cells moving in a
scroll travel at about 24 /zm rain -1, while the
whole slug travels in the order of 17/xm min -1
(at least that is the speed of the linear moving
prespore cells). To turn, it would mean that those
cells in the scroll would have to move faster for a
short period of time (about 1 min) when they
happen to be on one side of the slug. This seems
unlikely, leaving two alternative possibilities: one
is that the turning follows the organizer shift
hypothesis, for it would be compatible with cells
moving in a scroll, and the other is that there is
no scroll when the slug turns.
Another slug chemotactic agent has been proposed by Fisher et al. [25]. They show that slug
phototaxis is much less accurate in very dense
cultures as compared to sparse ones. By extraction from such dense cultures they have produced
a low molecular mass, but non-volatile substance
that repels slugs. To demonstrate this they grow
dense cultures on a filter paper and then put a
strip of this paper, which must contain what they
call the 'slug turning factor' on one side of a
group of migrating slugs that are emanating from
a circular group. They also show that in the
presence of their factor the accuracy of slug phototaxis is somewhat reduced. In both types of
experiments the effect of the factor is very slight
and requires statistical analyses to establish it. It
is difficult to know how this factor is affecting
orientation; perhaps it is another substance that
affects the internal pH of cells, but it is far less
effective than NH 3 and other small amines [23].
The key to the problem is probably not how
substances in the agar substratum or in the atmosphere above the culture affect orientation, but
rather what is happening in local regions inside
the slug tip.
Conclusion
It is remarkable that this seemingly simple bag
of amoebae can be so delicately coordinated that
it will go towards light of exceedingly low intensities and sense such minute heat gradients. Furthermore, we have evidence that its sensitivity to
very small differences in NH 3 is quite exceptional. It achieves this by a combination of (1)
cAMP chemotaxis of the amoebae within the slug
towards the apical pacemaker, (2) the confining
slime sheath, and (3) the adhesion and traction
between the cells. The evidence that factors that
affect the speed of cells play an important role as
well is encouraging. But despite all that we know,
I still find it difficult to understand how it can be
quite as sensitive as it i s - think of the slug's
amazing accomplishments, all achieved without a
brain, without sense organs, or without any limbs.
We still have a long ways to go before we can
understand how such a simple system can do such
clever things.
Acknowledgements
I am greatly indebted to, and wish to thank
E.C. Cox, V. Nanjundiah, E. Palsson, K. Inouye
and J. Williams for their exceedingly helpful comments on an earlier draft of this review.
References
1 In this brief review it will be impossible to cover all of the
many studies on slug migration, and therefore I will stress
what I think are the main points concerning the mechanisms of locomotion and orientation. Because of space,
reference will only be made to a few key papers which in
turn will serve as a source for other references. For work
done in my own laboratory I have resorted to the dubious
procedure of referring to a collection of our papers: Bonnet, J.T. (1991) Researches on Cellular Slime Moulds.
Indian Academy of Sciences, Bangalore, India.
2 Durston, A.J. and Vork, F. (1979) A cinamatographical
study of the development of vitally stained Dictyostelium
discoideum. J. Cell Sci. 36, 261-279.
3 Francis, D.F. (1959 and 1962) M.S. and Ph.D. theses.
University of Wisconsin, Madison, Wl.
4 Farnsworth, P.A. and Loomis, W.F. (1974) A barrier to
diffusion in pseudoplasmodia of Dictyostelium discoideum..
Dev. Biol. 41, 77-83.
5 Shaffer, B.M. (1962 and 1964) The Acrasina. Advan. Morphogenesis 2, 109-182 and 3, 301-322.
6 Odell, G.M. and Bonner, J.T, (1986) How the Dictyostelium discoideum grex crawls. Phil. Trans. R. Soc.
Lond. B 312, 487-525.
7 Yamamoto, M. (1977) Some aspects of behavior of the
migrating slug of the cellular slime mold Dictyostelium
discoideum, Devel. Growth and Differ. 19, 93-102.
8 Sternfeld, J. and David, C.N. (1981) Oxygen gradients
cause pattern orientation in Dictyostelium cell clumps. J.
Cell Sci. 50, 9-17.
9 Nanjundiah, V. and Saran, S. (1992) The determination of
spatial pattern in Dictyostelium discoideum. J. Biosci. 17,
353-394.
10 Inouye, K. and Takeuchi, I. (1979) Analytical studies on
migrating, movement of the pseudoplasmodium of Dictyostelium discoideum. Protoplasma 99, 289-304.
11 Inouye, K. and Takeuchi, I. (1980) Motive force of the
migrating pseudoplasmodium of the cellular slime mould
Dictyostelium discoideum. J. Cell Sci. 41, 53-64.
12 Wang, M. and Schaap, P. (1985) Correlations between tip
dominance, prestalk/prespore pattern, and cAMP- relay
efficiency in slugs of Dictyostelium discoideum. Differentiation 30, 7-14.
13 Siegert, F. and Weijer, C.J. (1992)Three dimensional
scroll waves organize Dictyostelium slugs. Proc. Natl. Acad.
Sci. USA 89, 6433-6437.
14 Cox. E.C. (1992) Modelling and experiment in developmental biology. Curr. Opinion Genet. Develop. 2, 647-650.
15 Steinbock, O., Siegert, F., Miiller, S.C. and Weijer, C.J.
(1993) Three-dimensional waves of excitation during Dictyostelium morphogenesis Proc. Natl. Acad. Sci. USA 90,
7332-7335.
16 Francis, D.W. (1964) Some studies on phototaxis of Dictyostelium. J. Cell. Comp. Physiol. 64, 131-138.
17 Kitami, M. (1982) The motive force of the migrating
pseudoplasmodium of Dictyostelium discoideum under
dark and light conditions. J. Cell Sci. 56, 131-140.
18 H~ider, D.P. and Hansel, A. (1991) Responses of Dictyostelium discoideum to multiple environmental stimuli.
Botan. Acta 104, 200-205.
19 Fisher, P.R. and Williams, K.L. (1982) Thermotactic behaviour of Dictyostelium discoideum slug phototaxis mutants. J. Gen. Microbiol. 128, 965-971.
20 Poff, K.L. and Skokut, M. (1977) Thermotaxis by pseudoplasmodia of Dictyostelium discoideum. Proc. Natl. Acad.
Sci. USA 74, 2007-2010.
21 Whitaker, B.D. and Poff, K.L, (1980) Thermal adaptation
of thermosensing and negative thermotaxis in Dictyostelium Exp. Cell Res. 128, 87-93.
22 Feit, I.N. and Sollitto, R.B. (1987) Ammonia is the gas
used for the spacing of fruiting bodies in the cellular slime
mold Dictyostelium discoideum. Differentiation 33, 193,196.
23 Kosugi, T. and Inouye, K. (1989) Negative chemotaxis to
ammonia and other weak bases by migrating slugs of the
cellular slime molds. J. Gen. Microbiol. 135, 1589-1598.
24 Bonner, J.T. (1993) Proteolysis and orientation in Dictyostelium slugs. J. Gen. Microbiol. 139, 2319-2322.
25 Fisher, F.R., Smith, E. and Williams, K.L. (1981) An
extracellular chemical signal controlling phototactic behavior by D. discoideum slugs. Cell 23, 799-807.