1. 1
THE EFFECT OF CONFINEMENT DURATION ON MOTILITY IN
DICTYOSTELIUM DISCOIDEUM COLONIES
LARRY O'CONNELL, CHRISTOPHE ANJARD
Département Biophysique, Institut Lumière Matière
Bâtiment Brillouin, 8 rue André-Marie Ampère
Campus de la Doua
69100 Villeurbanne, France
oconnel1@tcd.ie
Received 15/07/16
A protocol for production of individual, isolated, and densely packed (2510 cells/mm2
) colonies of
AX2 Dictyostelium discoideum is presented. The effect of confinement duration of D. discoideum on
subsequent spreading behaviour is then examined. A brief overview of the field and avenues for further
research are discussed.
Keywords: Dictyostelium discoideum, cell motility, model organism
1. Introduction
The individual and collective movement of cells is
a common theme in the study of a variety of fields as
diverse as immunology,1
cancer,2
and developmental
biology.2
There exist two broad classes of mechanisms by
which cells sense one another’s presence and modulate
the magnitude and orientation of their movement in
response.
The first class of mechanism is that of long range
sensing, which utilizes chemical factors that are
secreted and detected by the cells themselves. In the
second class of mechanism, physical contact between
the cells allows for short-range sensing of neighbours in
a cell’s immediate vicinity.
Previous work at ILM has made extensive
investigations into the spreading dynamics of D.
discoideum.3
This work did not, however, investigate
the effect of confinement duration on the cells. As
explained in section 1.2, there are several cell motility-
modulating systems interplaying in our D. discoideum
colonies. The design of the present experiment is such
that the long-distance ambient chemical-based effects
are diminished, while the contact effects are
emphasized as much as possible. This is achieved
through dense physical confinement of cells within a
small colony, while maintaining a total number of cells
that minimizes the concentration of any chemical
factors that may be present. The experiment then aims
to concentrate on the duration of confinement of cell
colonies and on the subsequent spreading behaviour.
By exploring the effect of confinement, if any, this
work hopes to probe the interplay of attractive and
repulsive contact-mediated signals acting on D.
discoideum and to elucidate further avenues of study.
1.1. Dictyostelium discoideum
Discovered in 1869, Dictyostelium is a genus of
eukaryotic, bacteriovorus amoeba that forms a large
component of soil microflora.4,5
Dictyostelids are also
known as slime moulds, although they are
phylogenetically distinct from fungi.6
Dictyostelium
discoideum is the most well-studied species of the
dictyostelid group.4
As an amoeboid, D. discoideum show phagocytic
behaviour, engulfing bacteria such as E. coli which
comprises its main food source.5
D. discoideum
amoebae are motile, crawling on surfaces through the

2. 2 Larry O'Connell
extension and retraction of pseudopodia – large
excrescences of cytoplasm formed by the coordinated
polymerization of actin microfilaments which are in
turn actuated by molecular motors such as myosins.
This interaction between actin and myosin creates a
protrusive front and a retractile rear, inducing a polarity
in the cell.3,7
D. discoideum is a well-established model
organism due to its relatively short life cycle,8
fully
sequenced genome,4
possession of many genes that are
homologous to those of humans, and ability to
differentiate a homogeneous population of cells into
distinct cell types.4,9
In addition, D. discoideum has an
easily manipulable genome, can be easily grown in
large quantities, and can be preserved for several years
through freezing.8,9
These qualities have made it an attractive candidate
for the study of developmental biology, signal
transduction, and – of particular relevance to the present
work – cell motility.
As social amoeba, they exhibit a remarkable ability
to alternate between unicellular and multicellular
forms, a property which is key to their role in diverse
fields of active research. D. discoideum, when in an
aggregative phase, exhibit many of the properties of a
developing mammalian embryo: polarity,
specialization of cells, self-regulation, and the use of
organizing centres.
1.2. D. discoideum as a model system
Short-distance signalling in D. discoideum occurs
through physical contact between cells. When two cells
meet, they physically block one another’s protrusions
into the contact zone. Force transduction is mediated by
transmembrane receptors upon physical contact by the
cells’ advancing protrusions.3,10
This is known as
“contact inhibition of locomotion” (CIL), a concept first
invoked in 1953 to explain the social behaviour of chick
heart fibroblasts in vitro.11
Recently, CIL has gained
renewed popularity as a field of interest.10,12
The differences in interspecies contact inhibition of
locomotion can promote the invasion of one tissue by
another.10
Indeed, inhibition of CIL in malignant cancer
cells is believed to contribute to their ability to invade
healthy tissues.13
In addition, previous work at ILM found an
unexpected contact-mediated effect which acted to
increase cell persistence over long timescales. The
persistence time is the period over which a cell moves
with a memory of its previous direction.3
This effect
was dubbed “contact enhancement of locomotion”
(CEL) in analogy to CIL. This CEL effect was theorized
to be potentially attributable to the accumulation of
motility modulating molecules, which are produced
transiently upon contact leading to a contact frequency-
dependent accumulation of motility.3
If this were the
case, we would expect to observe an increase in the
radial spreading velocity (vr) with increasing
confinement time (tc), concomitant with the increase in
persistence due to CEL.
In contrast, long-distance signalling mechanisms in
D. discoideum are based on chemical factors secreted
and detected by the cells themselves. While a “quorum
sensing factor” (QSF) has been found to reduce cell
motility and proliferation at high density and large
timescales (>150 minutes),14
many competing
endogenous chemotactic factors have also been
identified, such as ApraA, which is produced by D.
discoideum itself and acts to incipiently increase cell
motility as colony spreading begins.15
In the context of chemical-based cell-sensing
mechanisms, there is great interest in the insight gained
into the high motility of metastatic cancer cells through
the study of analogous model systems.2
For example, a
major problem in treating cancer lies in tumour
dormancy. Often, surgical removal of a tumour
stimulates cancerous cell proliferation in metastatic foci
in distant parts of the body.16,17
There is evidence that
there are chemical factors secreted by the tumour into
the bloodstream that inhibit both angiogenesis in the
distant foci18
and individual metastatic cell
proliferation.17
Elucidating the functioning of both
classes of cell-sensing mechanism, through the study of
model organisms such as those in the genus
Dictyostelium, could lead to the development of novel
therapies.19
1.3. The Dictyostelium life cycle
1.1.1 Vegetative phase
D. discoideum begins its life cycle when spores
hatch in warm, moist conditions to produce
myxamoebae. These myxamoebae phagocytize bacteria
in their environment, which they locate by travelling up

3. The Effect of Confinement Duration on Motility in Dictyostelium Discoideum Colonies 3
the folic acid gradient produced by the bacteria.7
D.
discoideum divides by mitosis and sequesters food
reserves for the aggregation phase of the life cycle.6
1.1.2 Aggregation phase
Starvation, due to depletion of bacterial “food” or
nutrients in the medium, marks the end of the vegetative
or trophic phase of the D. discoideum life cycle.5
Upon
starvation, D. discoideum will undergo a morphological
change where genes associated with growth are down-
regulated, while genes needed for aggregation are
induced. In D. discoideum and many other species of
Dictyostelium, cyclic adenosine monophosphate
(cAMP) is used as the chemotactic signalling
molecule.6,7
Spontaneous nucleation sites form around the cells
that are first to release a cAMP pulse. Upon detection
of this pulse, surrounding cells will emit then their own
pulse of cAMP and then move up the cAMP gradient
while simultaneously degrading the cAMP in their
vicinity. cAMP pulses will continue to be emitted every
six minutes and propagate outward from the nucleation
sites throughout the population. In this way, D.
discoideum synthesize, secrete, detect, and degrade
cAMP in order to regulate their aggregation by
chemotaxis.6,7
This pulsatile signalling is advantageous
for several reasons. A continuous cAMP signal would
eventually lead to a shallowing or destruction of the
chemical gradient as more is released, eliminating
directed cell motion. Furthermore, pulsatile signalling
necessitates a lower overall quantity of signalling
molecule, important in the case of D. discoideum since
adenosine triphosphate (ATP), a valuable cellular
molecule, must be sacrificed in the production of
cAMP.6
1.1.3 Migration and culmination phases
The aggregation continues until a motile
pseudoplasmodium or “slug” of approximately 100,000
individual cells is formed.5,8
For most species of
Dictyostelium, including D. discoideum, this slug
undergoes further development via differentiation of
individual cells into a fruiting body known as a
sorocarp, which releases hardened spores for transport
to other – hopefully more nutrient-rich – regions.6,8
This
latter stage is known as the culmination phase.9
2. Materials and methods
2.1 Stencil design and fabrication
Confinement of the colonies was achieved through
the use of a polydimethylsiloxane (PDMS) stencil
(Sylgard 184, 10:1 base:curing agent ratio). The stencil
took the form of a 390 µm diameter cylindrical cavity
in a 70 µm PDMS layer. Each experiment was
performed in duplicate with a pair of stencil cavities
separated by 9 mm (see Fig. 1 (c))
A hard mask was fabricated from monocrystalline Si
by normal photolithography processes. PDMS was
spun coated at 100 RPM for 10 s, 500 RPM for 10 s,
followed by 800 RPM for 60 s and then baked at 70 ºC
for 12 hours, to achieve a 70 µm thick layer of PDMS.
The stencils were then cut from the surface of the hard
mask.
2.2 Cell subculture
AX2 strain D. discoideum cells were observed to
have a doubling time of approximately 6-8 hours. In
order to maintain cells in a consistent vegetative state,
exhibiting no behaviour associated with high-density
regime, the cells were periodically subcultured in HL5
medium. The cells were inoculated into 4 ml sterile
Figure 1 - (A) Side view schematic diagram of the settling process before spreading begins. Confinement occurs
over this period. (B) Side view of colony spreading after stencil removal. (C) Top view of stencil geometry. Not to scale.

4. 4 Larry O'Connell
HL5 to a concentration of 1×103
cells/ml, and
subcultured to the same concentration once the culture
reached a concentration of approximately 5×105
ml-1
.
This concentration was then centrifuged at 2500 RPM
for 2 min (594 g,20
Biofuge Fresco, Heraeus
instruments), and resuspended in fresh HL5 to the
necessary concentration: 2.5×106
ml-1
for the 45 and 90
minute relaxation sequences and 5×106
ml-1
for the 20
minute relaxation sequences. Cells were maintained at
22.5 ºC at all times in an incubator and/or a
temperature-controlled room.
2.3 Experimental procedure
Stencils are placed on the base of a small 35 mm
Petri dish. 50 µl of sterile HL5 medium (Formedium,
Hunstanton, England) is placed on top of each stencil
cavity. The PDMS is sufficiently hydrophobic to
confine the 50 µl HL5 to a spherical drop on top of the
cavity. The dish is then placed under light vacuum for
15 mins to draw the HL5 medium into the stencil cavity,
creating a contiguous liquid column from the bulk
liquid down to the surface of the dish. The dish is
removed from the vacuum and the pure HL5 droplets
are removed. HL5 containing cultured axenic AX2 D.
discoideum (concentration ~2.5×106
cells/ml or ~5×106
cells/ml) is then similarly placed in droplets of 50 µl on
top of the cavities.
The cavity, now containing a contiguous water
column between the surface and droplet, allows cells to
accrete onto the dish surface, forming a negative image
of the stencil pattern (i.e., a disc-shaped colony, see Fig.
3.
The parameter varied during this study is that of the
confinement time (tc) of the cells at this point. The
stencil is left on the surface of the dish, confining the
cells to a circular region of 390 µm diameter (0.12 mm2
area). This tc is varied between 20 minutes and 90
minutes. The initial concentration is varied so as to keep
the number of cells in the colony at approximately 300,
as outlined below.
At the end of the confinement period, the dish is
filled with fresh HL5 medium and the stencil is slowly
peeled off. This must be done carefully, as moving the
PDMS layer too quickly can entrain the medium and
cause local turbulence above the colony, dislodging the
cells. The HL5 medium is again aspirated and replaced,
several times if needed, in order to remove superfluous
cells surrounding the medium. This removal of ambient
cells helps to preserve the isotropy of the environment
surrounding the colony.
The dish is kept covered and placed in a
temperature- and light-controlled housing containing
the microscope, at approximately 22.5 ºC. The colonies
are imaged under 20X magnification with a Nikon
brand objective lens and camera. The microscope is
deliberately underfocused such that each cell resolves
as a bright sphere, rather than having its true
morphology resolved. Automatic actuation of the stage
(for imaging several colonies simultaneously) is
permitted using an automated, computer-controlled X-
Y-Z stage. All image capture is managed using
Micromanager, a plugin for ImageJ. Exposure time is
15 ms, with 900 images taken at intervals of 20 s
(totalling a 5-hour runtime). The microscope shutter is
set to close between images in order to reduce light
Figure 2 - Snapshots of the spreading of a colony composed of 300 cells. (A) Cells at t=0 min (B) t=100 min and (C)
t=300 min. The contrast has been enhanced for better visualisation

5. The Effect of Confinement Duration on Motility in Dictyostelium Discoideum Colonies 5
fluence through the cells, as D. discoideum is known to
be phototrophic.3,8
2.4 Data treatment
ImageJ (v1.50i) was used to find the positions of all
cells in each image. There are several potential
strategies for obtaining the locations of the cells.
Previous work at ILM investigated the use of Edge
Detection, Binarisation-Thresholding, and ImageJ’s in-
built Find Maximum function.3
It was found that the
Find Maximum function was the most effective means
of locating cell positions, following selection of
appropriate tolerance levels.3
Find Maximum returns a
binary map of all pixels that are brighter than the
neighbouring 8 pixels by an amount greater than or
equal to the noise tolerance value. It is for this reason
that the contrast-enhancing effect of Fresnel fringes
upon underfocusing is important. Cells are deliberately
captured out of focus in order to allow the tracking
software to reliably find the cells’ positions (see Fig. 3
(b))
Find Maximum yields a position list for each
individual image. This list is then processed in
MATLAB by carrying out a least-squares regression
analysis to correlate the position of each cell in one
image to its position in the next. The parameters of this
processing allow a specified maximum distance
between frames before cells trajectories are assumed to
belong to separate cells. The parameters also permit a
trajectory to contain gaps of one frame without being
considered a separate trajectory. This allows the Find
Maximum function to fail to resolve the position for a
cell for one frame without the processing program
inferring separate trajectories.
The radial component of the velocity vector for each
cell vr was then extracted from the images and averaged
over all cells in a colony, yielding an averaged 〈𝑣𝑟〉
value that evolves over time.
3. Results and analysis
3.1 Confinement time results
The results obtained are shown graphically in Fig. 4,
while the key data points are given in Table 1. The
authors anticipated that there may exist a consistent
change in the temporal evolution of vr with increasing
tc.
Such a pattern is difficult to discern from the results
obtained. The peak vr value decreases between the 20
and 45 minute sequences, but then increases again
between the 45 and 90 minute sequences. However, the
variation between different colonies in each sequence
was significant and does not allow us to draw credible
conclusions about why the peak vr changes in such a
way as any correlation is likely to be spurious.
However, we do see in the first ~60 minutes a
correlation between the initial vr and tc. Increasing tc
seems to have the effect of increasing the initial colony
spreading speed. This would be consistent with the
postulated mechanism whereby there is an
accumulation within the cells of motility-modulating
molecules produced transiently upon contact. This may
indeed act to increase the initial spreading velocity of
the cells once the physical barrier to movement is
removed.
3.2 Settling
In order to ensure that a consistent number of
approximately 300 cells would settle within the
relaxation time (20, 45, or 90 minutes) it was necessary
to investigate the relationship between the initial
Figure 3 - (A) A typical well-formed colony of 239 cells
at beginning of experiment and (B) an overlaid example
of the binary map of cell positions output by Find
Maximum

6. 6 Larry O'Connell
concentration in the sample droplet, and the number of
cells present in the final colony. It was found that a
concentration of 2.5×106
ml-1
was optimal for the 45
and 90 min relaxation sequences, and 5×106
ml-1
was
optimal for the 20 min relaxation sequence.
4. Comparison with previous research
Previous work at ILM studying spreading of D.
discoideum used a more elaborate stencil arrangement
in order to confine the cells.3
Whereas in that work a
stencil was physically bonded to the PDMS stencil in
order to contain the settling sample liquid, this work
improved upon this protocol. It was found that the
wetting properties of PDMS created a sufficiently
hydrophobic surface to maintain a nearly spherical
sample droplet above the stencil (see Fig. 1 (a)). This
improvement meant that the time needed to produce a
suitable colony was significantly reduced, allowing the
experimenters to study the spreading behaviour at
smaller timescales. The minimum confinement time
achieved in this work was 20 minutes, compared with a
consistent 45 minute confinement time in d’Alessandro,
2016.3
The overall form of the temporal evolution found in
d’Alessandro, 2016, was successfully replicated in this
work. An initial, comparatively steep increase in vr is
observed, followed by a slower drop-off to a smaller
value around 0.2 µm/min.
Some of the results of our experiment can be
compared directly with previous work in d’Allesandro,
2016, namely the experiments that also featured a 45
minute confinement time. That work found a vr of ~1.8
µm/min for n=259 cells whereas this work found an
average vr of 1.0 µm/min with an average colony size
of n=285.
5. Discussion
What became obvious during experimentation was
that the recent history (previous ~48 hours) of the cells
used had a bearing on the results of the spreading
experiment. Initially, it had been thought that only the
cell concentration at the beginning of the experiment
would affect the outcome of the radial velocity
measurements. This was a “path independent”
assumption that may appeal to those in the physical
sciences more than those in the biological sciences. Due
to this assumption, early experiments saw cells either
diluted or concentrated to the appropriate level
immediately before commencing the experiment. It was
observed, however, that cells behaved differently
depending on whether they had been diluted or
concentrated prior to the beginning the experiment.
Figure 1 - Evolution of the radial component of the velocity vector vr. Each trace shows the average over all
experimental runs for the designated relaxation time. The standard deviation of each trace is shown flanking in grey
for each trace. 3916 cells in total were tracked.

7. The Effect of Confinement Duration on Motility in Dictyostelium Discoideum Colonies 7
Cells that had been allowed to reach a high
concentration of ~5×106
ml-1
before being diluted
exhibited both abnormal macro-scale colony spreading
and cellular scale behaviour. It was observed that small,
anomalous groups of 4-5 cells that had mutually
adhered were present in such colonies. The spreading of
recently dense cells exhibited a range of strange
behaviours including: incipient contraction, greatly
reduced motility, and divergence in the radial velocity
trends between colonies of the same initial conditions.
These observations informed the decision to instead
use cultures at no higher concentration than 5×105
ml-1
,
to centrifuge all samples identically, and then to
resuspend the cells to the concentration required. Thus,
all cells used have been kept below the ~10^6
concentration that seems to affect spreading behaviour,
and all cells have been similarly centrifuged.
5.1 Limits of present work
One limitation of this research is an inability to
control for the effect of centrifugal concentration on the
spreading behaviour. As explained above, it was
necessary to centrifuge all cells to the desired
concentration immediately before beginning the
experiment. This centrifugation (of approximately 594
g for 3 minutes) and resuspension may have an effect
on the cells, possibly acting to change their motility.
This is especially relevant since the class of effect
investigated in this study is specifically those which are
contact-mediated.
Additionally, in order to achieve the necessary
number of cells on the substrate surface for shorter
confinement times – and thus shorter available time for
accretion into the colony – much higher concentrations
were needed for the 20 min confinement experiments,
in the order of 5×106
ml-1
, compared to 2.5×106
ml-1
for
the 45 and 90 min confinement sequences. These higher
cell concentrations immediately prior to some
experimental runs could have had an effect on the
motility. Previous research has found a spontaneous
aggregation phenomenon when cells are plated at high
density.6
Further research may focus on the effect of
centrifugation on motility.
Acknowledgments
The author would like to thank Joseph D’Alessandro
for his advice and guidance over the course of this
project. The author also thanks Christophe Anjard and
Jean-Paul Rieu for the opportunity to work with the
Biophysics department at ILM.
This work was carried out at Institut Lumière
Matière, Lyon (ILM) as part of studies funded by the
Table 1 – Key data points: maximum, mean, and standard deviation of vr;
maximum, mean, and standard deviation of the timing of the peak height.
Time
Cell
count
Peak v
(µm/min)
Mean peak
v (µm/min)
Std. dev. v
Peak time
(min)
Mean peak
time (min)
Std. dev.
peak time
328 0.98 83
277 1.19 102
337 1.36 83
335 1.39 87
372 1.29 109
339 1.42 105
318 1.02 65
265 0.93 66
300 1.10 71
257 1.10 101
298 1.60 82
278 1.36 74
275 1.42 74
1.27 94.80.15 10.8
90
45
20
1.46 76.70.10 3.8
1.04 75.80.07 14.8

8. 8 Larry O'Connell
Programme Avenir Lyon Saint-Etienne (PALSE)
scholarship programme.
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