A secreted protein is an endogenous chemorepellant in
Jonathan E. Phillipsa,band Richard H. Gomerb,1
aDepartment of Biochemistry and Cell Biology, Rice University, Houston, TX 77005 andbDepartment of Biology, Texas A&M University, College Station, TX
Edited by Herbert Levine, University of California at San Diego, La Jolla, CA, and approved May 22, 2012 (received for review April 16, 2012)
Chemorepellants may play multiple roles in physiological and
have been identified, and how they function is unclear. We found
that the autocrine signal AprA, which is produced by growing Dic-
tyostelium discoideum cells and inhibits their proliferation, also
show directed movement outward from the colony, whereas cells
lacking AprA do not. Cells show directed movement away from
a source ofrecombinant AprA and dialyzed conditioned media from
The secreted protein CfaD, the G protein Gα8, and the kinase QkgA
are necessary for the chemorepellant activity of AprA as well as its
proliferation-inhibiting activity, whereas the putative transcription
factor BzpN is dispensable for the chemorepellant activity of AprA
but necessary for inhibition of proliferation. Phospholipase C and
PI3 kinases 1 and 2, which are necessary for the activity of at least
one other chemorepellant in Dictyostelium, are not necessary for
recombinant AprA chemorepellant activity. Starved cells are not re-
pelled by recombinant AprA, suggesting that aggregation-phase
cells are not sensitive to the chemorepellant effect. Cell tracking
indicates that AprA affects the directional bias of cell movement,
but not cell velocity or the persistence of cell movement. Together,
our data indicate that the endogenous signal AprA acts as an auto-
crine chemorepellant for Dictyostelium cells.
autocrine signaling|signal transduction|chemorepellent|gradient
prokaryotic cells (1). In eukaryotes, chemotaxis proceeds through
extracellular signal sensing and polarization of the actin cytoskel-
eton, resulting in cellular extensions (pseudopods) that facilitate
movement (2). Many chemoattractants, or signals that cells move
toward, have been identified (3–6), and many molecular compo-
nents involved in directional sensing and regulation of actin dy-
namics have been characterized (7). An alternative chemotactic
process involves chemorepellants, signals that cells move away
as chemorepellants at high concentrations (8, 9), and the protein
semaphorin III acts as a chemorepellant in the context of neuronal
growth cone guidance (10). Chemorepellants may function in the
resolution of inflammation (11), gastrulation (12), the pathoge-
nicity of the parasite Entamoeba histolytica (13), and metastasis
(14). However, few endogenous chemorepellants have been iden-
tified, and relatively little is known regarding their mechanism
The eukaryote Dictyostelium discoideum is an excellent model
for the study of chemotactic processes. In the presence of
nutrients, Dictyostelium exist as unicellular amoebae that re-
produce by fission. When starved, cells secrete and respond to the
cAMP involves G protein-coupled receptors (16), heterotrimeric
G proteins (17, 18), Ras (19, 20), PI3 kinase (PI3K) (21), phos-
Dictyostelium chemorepellants. For example, when a small spot of
hemotaxis, or directed movement in response to a chemical
gradient, is an ancient and critical behavior of eukaryotic and
high-density cells are placed next to each other, cells show
a greater degree of movement away from the adjacent spot than
toward it (25). In addition, the synthetic cAMP analog 8CPT-
cAMP induces negative chemotaxis through localized phospholi-
pase C inhibition (26). However, no endogenous chemorepellants
have been identified.
AprA is an autocrine-signaling protein produced by vegetative
Dictyostelium cells that inhibits cell proliferation (27, 28). As the
local cell density increases, the concentration of AprA concomi-
tantly increases, resulting in the inhibition of proliferation at high
cell density and thus establishing a threshold for the maximum
density of cells. AprA shows saturable binding to cells (27) and,
although no receptor has been identified, AprA requires the het-
erotrimeric G protein complex components Gα8 and Gβ to inhibit
proliferation, to induce high-affinity GTP binding to membranes
(29), and for the GTP analog GTPγS to inhibit the binding of
recombinant AprA (rAprA) to cell membranes (29). Inhibition of
the ROCO kinase QkgA (31), and the putative transcription factor
BzpN (32), implicating these proteins in signal transduction by
AprA. Interestingly, although aprA−cells are able to chemotax
toward cAMP, as evidenced by the ability of the cells to aggregate,
and aprA−cells show random motility like that of wild-type cells,
colonies of aprA−cells show a reduced rate ofexpansion compared
with wild type (31). In this report, we provide evidence indicating
that AprA functions as an autocrine chemorepellant for vegetative
Wild-Type but Not aprA−or cfaD−Cells Show Directed Movement
Away from Regions of High Cell Density. We previously found that
colonies of aprA−and cfaD−cells expand less rapidly than wild-
type cells on a lawn of bacteria, although these mutants show no
difference in random cell motility at low cell density compared
with wild type (31). To test whether wild-type cells show a directed
movement away from areas of high cell density, we established
displacement of individual cells at the colony edges by video mi-
croscopy. These cells showed an average displacement away from
the cell colony (Fig. 1), with 94 ± 6% of measured cells showing
10 cells per experiment). For aprA−or cfaD−cells, although cells
were motile, the average displacement of cells was not strongly
cfaD−cells showing displacement away from the colony. These
Author contributions: J.E.P. and R.H.G. designed research; J.E.P. performed research; J.E.P.
and R.H.G. analyzed data; and J.E.P. and R.H.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 3, 2012
| vol. 109
| no. 27www.pnas.org/cgi/doi/10.1073/pnas.1206350109
results show that wild-type but not aprA−or cfaD−cells show
a directed movement away from regions of high cell density, in-
dicating that AprA and/or CfaD are required for directed move-
ment away from a cell colony.
Cells Show Directed Movement Away from a Source of AprA but Not
but not aprA−or cfaD−cells, away from a colony is that AprA and/
or CfaD may be acting as an autocrine chemorepellant, present at
high concentrations where cell density is high and facilitating the
assay (33), which consists of placing a layer of agarose over a uni-
form field of cells, adding rAprA or rCfaD to a well in the agarose,
and examining the displacement of individual cells adjacent to the
well. When medium alone was added to the well, cells adjacent to
the well showed no significant bias in displacement in the direction
away from the well (Fig. 2A), with 56 ± 4% of cells showing dis-
the well, cells showed an average displacement away from the well,
and 73 ± 9% of cells moved away from the well. When rCfaD was
added to the well, no significant bias in displacement wasobserved.
We examined actin and myosin localization in cells in a rAprA
gradient and invariably detected actin localization at the leading
edge and myosin II localization at the trailing edge (Fig. S1), sug-
gesting that cells that show directed movement away from rAprA
move with the classical mechanism of actin-rich protrusions at the
leading edge of the cell and myosin II-mediated contraction at the
rear (2). These results indicate that AprA functions as a chemo-
repellant and suggest that CfaD does not share this function. Be-
cause rAprA and rCfaD were produced in identical protocols and
rCfaD does not have chemorepellant activity, the chemorepellant
activity of AprA is likely not due to a contaminant from protein
expression and purification.
The extracellular concentration of AprA increases as a function
of cell density in shaking culture and reaches a maximum con-
centration of 300 ng/mL (27). To test the effect of different con-
assay (34), which is a more tractable assay for chemotaxis than the
previously described under-agarose assay. We tested the response
of cells to the chemoattractant folate and to rAprA and observed
chemotaxis toward folate and away from rAprA (Fig. S2), showing
that cells used in this assay are chemotaxis competent and che-
morepulse from rAprA. A source of rAprA at a concentration of
20 ng/mL was sufficient for chemorepellant activity (Fig. 2B).
These results indicate that the chemorepellant effect of AprA
occurs at physiological concentrations.
Chemorepellant Activity Present in Dialyzed Conditioned Media from
Wild-Type Cells Is Reduced in aprA−Conditioned Media. If AprA
functions as a chemorepellant, then conditioned media from wild-
be reduced or absent in conditioned media from aprA−cells. To
away from a colony of cells. A spot of cells was allowed to settle in a cell
culture chamber, and then the chamber was filled with media. The edge of
the colony was filmed, and the movement of individual cells was followed
over a period of 5 h. The average displacement of cells in the direction away
from the colony is shown. Values are mean ± SEM; n = 3, with the dis-
placement of at least 10 cells per genotype measured for each independent
experiment. The difference between wild type and either aprA−or cfaD−is
significant (P < 0.001, one-way ANOVA, Tukey’s test).
Wild type, but not aprA−or cfaD−cells, show a directed movement
buffer, rCfaD, or rAprA was added. Cells adjacent to the well were filmed, and the displacement of individual cells in the direction away from the well over 2 h was
measured. Values are mean ± SEM; n ≥ 4, with the displacement of at least 10 cells per genotype measured for each independent experiment. The differences
between the displacements for AprA and either buffer or CfaD are significant (P < 0.01, one-way ANOVA, Dunnett’s test). (B) Wild-type cells adjacent to a source of
rAprA at the indicated concentration in HL5 media were filmed for 1 h, and the displacement of individual cells away from the rAprA source was measured. The
Phillips and GomerPNAS
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| vol. 109
| no. 27
aprA−cells, or fresh media using the under-agarose assay. Condi-
tioned media from both wild-type and aprA−cells at high density
had a chemorepellant effect (Fig. S3). However, whenconditioned
media was dialyzed against fresh media using a 10-kDa cutoff
membrane and then used in the under-agarose assay, conditioned
media from wild-type cells caused a significant bias in cell dis-
placement away from the conditioned media compared with fresh
media (Fig. 2C), whereas conditioned media from aprA−cells did
not show a significant effect on cell displacement. These results
indicate that something larger than 10 kDa that is present in wild-
type conditioned media but not significantly present in aprA−
conditioned media functions as a chemorepellant, supporting the
hypothesis that AprA itself is a chemorepellant. In addition, a
chemorepellant that is smaller than 10 kDa is present in high-
density aprA−conditioned medium.
AprA Shows Chemorepellant Activity in the Absence of Rich Media.
An autocrine chemorepellant could potentially work by degrading
or inactivating a chemoattractant present in the surrounding area,
causing the chemoattractant concentration to be low in areas of
high cell density and high elsewhere. This would then create
areas of high cell density. Because at least one compound in rich
media, folate, functions as a Dictyostelium chemoattractant (35),
this model of chemorepulsion could potentially be occurring un-
der our experimental conditions. We thus examined whether rich
media is necessary for AprA chemorepellant activity by assaying
for chemorepellant activity using cells in buffer. Under these
conditions, we still observed a chemorepellant effect of rAprA
that was not significantly different from that observed in rich
media (t test; Fig. 3 A and B); 48 ± 5% of cells measured showed
movement away from buffer (with buffer also in the opposite
chamber), whereas 81 ± 4% of cells showed movement away from
rAprA in buffer (P < 0.01, t test, n = 3). These results show that
the chemorepellant effect of AprA does not require rich media,
strongly suggesting that AprA does not function as a chemo-
repellant by inactivating a chemoattractant.
AprA Shows No Detectable Chemorepellant Activity on Starved Cells.
AprA is most strongly expressed during vegetative growth, al-
though expression is also detected during aggregation and de-
velopment (28). To examine whether AprA affects the movement
of cells at the aggregation stage, we starved wild-type cells for 5 h
in buffer to induce the transition from growth to development and
then assayed the chemorepellant effect of rAprA on these cells.
Under these conditions, no bias in the direction of cell movement
was evident(Fig. 3A). This result suggests that rAprA does not act
as a chemorepellant for cells during the aggregation stage.
Chemorepellant Effect of AprA Requires CfaD, QkgA, and Gα8, but
Not BzpN, Phospholipase C, or PI3 kinases 1 and 2. The G protein
Gα8, the kinase QkgA, and the putative transcription factor BzpN
are necessary for the proliferation-inhibiting activity of AprA (29,
the secreted protein CfaD is necessary for AprA activity (30). To
test whether these proteins are necessary for the chemorepellant
activity of AprA, we examined whether cell lines mutant for these
proteins were repelled by rAprA in an Insall chamber assay. We
saw that wild-type cells adjacent to a source of rAprA showed
a significant bias in displacement away from rAprA (Fig. 3B and
Movies S1 and S2). Similarly, aprA−and bzpN−cells showed di-
rected movement away from a rAprA source. In contrast, gα8−,
qkgA−, and cfaD−cells showed no significant bias in displacement
and CfaD are necessary for the chemorepellant effect of AprA,
whereas BzpN is not. Consistent with our interpretation of AprA
chemorepellant activity acting to disperse cell colonies, we found
that, like aprA−, cfaD−, and qkgA−cells, gα8−cells showed a re-
duced spreading at the edge of cell colonies compared with wild
type, whereas bzpN−cells did not (Fig. S4).
The synthetic cAMP analog 8CPT-cAMP acts as a chemo-
repellant for starved Dictyostelium cells, and this chemorepulsion
requires phospholipase C (PLC) and the activity of PI3 kinases 1
and 2 (26). We observed that both plc−and pi3k1−/2−cells showed
movement away from a rAprA source in a manner similar to wild-
type cells (Fig. 3B). We further examined the role of PI3K activity
in AprA-mediated chemorepulsion by using cells expressing cyto-
solic regulator of adenylate cyclase (CRAC)-GFP, a marker for
PI3K activity at the cell cortex, which localizes to the upgradient
side ofa cell during chemotaxis towardcAMP (36).When situated
in a gradient of rAprA, cell tracking over a 5-min period revealed
that 54% of cells expressing CRAC-GFP moved down the rAprA
gradient, 27% of cells moved up the gradient, and 19% of cells
moved roughly parallel to the gradient, suggesting that rAprA has
a chemorepellant effect on these cells. When CRAC-GFP was
imaged, the majority of cells showed cytosolic CRAC-GFP local-
ization, and localization did not correlate with the direction of the
rAprA gradient for the whole-cell population or for the subset of
cells moving down the rAprA gradient (Fig. S5). This result indi-
cates that PLC and PI3K activity are not necessary for the che-
morepellant activity of AprA and that AprA and 8CPT-cAMP
function as chemorepellants through distinct mechanisms.
and requires the proteins Gα8, QkgA, and CfaD but not BzpN or PLC for
activity. (A) Cells were placed adjacent to a source of rAprA or buffer using
an Insall chamber and filmed for 1 h. The displacement of individual cells in
the direction away from the rAprA source was then measured. Values are
mean ± SEM; n = 3. The difference between the indicated conditions is
significant (P < 0.05, one-tailed t test). The 5-h starved WT condition is not
significantly different from the value 0 (P > 0.05, paired t test). (B) Cells in
HL5 media were placed adjacent to a source of rAprA or media using an
Insall chamber and filmed for 1 h. The displacement of individual cells in the
direction away from the rAprA source was then measured. *P < 0.05, **P <
0.01, and ***P < 0.001 (t test). The differences in average displacement
between genotypes that show a significant chemorepellant response are not
significant (one-way ANOVA, Tukey’s test).
AprA functions as a chemorepellant in the absence of rich media
| www.pnas.org/cgi/doi/10.1073/pnas.1206350109 Phillips and Gomer
AprA Biases the Direction of Cell Movement but Does Not Affect
Average Cell Speed. To gain insight into the mechanism by which
AprA might affect cell movement, we tracked the movement of
wild-type cells in a gradient of rAprA over 1 h and examined the
parameters of cell movement. Whereas no bias in the direction
of movement was evident for a media control (Fig. 4A), cells
tended to move away from the rAprA source, and the center of
mass of the cell endpoints indicated a bias away from the rAprA
source (Fig. 4B). Tracks of cells in a gradient of rAprA showed
a negative forward migration index (FMI) that was significantly
different from the FMI for the media control (Table 1), sup-
porting the interpretation of AprA as a chemorepellant. The
average speed of cells in a gradient of rAprA was not signifi-
cantly different from the control (Table 1) and was consistent
with previous measurements of randomly motile vegetative cells
(37, 38), indicating that AprA does not stimulate an increase in
the average speed of cells. Supporting this conclusion, cells in
a uniform concentration of 2 μg/mL rAprA showed a displace-
ment of 152 ± 33 μm over an interval of 2 h, whereas cells in
media showed a displacement of 157 ± 19 μm; the difference is
not significant. Cells in a gradient of rAprA showed a signifi-
cantly higher directionality (a measure of how directed, as op-
posed to random, the movement of a cell is) than the control
(Table 1), further indicating that AprA affects the direction of
cell movement. Finally, we examined whether the distribution of
the endpoints of cell tracks was nonrandom by performing
a Rayleigh test. The distribution for cell endpoints adjacent to
a rAprA source was significantly biased, whereas the distribution
of endpoints for the media control was not (Table 1). Together,
these data indicate that AprA regulates the directionality of cell
movement so that cells move away from high AprA concen-
trations and that this regulation does not involve a change in
average cell speed.
AprA Affects Directionality but Not Persistence of Cell Movement. To
further characterize how AprA affects movement, we used the
cell-tracking data to examine how AprA might affect the di-
rectional persistence of cells. Movies of cells were processed into
35-s intervals, and the displacement of cells in the direction of the
AprA source was determined for all cells and all intervals. Most
commonly, cells showed no change in position over a 35-s interval
(Fig. S6). For the control condition, the distribution of displace-
ments revealed no bias in movement, whereas for cells in a gradi-
ent of rAprA a bias in displacement in the direction away from
rAprA was evident (Fig. S6 and Table 2). We then examined the
average displacement in all intervals in the direction toward or
away from the rAprA source to determine whether rAprA might
cause an increase in cell velocity in certain directions. There was
no significant difference in the average displacement for cells
moving away from the source between the rAprA and the control
condition, and there was no significant difference in the average
displacement for cells moving toward the source between the two
conditions (Table 2). These results suggest that AprA does not
significantly increase the velocity of cells in a directional manner.
To determine the effect of rAprA on the persistence of cell
movement, we first defined PAas the probability that a cell would
move away fromrAprA,andPTastheprobabilitythat a cell would
move toward rAprA. As shown in Table 2, PAand PTare roughly
the same for the media control and significantly different in
a gradient of rAprA. We then measured, for each cell, PA2, the
percentage of intervals where the cell moved away from rAprA in
one interval and away again in the next interval. Because the
probability that a cell will move away is different depending on
whether there is a gradient of rAprA, we calculated NA2 = PA2/
(PA)2, or the observed probability of two subsequent movements
away from rAprA divided by the expected probability that such an
eventwouldhappenatrandom.NA2thus representsa normalized
probability that a cell will move away from rAprA in two sub-
sequent time intervals, with higher values indicating more persis-
tence in movement than would be expected at random. We found
that NA2 was not statistically different between the rAprA gra-
dient condition and the control and that the corresponding NT2
(the normalized probability of a cell moving toward rAprA in two
gradient and control (Table 2). We similarly calculated the nor-
three or four consecutive intervals as NA3 = PA3/(PA)3or NA4 =
PA4/(PA)4. For both control and rAprA gradient conditions, NA4
was greater than NA3, and NA3 was greater than NA2, suggesting
expected at random compared with shorter ones. We observed no
a cell moved away in three or four consecutive time intervals be-
tween rAprA gradient and control conditions. Similarly, the cor-
responding NT3 and NT4 values are not significantly different
betweenrAprAgradient and controlexperiments.Together,these
results suggest that the chemorepulsion mediated by rAprA is not
due to an altered persistence in movement away from or toward
the source of rAprA.
chamber; cells were tracked over a 1-h period, and tracks were graphed. The AprA source is on the right side of the origin. Red dots represent the final
position of cells, and green dots are the averaged center of mass for all tracks. The tracks are a compilation of three independent experiments with at least
seven tracks per experiment.
Tracking of cells adjacent to an AprA source. Wild-type cells in the absence (A) or presence (B) of a rAprA gradient were filmed using an Insall
Phillips and GomerPNAS
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| vol. 109
| no. 27
Colonies of aprA−cells expand less rapidly than wild-type colo-
nies, despite the fact that aprA−cells proliferate more rapidly
than wild-type cells (28, 31). Our data support the hypothesis
that rAprA functions as an autocrine chemorepellant for Dic-
tyostelium cells and that this chemorepellant function may fa-
cilitate the spreading of cell colonies.
We found that dialyzed conditioned media from wild-type but
not aprA−cells has chemorepellant activity. However, when con-
ditioned media was taken directly from cells, filter-sterilized, and
assayed, conditioned media from aprA−cells showed chemo-
repellant activity. This result suggests that a small molecule or
molecules that have chemorepellant activity accumulate in the
media of high-density cultures of aprA−cells. Alternatively, con-
ditioned media from high-density cells may be depleted of
nutrients, and using this depleted media could establish a gradient
moving away. Regardless, this chemorepellant effect is likely not
functioning physiologically in the movement of cells away from
movement away from a colony (Fig. 1).
Multiple linesof evidence suggest that AprA actsas a ligand for
a G protein-coupled cell-surface receptor as opposed to inter-
acting with another extracellular factor or factors (27, 29). We
found that rAprA shows chemorepellant activity in the absence of
rich media, indicating that AprA does not function by modifying
factors in media with chemokinetic properties. This result further
suggests that AprA acts as a ligand as opposed to modifying an
We found that the G protein Gα8 and the kinase QkgA are
necessaryfor the chemorepellant activity of AprA as well as for its
proliferation-inhibiting activity, but that that the putative tran-
scription factor BzpN is dispensable for AprA chemorepellant
activity despite being necessary for inhibition of proliferation.
These results suggest that AprA inhibits proliferation and induces
chemorepulsion using partially overlapping signal transduction
pathways and that the signal transduction branches, with BzpN
being a component of the proliferation-inhibiting pathway but not
the chemorepellant pathway.
Whereas the previously reported Dictyostelium chemorepellant
8CPT-cAMP requires PLCand PI3 kinases 1 and2 for activity, we
found that these proteins are not required for the chemorepellant
activity of rAprA. Furthermore, whereas 8CPT-cAMP acts on
starved cells (26), rAprA shows no chemorepellant activity on
starved cells. These results suggest that these chemorepellants
likely function through different mechanisms and that, when cells
We further investigated the role of PI3-kinase activity on che-
morepulsion and saw that CRAC-GFP localization did not cor-
respond to a rAprA gradient, suggesting that polarized PI3-kinase
activity is not essential for rAprA chemorepellant activity.
We tracked the movement of cells in a gradient of rAprA and
found that cells showed a negative migration index and more di-
rected movement than control cells, although the difference in
average speed for cells in a rAprA gradient and control cells was
not significant. We also found that AprA does not affect the per-
our data indicate that rAprA functions as a chemorepellant by
gradient, and not by affecting the speed of cells or the persistence
of cell movement.
A Dictyostelium autocrine chemorepellant mechanism mayhave
been selected for because it spreads out groups of vegetative cells,
as cells would tend to move away from areas of high cell density by
moving down an AprA gradient. This movement would result in
coverage of a larger area by a population of cells and thus provide
access to a larger quantity of nutrients, facilitating growth. Such an
effect seems evident during growth of wild-type or aprA−cells on
lawns of bacteria, as wild-type colonies expand and clear the bac-
terial lawn more rapidly than aprA−cell colonies (31). The pro-
liferation-inhibiting activity of AprA (28) may act in cooperation
and cell distribution
Effect of AprA on FMI, cell speed, cell directionality,
Rayleigh test (P value)
−0.01 ± 0.04
4.9 ± 0.2
0.21 ± 0.03
−0.18 ± 0.06*
5.0 ± 0.2
0.34 ± 0.03***
The data from Fig. 4 were analyzed to calculate the forward migration
index, or FMI (a measure of cell movement in the direction of a gradient in
respect to total cell movement, with zero indicating no movement in the
direction of the gradient), cell speed, directionality (the ratio of the Euclid-
ean distance to the total distance traveled), and P values for the Rayleigh
test, a test for nonrandom distributions of cell endpoints. *P < 0.05 indicates
a significant difference; ***P < 0.001 (t test).
Table 2.Effect of AprA on the direction of cell displacement and cell persistence over 35-s intervals
Media control rAprA
Probabilities of cell displacement over one 35-s interval Displacement away from source (PA)
Displacement toward source (PT)
Displacements away from source
Displacements toward source
Away from source (NA2)
0.27 ± 0.02
0.28 ± 0.02
0.46 ± 0.01
3.3 ± 0.1
3.1 ± 0.1
1.3 ± 0.2
0.35 ± 0.01**
0.20 ± 0.02*
0.44 ± 0.01
3.4 ± 0.1
3.1 ± 0.1
1.4 ± 0.1
Average displacement of indicated subset (μm)
Normalized probability of two subsequent movements in the
Toward source (NT2)
Away from source (NA3)
1.4 ± 0.1
2.0 ± 0.7
1.7 ± 0.2
2.0 ± 0.1Normalized probability of three subsequent movements in the
Toward source (NT3)
Away from source (NA4)
2.1 ± 0.1
3.7 ± 1.7
2.8 ± 0.7
2.8 ± 0.1Normalized probability of four subsequent movements in the
Toward source (NT4)4.8 ± 0.6 5.5 ± 2.1
The data from cell tracking are shown here as the probability that, in a 35-s interval, a cell will show displacement away from the rAprA source, toward the
source, or no displacement. Values are mean ± SEM from three independent experiments. *P < 0.05 indicates that the differences between control and rAprA
gradient conditions are significant; **P < 0.01 (t test). For the control, PAand PTare not significantly different, whereas in a gradient of rAprA, PAand PTare
significantly different with P < 0.01 (t test). For all normalized probabilities of subsequent movements in the same direction, differences between the control
and rAprA conditions are not significant (P > 0.05, t test).
| www.pnas.org/cgi/doi/10.1073/pnas.1206350109Phillips and Gomer
with this process by preventing proliferation at high cell density, Download full-text
and thus conserving local resources, giving high-density cells the
opportunity to spread before being depleted of nutrients and
Few endogenous chemorepellants have been identified, and
their mechanisms of action are largely unknown. We have identi-
fied an endogenous chemorepellant in D. discoideum and have
identified conserved proteins necessary for chemorepellant func-
Dictyostelium, a highly tractable model organism, has been im-
mensely useful in elucidating conserved chemotactic mechanisms.
its mechanism of action could shed light on conserved mechanisms
of chemorepulsion, perhaps providing insight or therapeutic
approaches for disease states in which chemorepellants play a role.
Materials and Methods
Cell Culture and Recombinant Protein Purification. The strains Ax2 (wild type),
aprA−[DBS0235509 (28)], cfaD−[DBS0302444 (30)], qkgA−[DBS0236839
(39)], bzpN−(32), gα8−[DBS0236107 (40)], plc−[DBS0236793 (26)], pi3k1−/2−
[DBS0236766 (41)], and Ax2/CRAC-GFP [DBS0235626 (36)] were grown in
axenic shaking culture as described previously (42) except that Formedium
HL5 media was used (Formedium). Recombinant AprA and CfaD were
expressed and purified from Escherichia coli as described previously (28, 30).
Insall Chamber Assay. TomeasuretheeffectofAprAoncelldisplacementusing
HL5 media, and then 300-μL volumes of the dilution were grown on 22- × 22-
gift from Robert Insall (Beatson Institute for Cancer Research, Glasgow, UK),
consisting of two concentric square depressions separated by a bridge (34).
Both depressions and the bridge were filled with HL5 media, and then the
mediawas removed from the coverslips,whichwerethenplaced facedown on
the chamber. Media was then removed from the outer chamber and was
replaced by either rAprA in HL5 or HL5 alone. Cells located on the bridge be-
tweenthe square depressions were then filmedusinga 10× objective. After an
initial 20-min period, the displacement of at least 10 individual cells per ex-
periment was measured over a period of 1 h. The displacement of cells in the
direction away from the rAprA source was then calculated by vector de-
composition. To analyze cell displacement in the absence of rich media, cells
weregrownoncoverslips for 1h asdescribed above,thenmediawas removed,
described above, except that PBM was used in place of HL5 for all steps.
For measurement of cell displacement at colony edges; under-agarose
assays; measurement of cell movement in uniform rAprA concentrations; cell
tracking; imaging of GFP fusion protein localization in live cells; and statistics,
see SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. Robert Insall for the gift of Insall
chambers, Dr. David Knecht for providing cells expressing the Lifeact-GFP
fusion protein, Alex Constantine for assistance with under-agarose and Insall
chamber assays, and Sarah Herlihy for comments on the manuscript. This
work was supported by National Institutes of Health Grant GM074990.
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Phillips and GomerPNAS
| July 3, 2012
| vol. 109
| no. 27