Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Supplementaryfigures
10
A
B
100 µM ATP
100 µM ATP
10
8
8
6
6
4
4
2
2
10
C
8
Ratio (R/Ro)
YC3.60
D
10 µM ATP
10 µM ATP
10
8
6
6
4
4
2
2
10
E
8
F
1 µM ATP
1 µM ATP
10
8
6
6
4
4
2
2
10
G
8
H
0.5 µM ATP
0.5 µM ATP
10
8
6
6
4
4
2
1
2
1
0
20
40
Time (s)
60
0
20
40
Time (s)
60
Fig.S1.InDictyosteliumcells,cameleonYC2.60ismoresuitedtomeasureATP
mediatedchangesincytosolicCa2+thanYC3.60(A)FRETmeasurementsofchanges
incytosoliccalcium([Ca2+]c)withYC2.60(A,C,EandG)showthatitgenerateslarger
changesinratioandismoresensitivetosub-maximaldoesofATPcomparedto
YC3.60(B,D,FandH).ThemeanandS.E.M.oftheratiofromupto10aggregationcompetentAx2cellsareplottedineachpanelandtheresultsarerepresentativeof
atleast3independentexperiments.Whenachangeinratiowasobserved>95%of
thecellsrespondedwhereasinpanels(F)and(H)noneofthecellsresponded.
Biology Open • Supplementary information
YC2.60
Normalised Response
100
50
0
-8
-7
-6
-5
-4
100
50
0
-8
-3
-7
Normalised Response
Normalised Response
ATP (M)
100
50
0
Normalised Response
-11 -10 -9
-8 -7 -6
cAMP (M)
-5
-4
-6
-5
-4
ADP (M)
-3
-8
-7
DIF-1 (M)
-6
150
100
50
0
-10
-9
150
100
50
0
-10
-9
-8
-7
DIF-2 (M)
-6
Fig.S2.Dose-responsecurvesforATP,ADP,DIF-1,DIF-2andcAMPobtainedusing
aggregation-competentcells.Thepercentageofcellsrespondingateach
concentrationwhereasfollows.ATP100µM94%,30µM92%,10µM83%,5µM
87%,2.5µM77%,1µM84%,500nM68%,250nM63%,125nM17%,62.5nM5
%and31.25nM0%.ADP1mM84%,100µM84%,30µM95%,10µM96%,5µM
77%,2.5µM 93%,1µM89%,500nM 72%,250nM5%,125nM6%and62.5nM
11%.cAMP100µM93%,10µM100%,1µM100%,500nM82%,250nM97%,
100nM89%,10nM91%,1nM48%,100pM26%,50pM20%and10pM0%.
Biology Open • Supplementary information
Normalised Response
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
DIF21µM93%,500nM97%,250nM69%,100nM98%,50nM90%,25nM74%,
10nM56%,1nM50%,500pM33%and100pM47%.
DIF1250nM74%,100nM78%,50nM57%,25nM71%,10nM32%,5nM33%,1
nM22%,500pM50%and100pM0%.
DIF21µM93%,500nM97%,250nM69%,100nM98%,50nM90%,25nM74%,
Biology Open • Supplementary information
10nM56%,1nM50%,500pM33%and100pM47%.
C
40
60
10 µM ATP
10 µM ADP
10 µM cAMP
80
100
Time (s)
D
6
4
4
2
2
0
0
Biology Open • Supplementary information
20
A
x
M 2
sc
M S
sc
S
B
10
P
M
P
D
A
AT
P
AT
P
cA
µM
µM
30
µM
µM
A
A
x
M 2
sc
M S
sc
S
0
30
1
Stimulated Ca2+ uptake
(nmoles/mg protein)
6
A
x
M 2
sc
M S
sc
S
Stimulated Ca2+ uptake
(nmoles/mg protein)
6
A
x
M 2
cl
M N
cl
N
A
x
M 2
cl
M N
cl
N
6
A
x
M 2
cl
M N
cl
N
Stimulated Ca2+ uptake
(nmoles/mg protein)
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Ax2
MscS-/MclN-/TrpP-
4
2
0
Ax2
TrpP-
4
2
0
120
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Fig.S3.IdentificationofTrpPasthemediatorofATPstimulatedcalciumuptake
using45Ca2+.(A)AmscS-/mclN-/trpP-triplemutantwastestedforATP,ADPand
cAMPstimulatedcalciumuptakefor1minute.ThemutantandtheAx2parenthad
similaruptakesofCa2+stimulatedbycAMP,butthemutantrespondedmuchless
welltoATPandADPthanAx2cells.(B)ThedefectinATP-stimulatedCa2+uptakeis
theresultofablationoftrpP.TimecourseofATP(30µM)stimulatedCa2+uptakein
Ax2(opencircles)andtrpP-nullcells(solidcircles).ThemclA-andmscS-nullcells
accumulateCa2+tosimilarlevelswhenstimulatedwithATP,ADPandcAMP
comparedtoAx2(C,D).TwoindependentmclN-clones(C)andmscS-clones(D)are
shown.Uptakewasmeasuredfor1minuteafterstimulation.Allexperimentsused
aggregation-competentcellsandarerepresentative.Thebufferusedtomeasure
calciumuptakewasspikedwith45Ca2+anduptakemeasuredessentiallyasdescribed
Biology Open • Supplementary information
inTraynoretal.,(2000).
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Vegetative cells
TrpP-/TrpPOE
Ratio (R/Ro)
10
A
30 µM ATP B
30 µM ATP
10
8
8
6
6
4
4
2
1
2
1
C
8
100 nM DIF-1 D
100 nM DIF-1
8
6
6
4
4
2
1
2
1
E
8
100 µM Folate F
100 µM Folate
8
6
6
4
4
2
1
2
1
100
0
20
40
60
80
0
20
40
60
80
Time (s)
Fig.S4.CalciumresponsestoATP,DIF-1andfolatebyvegetativetrpP-nulland
trpP-/TrpP-complementedcells.VegetativecellsexpressingthecameleonYC2.60
FRETreporterfor[Ca2+]cwerestimulatedwithligandorbufferandtheratiometric
changesinfluorescencemeasured,witheachpanelshowingthemeanratio±SEM
(greybars)of6-12cells.Thedataisrepresentativeofatleast3independent
experiments.Thearrowindicateswhenthecompoundwasadded.
Biology Open • Supplementary information
TrpP-
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
5 µm
Fig.S5.LocalizationofTrpPproteininvegetativecells.ExpressionofaTrpP-GFP
fusionproteindrivenbythetrpPpromoterin trpP-cells.Atypicalcellisshown.The
toppanelsarefluorescenceimagesfrom3differenttimesandthebottompanelsare
thecorrespondingDICimages.TrpP-GFP(plasmidpDT68)ispresentcontinuously on
Biology Open • Supplementary information
theplasmamembrane,thoughcytoplasmicpunctaarealsoevident.
t
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
TrpP-/IplA-
TrpP10
A
10
30 µM ATP
8
8
6
6
4
4
2
2
10
C
D
30 µM ADP
10
10 µM cAMP
8
8
6
6
4
4
2
2
10
E
Buffer
10
F 5 µM Calmidazolium
8
8
6
6
4
4
2
1
2
1
100
0
20
40
60
80
0
20
40
60
80
Time (s)
Fig.S6.AdelayedcalciumresponseremainsintrpP-cellsanddependsonIplA.
AdditionofATP,ADPorbufferalonecansometimeselicitadelayedincreasein
[Ca2+]cintrpP-cells,whichdependsonIplA.(A,C,E)residualresponsesintrpP-null
cellstoATP,ADPortobufferalone;(B,D)abolitionofresponsestoATPandADPin
trpP-/iplA-doublenullcells;(F)controlshowingthattrpP-/iplA-doublenullcellscan
stillrespondtothecalmodulinantagonistcalmidazolium(5µM).ParentalAx2cells
respondsimilarlytocalmidazolium.Aggregation-competentcellsexpressingthe
cameleonYC2.60FRETreporterfor[Ca2+]cwerestimulatedwithligandorbufferand
theratiometricchangesinfluorescencemeasured,witheachpanelshowingthe
meanratio±SEM(greybars)of6-12cells.Thedataisrepresentativeofatleast3
independentexperiments.Thearrowindicateswhenthecompoundwasadded.
Biology Open • Supplementary information
Ratio (R/Ro)
B
30 µM ATP
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
IplA10
A
30 µM ATP
B
1 mM GABA
C
1 mM L-Glu
8
6
4
2
Ratio (R/Ro)
10
8
6
4
2
10
6
4
2
1
0
20
40
60
80
100
Time (s)
Fig.S7.GABAandL-Glutamateinduced[Ca2+]cresponsesaredependentonIplA.
(A)iplA-nullcellsproducethetypicalrapidresponseto30µMATP,whereas(B)1
mMGABAand,(C)1mML-Glutamatefailtoevokeanyresponse.Thearrows
indicatewhenthecompoundwasaddedtoaggregation-competentiplA-cells.All
thecellsanalysed(7)in(A)respondedwhereasnoneofthecells(6and7)responded
in(B)or(C).Theresultsarerepresentativeof3experiments.
Biology Open • Supplementary information
8
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
TrpP-
B
MlcE-
-0.60 s
6.96 s
6.00s
13.34 s
12.00s
16.82 s
24.00 s
35.96 s
36.00 s
64.38 s
66.00 s
∆
∆
-0.58 s
Biology Open • Supplementary information
A
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Fig.S8.ATPinducesblebbingintrpP-cellsandisdependentonthemyosin
essentiallightchain,MlcE.(A)ConfocalflourescenceandDICimagesofatrpP-cell
stimulatedwith30µMATPatt0.ThecellexpressestheF-actinreporterGFP-ABP120.
Blebsaremarkedwithwhitearrowheadswhereasactinscarsareindicatedbyred
arrowheads.Thefirstinfocusblebisobservedafter~14secondsandthelastblebs
areformedat~36secondspoststimulation.(B)ConfocalDICimagesofmlcE-null
cellsstimulatedwith100µMATPasin(A).Therearenoobservableblebsevenafter
66secondsofstimulation.Allcellsusedareaggregation-competentandinMKC
Biology Open • Supplementary information
buffer.Timesandthescalebars(10µm)areindicatedinwhite.
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
120
No Flow
100
80
80
60
60
40
40
20
20
y (µm)
y (µm)
100 A Ax2
0
-20
-20
-40
-60
-60
-80
-80
-100
-100
120
50
0
x (µm)
50
C TrpP-
100
-120
120
No Flow
100
80
80
60
60
40
40
20
20
y (µm)
y (µm)
100
100
0
-20
-60
-60
-80
-80
-100
-100
50
0
x (µm)
50
100
50
0
x (µm)
50
D TrpP-
100
Flow
-20
-40
100
100
0
-40
-120
Flow
0
-40
-120
B Ax2
-120
100
50
0
x (µm)
50
Velocity (µm min-1) ± s.e.m Directionality
Ax2
No flow
4.20 ± 0.28
0.18 ± 0.02
(n=33)
Ax2
Flow
5.70 ± 0.52
0.44 ± 0.06
(n=33)
TrpP- No flow
4.28 ± 0.20
0.20 ± 0.02
(n=28)
TrpP- Flow
4.68 ± 0.23
0.51 ± 0.05
(n=28)
Fig.S9.RheotaxisofvegetativeparentalAx2andmutanttrpP-nullcellsexposedto
asheerstressof3Pa.Cellstracksandmovementparametersaregiven.
100
Biology Open • Supplementary information
120
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Supplementarytables
TableS1.Theresponseofindividualcellsto30µMATPorbuffer
Ax2+30µMATP
Ax2+Buffer
TrpP-+30µMATP
TrpP-+Buffer
Fastresponse
NoResponse
LateResponse
115
0
0
0
5
55
95
88
5
5
21
10
Biology Open • Supplementary information
CalciumresponsestoATPorbufferaloneinindividualcellsweredetectedby
imagingwiththecameleonYC2.60FRETreporter.MostAx2cellsrespondrapidlyto
ATPasshowninFig.1ofthemaintext(‘fastresponse’)butTrpP-cellsneverrespond
inthisway.However,bothAx2andTrpP-cellsmoreoccasionallyproduceadelayed
responsetoATPortobuffer(‘lateresponse’)andsomedonotrespondatall(‘no
response’)astabulatedhere.ThefrequencyofthelateresponseinTrpP-cellswas
notstatisticallydifferentwithorwithoutATP(theproportionofrespondingcellswas
usedtogenerateaZ-scorewithatwo-tailedhypothesisgivingapvalueof0.101).
OnthisbasisitisnotnecessarytoinvoketheexistenceofafurtherATPreceptorto
explaintheseobservations.
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
TableS2.ChemotaxisofAx2parentalandtrpPnullcellstocAMP,withorwithouta
uniformconcentrationofATP,orunder0.4%agar
Speed
(µm/min)
Persistence
Chemotactic
index
N
Gradient
Uniform
Ax2
12.4±3.7
0.82±0.09
0.81±0.09
18
cAMP
TrpP-
12.6±2.1
0.82±0.08
0.81±0.06
20
0-1000nM
Ax2
12.7±4.9
0.70±0.29
0.65±0.25
16
cAMP
TrpP-
13.1±4.3
0.70±0.17
0.70±0.17
23
0-100nM
Ax2
11.5±4.9
0.65±0.13
0.72±0.14
15
cAMP
TrpP-
15.4±4.3
0.69±0.16
0.74±0.11
23
0-25nM
Ax2
15.0±5.8
0.63±0.17
0.68±0.18
17
cAMP
TrpP-
16.3±5.2
0.68±0.16
0.72±0.12
14
100-200
nM
Ax2
8.16±2.0
0.69±0.19
0.68±0.16
23
cAMP
ATP
TrpP-
7.64±2.2
0.71±0.18
0.74±0.13
20
0-100nM
50µM
Ax2
11.2±4.3
0.69±0.20
0.72±0.18
21
cAMP
ATP
TrpP-
13.8±3.3
0.73±0.12
0.75±0.13
27
0-100nM
100µM
Ax2
10.4±3.7
0.78±0.11
0.60±0.23
14
cAMP
under
TrpP-
8.42±1.48
0.67±0.15
0.51±0.2
11
0-4µM
agar
ChemotaxistocAMPwasdeterminedbytime-lapsefilmingofaggregationcompetentcellseitherunderbufferinaDunnchamber,orunderagaroseas
describedbyZatulovskiyetal,2014.Cellswereexposedtothestatedgradientsof
cAMP,withorwithouttheindicatedconcentrationofATPuniformlyinMKCbuffer.
Noneofthedifferencesbetweenparentandmutantinspeedorchemotacticindex
withinthesameexperimentweresignificant(two-tailedt-test)
Biology Open • Supplementary information
Strain
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
TableS3.RandommovementparametersofAx2parentalandtrpP-cellswithor
withoutauniformconcentrationofATP
Strain
Genotype
1
Ax2
2
HM1531 trpP-
3
Ax2
4
HM1532 trpP-
5
Ax2
6
HM1531 trpP-
7
Ax2
8
HM1531 trpP-
9
Ax2
10
HM1532 trpP-
11
Ax2
12
HM1531 trpP-
13
Ax2
14
HM1532 trpP-
Stage
Addition
wild-type veg
Speed
Persistence
(µm/min)
N
5.71±2.3
0.42
26
veg
6.12±1.7
0.70
27
wild-type veg
5.36±2.0
0.52
23
5.17±2.4
0.59
24
5.29±1.6
0.51
27
100µM 5.51±2.1
0.52
26
10.9±2.9
0.53
25
agg-comp
9.78±4.5
0.44
22
wild-type agg-comp
9.47±4.6
0.40
24
7.59±4.9
0.35
22
11.7±3.6
0.46
26
100µM 8.64±3.5
0.33
26
0.43
31
0.27
28
veg
wild-type veg
veg
ATP
wild-type agg-comp
agg-comp
wild-type agg-comp
agg-comp
wild-type agg-comp
agg-comp
ATP
ATP
11.3±3.7
100µM 7.56±3
Randomcellmovement(intheabsenceofchemoattractantgradients)was
determinedfromtime-lapsemoviesofvegetativeoraggregation-competent(vegor
agg-comp)cellseitherwithorwithoutauniformconcentrationof100µMATPinthe
medium.TwoindependenttrpP-cloneswereused.Thelackofdifferenceinspeed
betweenwild-typeandmutantcomesfromcomparisonsinthesameexperiment(eg
comparerows1&2)noneofwhichwerestatisticallysignificant(two-tailedt-test).
Whilethe(lackof)effectofATPonthespeedofthewild-typeAx2comesfrom
comparisonswithandwithoutATP(comparerows1&5;2&6;7&11;9&13),
noneofwhichweresignificant.
Biology Open • Supplementary information
No.
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
TableS4.GrowthofparentalAx2andtrpP-nullcellsinliquidmedium
Strain
Genotype
MGT(hr)
SEM
N
Ax2
wild-type
9.49
0.23
12
HM1531
trpP-
21.4
3.0
6
HM1532
trpP-
9.18
0.68
9
HM1578
trpP-
14.0
0.17
3
HM1579
trpP-
8.46
0.09
3
HM1822
trpP-
8.86
0.65
6
HM1823
trpP-
8.94
0.61
6
Biology Open • Supplementary information
CellsweregrowninHL5mediumat220Cwithshakingat180rpmandtheirnumbers
measuredtwicedailyusingaCoulterparticlecounter.Themeangenerationtime
(MGT)wasdeterminedduringlogphase.
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
Strain
Genotype
Parent
Source
Ax2(Kay)
Wild-type
HM1486
iplA-
Ax2(Kay)
thiswork
HM1691
gpbA-
Ax2(Kay)
P.Devreotes/J.
Borleis
HM1317
mscS-
Ax2(Kay)
thiswork
HM1449
mclN-
Ax2(Kay)
thiswork
HM1492
mscS-,mclN-,
trpP-
Ax2(Kay)
thiswork
HM1493
mscS-,mclN-,
trpP-
Ax2(Kay)
thiswork
HM1531
trpP- Ax2(Kay)
thiswork
HM1532
trpP-
Ax2(Kay)
thiswork
HM1578
trpP-
Ax2(Kay)
thiswork
HM1579
trpP-
Ax2(Kay)
thiswork
HM1822
trpP-
Ax2(Kay)
thiswork
HM1823
trpP-
Ax2(Kay)
thiswork
HM1578
trpP-,iplA-
HM1531
thiswork
HM1579
trpP-,iplA-
HM1531
thiswork
Biology Open • Supplementary information
TableS5.Strainsusedinthiswork
Biology Open (2017): doi:10.1242/bio.020685: Supplementary information
TableS6.Primersused
GGTGAAGGTGATGCTGAGACTGATGATG
CTTGTAAATTGTAAAGAACAATGTTGAC-3’
TAAGGGCCCAATGAATAGGTTGGGAGTGATAATACTGGATC
CTAGGGCCCTAACAGCTTCAAGTCTTTGAGAGAAAACTGTC
GTAGCGGCCGCAATGGTAGTATAAATGGTAGTAATCATATC
GTAGCGGCCGCAATGGTAGTATAAATGGTAGTAATCATATC
AAAGGATCCAAAGGATGAATACATTAAAGAGGACAGTTTTCTCTC
TTACTCGAGTAAAGGTGTATTAGTACCACCAGATAAATTGATATG
AATCTCGAGATGAGTAAAGGAGAAGAACTTTTCACTGGAG
AGAGTCGACTTATTTGTATAGTTCATCCATGCCATGTGTAATC
GACTTGAAGCTGTTAAAGCTTTAGAGAATAAAACCAG
CTGGTTTTATTCTCTAAAGCTTTAACAGCTTCAAGTC
TTTGTAGACGACGAACTAGAATGATTACATTCYC367
GGCTAGCATGACTGGTGGACAGCAAATGGGTCGCAATGG
GTGCGGCCGCAAGCTTGTCGACGACTAGTTTATTCAATGTTGTGTC
AAACTCGAGGACGAACTAGAATGATTACATTCACC
CATGGATCCTGAAGGTGTATTAGTACCACCAGATAAATTG
CACCTATTTGAGTGTATCCC
CAGTCTGTTTTTCTTCATCAGATG
CATCATGTTGTGATGGTGTTTGTACCTC
CAGAGATTAAATTAGAACCACTTGTGATAAC
CCAGTTTGTGCTTCAGTAGATGTC
GCAACAACAGTTGAAGCAGAACC
GTACTGTGAAGGAAAGATGAAAAGG
CCTATGGACCTTAGCGCTC
GAAGAAGATATTGCAATTCGTG
CCAGCATTTTGAATTTCACC
CTAGATGGGAGAGAACAATG
GTGATTGTGGCATACCAGTACTACC
Restrictionendonucleasesitesareinboldtext.
Biology Open • Supplementary information
PC2S26
PC2S27
PC2KO1
PC2KO2
PC2KO3
PC2KO4
PCL5
PCL3
RAGFP9
RAGFP10
PC2S47
PC2S48
PC2S37
YC367
YC368
PC2S69
PC2S70
cAR1F
cAR1R
ecmAF
ecmAR
pspAF
pspAR
rnlAF
rnlAR
spiAF
spiAR
trpPF
trpPR