Single Cell Calcium Measurements –

A Tool for Modeling and Revealing Heterogeneity of Cellular

ResponsesGrischa ChandyMicroscopy Lab - Stanford

Poster

4)     Single-Cell Calcium Measurements Reveals Heterogeneity of Ligand Induced Responses in RAW 264.7 Cells. Mary Verghese, Michal Ronen, Elizabeth Gehrig, Bob Sinkovits, Joelle Zavzavadjian, Leah Santat, Jamie Liu, Estelle Wall, Kavitha Dhandapani, Iain Fraser, Robert Rebres,Tamara Roach, and Grischa Chandy

Microtubules in RAW Cell

340 and 380 nm excitationSample 1/ second

Experimental protocol and time course3

40

/38

0 R

ati

o

Fura-2 LoadedCells

On Microscope510 nm emission

Experimental protocol and time course

Automatic cell detection

34

0/3

80

Rati

o

0

50

100

150

200

0 100 200 300 400 500 600 700 800

Time (s)

D [

Ca

2+

]

R44

R569

R23

Experimental protocol and time course

Stores release

Peak = max between ligand and 300 seconds

•Sustained = max between 240 and 480 or 600 seconds•Secondary Responses

Basal = average between 5 and 35 seconds

Thaps, Iono, EGTAManual ligand addition. Ligand is added 60 seconds after start of recording

UDP and C5a Response

Experimental Analysis

Gross Analysis - Average response- % Cells that respond

> 2 fold peak/basal-Slope based identification is more sensitive

- Histogram of Delta (Peak – Basal)- % of cells that had secondary activity- Ionomycin / Thapsigargin peak (Ca2+ store size)

Many other features all with frequency distributionsDelay times, rise times, decay rates, sustained levels, shape of

primary and secondary peaks, more

0

50

100

150

200

250

300

350

0 120 240 360 480 600 720Time (s)

[Ca

2+

] n

M

R3 (FMV040304002 10 uM UDP)

R217 (FMV040114007 100 nM c5a)

Time (s)

0

10

20

30

40

50

60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Fold (peak/basal)

% o

f c

ells

FMV040304002 (10 uM UDP)

FMV040114007 (100 nM C5a)

Responding Cells

Overlap

Fold (Peak/Basal)

[Ca

2+] i n

M

% o

f cells

Dose Response Curves

•Linear range of UDP dose response % of cells responding is between 1-100 nM (EC50% ~3 nM)

•Linear range of average response is between 3 and 1000 nM (EC50% ~80 nM).•Similar to macrophage labs reported value of 200 nM

•C5a has similar EC50% for activating delta Ca2+ and % of cells responding (Data on poster).

0

50

100

150

200

250

300

-14 -12 -10 -8 -6 -4

Ave

rag

e D

[C

a2+]

Am

pli

tud

e0

20

40

60

80

100

% o

f ce

lls

>2

fold

Delta Calcium

% cells

Avera

ge D

[C

a2

+]A

mp

litu

de

Log ([UDP] M)

% o

f ce

lls >

2 f

old

Buffer

0

50

100

150

200

250

300

0 120 240 360 480 600

Time (s)

D A

vera

ge

[Ca

2+]

nM

FMV040304001-7

Time (s)

D A

vera

ge [

Ca

2+]

nM

Oscillating Cells Averages and Examples of Single Cell Responses

•Higher doses of C5a in the absence of extracellular Ca2+

•Secondary responses continue in the absence of [Ca2+]o

•Found with lower doses of UDP (10-30% vs 0-1%)•Secondary responses are present in the absence of [Ca2+]o

0

20

40

60

80

100

120

140

160

180

200

0 120 240 360 480 600

Time (s)

[Ca2

+]

average

R152

R53

R131

FMV040304005 10 nM UDPFMV031125001

0

200

400

600

800

1000

1200

0 120 240 360 480 600 720 840 960

Time (s)

[Ca2

+]

average

R123

R111

Time (s) Time (s)

[Ca

2+]

nM

[Ca

2+]

nM300 nM C5a

2 mM EGTA

10 nM UDP

-25

25

75

125

175

0 120 240 360 480 600 720tim e (s)

D A

ve

rag

e [

Ca2+ ]

nM

FMV 040409003 (10 uM U D P )

FMV 040409004 (10 uM U D P +E GTA)

0

200

400

600

0 200 400 600

D [Ca2+] nM

Ma

x R

ele

as

e o

f s

tore

[C

a2

+ ] n

M

0

200

400

600

0 200 400 600

D [Ca2+] nM

Ma

x R

ele

as

e o

f s

tore

[C

a2

+ ] n

M

Relationship Between Initial Response and Store Depletion

UDP (N>2400) C5a (N>2300)

Pertubation?

D Ca2+ nM D Ca2+ nM

Sto

re [

Ca

2+]

nM

Sto

re [

Ca

2+]

nM

Time (s)

D A

vera

ge [

Ca

2+]

nM

+/- EGTA10 M UDP Ca2+

RNAi PerturbantsLentiviral Transduced shRNA Expressing Cells

3 Examples

-10

50

110

170

0 60 120 180 240 300Time (s)

D [

Ca

2+

] n

M

FMV040414002 (Control; N=283; 99%>2)

FMV040414010 (Galphaq; N=134; 28%>2)

Time (s)

D A

vera

ge[C

a2

+]

nM

Gq10 nM UDP

0

10

20

30

40

50

0 50 100 150 200 250 300 350 400D [Ca2+] Amplitude

% o

f c

ells

Control (N=283; 233 +/- 4 ; FMV040414002)

Galphaq (N=134; 51 +/- 6; FMV040414010)

100 nM UDP

IB: Gq

UGI2

P_In

f13

Grk

2_In

f13

Gq_

Inf1

3

75

50

37

Mol. Bio. Lab

-10

50

110

170

0 60 120 180 240 300

Time (s)

D A

vera

ge

[Ca2+

] nM

FMV040414004 (Control; N=302; 50.6%>2)FMV040414012 (Galphaq; N=133; 9.7%>2)

D A

vera

ge[C

a2

+]

nM

Time (s)

80% KD 70% KD

78% KD%

of

Cells

D [Ca2+] Amplitude

Gq - Examples of Single Cell Traces.10 nM UDP

•Though there is robust KD of Gq. Significant fraction of cells still have robust response to 100 nM UDP• Low Levels of Gq are sufficient for signaling• Alternate pathways (G11)• Other more complicated scenarios

100 nM UDP

-10

10

30

50

70

90

110

130

150

0 120 240 360 480 600

Time (s)

D A

vera

ge [C

a2+ ]

nM

R98R113R42R162FMV040414012 (Galphaq; N=133; 9.7%>2)

Time (s)

D A

vera

ge[

Ca2+

] n

M

-10

50

110

170

230

0 120 240 360 480 600

Time (s)

D [C

a2+]

nM

R135R94R13R39Galphaq (FMV040414010; N=134; 28%>2 )

Time (s)

D A

vera

ge[

Ca2+

] n

M

-10

0

10

20

30

40

50

60

70

0 120 240 360 480 600

Time (s)

D A

ve

rag

e [

Ca

2+

]

Galphai2 (58%>2; FMV040318012)

Control (20%>2; FMV040318014)

•Increase response of G2 knockdown cells readily apparent with 10 nM UDP but not seen with 1 uM.

0

10

20

30

40

50

60

D [Ca2+] Amplitude

% o

f C

ells

Contrtol (FMV040318014; 27 +/- 1; N=420)

Galphai 2 (FMV040318012; 81 +/- 3; N= 478)

Gi2 – 10 nM UDP

UGIP

Con

trol

PTEN

con

trol

Gi2

_shR

NA

Gi3

_shR

NA

IB: G alpha i2

Mol. Bio. Lab

3 fold enhancement 3 fold enhancement

D [Ca] Amplitude

% o

f C

ells

D A

vera

ge [

Ca

2+]

Time (s)

D A

vera

ge [

Ca

2+]

Arrestin3 – 100 nM C5a

-10

40

90

140

190

240

290

0 120 240 360 480 600

Time (s)

D A

vera

ge [

Ca2

+]

R262R199R259Arrestin3 (FMV040430003)

IB: Arr3

49

38

Mol. Bio. Lab

Lots of secondary peaks and oscillating cells

Control Arr3 Gb5

-10

0

10

20

30

40

50

60

70

0 120 240 360 480 600Time (s)

D A

vera

ge

[Ca2

+]

Control (FMV040430001)

Arrestin3 (FMV040430003)

Summary of Lentiviral mediated RNAi

•Calls and values based on Delta calcium and % of cells responding being more than 20% +/- of control

C5aR - 0.4* 0‡ 0.9‡

P2Y2R ++ 1.5* 0 0.8*

Gi2 ++ 2.1* + 1.3

Gq 0 1.2* - - 0.6

G2 - 0.0* 0.8‡

- 0.5* + 1.2

Arr3 ++ 1.3* ++ 1

Grk2 - /+  2.1* - 0.76*

PTEN - 1.5* - /+ 1

SHIP1 0 1.3* 0 1

IP3R2 + 0.9* 0 1.1

Single

C5a UDP or UTP‡

Pop.

Single

Pop.

Single Cell Confidence Level- = or + = one dose or data set0 no change-- or ++ = >1 dose or data set

Population Response ThresholdsElevated = >1.5No change = 0.7-1.4Decreased = <0.7* = p<0.05

Correlation between D Calcium and % respondingAll experiments pooled.

0

50

100

150

200

250

300

350

400

450

500

0 20 40 60 80 100

% of Cells >2 Fold

Ave

rag

e D

[Ca

2+] A

mp

litu

de

C5a

UDP

P2Y2Arr3

10 nM, Vector Control

10 nM, Gai2

•Average increased response due to additional cells being activated

•Average increased response due to increased response in each cell•Influx??

Calcium dynamics - Hypothesis

CCE/SOCNa/CaExchanger

R G PLC PIP2

DAG

IP3

IP3R

ER

C5a orUDP

Ca2+

ATPase

PKC

Ca2+

Oscillations

Arrestin

CCE/SOCNa/CaExchanger

R G PLC PIP2

DAG

IP3

IP3R

ER

UDP

Ca2+

ATPase

PKC

Ca2+

Sustained Ca2+

Arrestin

Calcium dynamics - Hypothesis

Summary

•Cells are heterogeneous•Single cells information is very different from averages•Possible differential dose dependent effects

•Unexpected shRNA phenotypes with low dose UDP

•We can provide detailed single cell data that can be used to constrain Ca2+ model (influx, efflux rates, frequency).

Verification Needed

•More experiments needed on controls – Error•Other perturbations (direct use of duplex, dominant negative and WT cDNAs)

Mary Verghese– Performed all experiments, ~200 uploaded to UCSDLiz GehrigNancy O’RourkeTobias Meyer

Molecular Biology Lab

Macrophage Lab

Michal Ronen – Data AnalysisBob Sinkovits – Bioinformatics Lab