Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
1The screen versions of these slides have full details of copyright and acknowledgements
1
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal Ph.D.With Long Cai in the Elowitz Lab
Divisions of Biology and Applied PhysicsCalifornia Institute of Technology
2
Tracking Gene Expression
Elowitz & Leibler, Nature, 403, 335-338, 2000
3
Tracking Post-Translational Modifications
X X*
X-GFP Protein Fusion
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
2The screen versions of these slides have full details of copyright and acknowledgements
4
Calcium Signaling in YeastHigh Ca2+
High Na+ Elevated Temperature
Pheromone
Cna1 Cna2
Cnb1
Tpk1
Plasma Membrane
Calmodulin/[Ca2+]cyto Calcineurin
FK506
Crz1P
Crz1 Nmd5
Nuclear Membrane
Crz1Pho85Hrr25
Crz1PMsn5
Target Promoters
Cmd1Cmd1Ca2+
5
Calcium Controls Crz1 Localization
0 mM Extracellular Calcium
200 mM Extracellular Calcium
S288c MatA Crz1-GFP
6
Crz1 Localization Dynamics
S288c MatA Crz1-GFP, 150 mM extracellular calcium
Cells attached with ConA, 10 sec frame rate, 6 hours duration, played at 300x
Technical assistance from Kurt Thorn and Chin-Lin Guo
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
3The screen versions of these slides have full details of copyright and acknowledgements
7
Extracting Localization Traces
S288c MatA Crz1-GFP, 200 mM extracellular calcium, Software from Sharad Ramanathan
Frames displayed here are separated by 4.5 min, but actual time resolution is higher
8
Loca
lizat
ion
Initial Observations - Adaptation
9
τdelay
S288c MatA Crz1-GFP
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
4The screen versions of these slides have full details of copyright and acknowledgements
10
Initial Observations - Autocorrelation
S288c MatA Crz1-GFP, 200 mM extracellular calcium
N = 85 cells
11 S288c MatA Crz1-GFP
τcluster
12
Calcium Increases Crz1 Localization
w303a Crz1-GFP
10 mM Calcium 50 mM Calcium 100 mM Calcium
150 mM Calcium 200 mM Calcium
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
5The screen versions of these slides have full details of copyright and acknowledgements
13
Crz1 Localization is Frequency-Modulated
14
FM vs. AM Radio
http://bendeily.com/id67.html https://en.wikipedia.org/wiki/File:Amfm3-en-de.gif
15
AM
FM
Signal
FM vs. AM Localization
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
6The screen versions of these slides have full details of copyright and acknowledgements
16
Crz1 Encodes Signals via FM[C
a2+ ]
extra
cellu
lar
Loca
lizat
ion
time
time
17
Transcriptional Consequences of Crz1 Bursts
2X CDRE plasmid adapted from Martha Cyert
18
-60 -40 -20 0 20 40 60
-0.1-0.05
0.050.1
0.150.2
0 25 50 75 100 125 150
50
100
150200
250 Crz1 Cmk2
Crz
1 Lo
caliz
atio
n Cm
k2 Expression C
orre
latio
n
w303a Crz1-cherry, Cmk2-Venus, 300mM extracellular calcium
Consequences of Bursts
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
7The screen versions of these slides have full details of copyright and acknowledgements
19
Crz1
A B
Prom
oter
Inpu
t Fun
ctio
ns
[Crz1]nuc
BA
Consider Two Target Geneswith Different Input Functions
20
AM Regulation Would Produce Disproportional Expression
Loca
lizat
ion
Time
Low Ca2+
Medium Ca2+
High Ca2+
Loca
lizat
ion
Fraction of Time
Pro
mot
er In
put F
unct
ions
[Crz1]nuc
BA
Nor
mal
ized
Exp
ress
ion
[Ca2+]
Promoter APromoter B
21
FM Regulation Produces Proportional Expression
Loca
lizat
ion
Time
Low Ca2+
Medium Ca2+
High Ca2+
Loca
lizat
ion
Fraction of Time
Pro
mot
er In
put F
unct
ions
[Crz1]nuc
BA
Nor
mal
ized
Exp
ress
ion
[Ca2+]
Promoter APromoter B
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
8The screen versions of these slides have full details of copyright and acknowledgements
22
[Crz1]nuc
Crz1
A BBA
Pro
mot
er A
ctiv
ity(T
rans
crip
tion)
[Ca2+]
Nor
mal
ized
Exp
ress
ion
Promoter APromoter B
Nor
mal
ized
Exp
ress
ion
[Ca2+]
AM
FM
FM, but not AM, Causes Proportional Expression
Promoter APromoter B
23 Crz1-cherry, pCDRE(1-4X)-Venus, CDRE plasmids adapted from Martha Cyert
Synthetic Promoters
24
Natural Targets of Crz1
Yoshimoto, et al., JBC, 2002
• 163 Crz1 targets
• 83 in GFP library
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
9The screen versions of these slides have full details of copyright and acknowledgements
25
Natural Target Selection Criteria
S288c GFP strains, extracellular calcium, 1 ng/ml FK506
64/83 targets ‘visible’ by flow cytometry
40/64 targets passed FK506 test
Exp
ress
ion
leve
l (a.
u.)
1618202224262830
ARK1
0mM 200 200+FK506
400 400+FK506
0mM 200 200+FK506
400 400+FK506
CMK2
5
10
1520
25
30
Exp
ress
ion
leve
l (a.
u.)
26
10 Natural Targets
27
All Natural Genes
Nor
mal
ized
Exp
ress
ion
Extracellular Calcium (mM)
100 200 300 400- 0.20.20.40.60.81 YOR220W
100200 300 400
0.20.40.60.81 YLR414C
100 200 300 400-0.250.250.5
0.751
1.25 RVS161
1002003004000.20.40.60.8
1YLR194C
100 200 300 400-0.2
0.20.40.60.81 YPL233W
100200300400
0.20.40.60.81 YBR071W
100 200 300 400-0.20.20.40.60.8
1 YBR005W
1002003004000.20.40.60.8
1 YGR268C
100 200 300 400- 0.20.20.40.60.81 YJL016W
100 200 300 400-0.20.20.40.60.81 YHR138C
100 200 300 400
0.20.40.60.81 YLR327C
1002003004000.20.40.60.81
ENA1
100 200 300 4000.20.40.60.81 ENA5
1002003004000.20.40.60.81 ENA2
100 200 300 400-0.20.20.40.60.81 PMC1
100 200300 4000.20.40.60.81 BOP1
1002003004000.20.40.60.81 SOK2
100200300400
0.20.40.60.81 GYP7
100200300400
0.20.40.60.81 DOA1
100 200 300 400-0.4-0.20.20.40.60.81 PRB1
100 200 300 400-0.2
0.20.40.60.81 YPS1
100 200 300 400
0.20.40.60.81 PUT1
100 200 300 400-0.2
0.20.40.60.81 CMK2
100 200 300 4000.20.40.60.81 NPT1
100 200 300 4000.20.40.60.81 YHR097C
100200 300400
0.20.40.60.81 SCW10
100 200 300 400-0.2
0.20.40.60.81 MPD1
100 200 300 400-0.20.20.40.60.8
1 YOR385W
1
0.20.40.60.81
1.2
1002003004000.20.40.60.8
MEP1
100 200300 400- 0.20.20.40.60.81 YBR287W
100200 300400
YAR027W
100 200 300 4000.20.40.60.81
YLR350W
100 200 300400-0.20.20.40.60.81
YJL171C
100 200 300 400-0.20.20.40.60.81 COS1
100 200 300 400
0.20.40.60.81
APE2
100 200 300 400
-1- 0.5
0.51
ARO10
100 200 300 400-0.4-0.20.20.40.60.81
CUP2
100 200 300 400-0.4-0.20.20.40.60.81
MET14
100 200 300 400-0.2
0.20.40.60.81
MLF3
100 200 300 400-0.4-0.20.20.40.60.81
YDR319C
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
10The screen versions of these slides have full details of copyright and acknowledgements
28
‘Failed’ Natural Genes
Nor
mal
ized
Exp
ress
ion
Extracellular Calcium (mM)
0.25 100 200 300 400-
0.250.5
0.751
1.25 YPK1
100 200 300 400- 0.5
0.51
1.5 SAC6
100 200 300 400-0.5
-0.25
0.250.5
0.751 ARK1
100 200 300 400
0.20.40.60.81 ICL2
100 200 300 400- 0.4-0.20.20.40.60.81 MET3
100 200 300 400- 0.2
0.20.40.60.81 COS6
100 200 300 400-0.2
0.20.40.60.81
1.2 ERG26
100 200 300 400-0.2
0.20.40.60.81 YLR257W
100 200 300 400-0.2
0.20.40.60.81
YDL173W
100 200 300 400
0.20.40.60.81
TIS11
100 200 300 400-0.2
0.20.40.60.81
YDL010W
100 200 300 400- 1
12
YDR210W
100 200 300 400-0.2
0.20.40.60.81
PHM7
100 200 300 400- 0.2
0.20.40.60.81 KRE6
100 200 300 400- 0.2
0.20.40.60.81 YAL053W
100 200 300 400
-1-0.5
0.51 YOR273C
100 200 300 400-0.2
0.20.40.60.81 PLB3
100 200 300 400-0.2
0.20.40.60.81 DFG5
100 200 300 400-0.2
0.20.40.60.81 SUR1
100 200 300 400-0.2
0.20.40.60.81 CSG2
100 200 300 400
0.20.40.60.81 YML125C
100 200 300 400
0.20.40.60.81 YAR028W
100 200 300 400
0.20.40.60.81 PRM8
100 200 300 400-0.2
0.20.40.60.81
YIP3
29
10 Natural Targets
30
Overexpression Perturbation
[Crz1]nuc
Promoter A
Promoter B
Rel
ativ
e Fr
eque
ncy
Prom
oter Input Function
Over-express
Wild-type
Fold Change
His
togr
am
Fold Change
His
togr
am
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
11The screen versions of these slides have full details of copyright and acknowledgements
31
Overexpression Data
Crz1over plasmid provided by Kyle Cunningham
32
Natural Targets of Crz1
Yoshimoto, et al., JBC, 2002
• 163 Crz1 targets
• 83 in GFP library
33
0 25 50 100 150 200 300 4000 25 50 100 150 200 300 4000 25 50 100 150 200 300 400
Calcium-Induced Non-Crz1 Targets
708090
100
140150160170180190200
100110120130140150160
ACB1 YLR231CYLR354C
Expr
essi
on
Extracellular Calcium (mM)
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
12The screen versions of these slides have full details of copyright and acknowledgements
34
Calcium Signaling in YeastHigh Ca2+
High Na+ Elevated Temperature
Pheromone
Cna1 Cna2
Cnb1
Tpk1
Plasma Membrane
Calmodulin/[Ca2+]cyto Calcineurin
FK506
Crz1P
Crz1 Nmd5
Nuclear Membrane
Crz1Pho85Crz1
PMsn5
Target Promoters
Cmd1Cmd1Ca2+
Hrr25
35
Calcium Pulses in Mammalian Cells
Rooney et al., JBC 1989
36
Intracellular Calcium Measurements
CFP YFPCalmodulin
Ca2+
FRET
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
13The screen versions of these slides have full details of copyright and acknowledgements
37
Intracellular Calcium Measurements (2)
S288c MatA Crz1-GFP, 200mM Calcium, Cameleon (YC 2.12) plasmid provided by Jochen Stadler
38
Sample FRET Traces
w303a Crz1-cherry, pADH1-Cameleon (YC2.12) on plasmid; plasmid provided by Jochen Stadler
100 150 200 250 300
0.20.40.60.8
11.2
100 200 300 400 500 0
00.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
0.20.40.60.8
11.2
100 150 200 250 300 100 200 300 400 500
100 200 300 400 500100 150 200 250 300
100 150 200 250 300 100 200 300 400 500 100 200 300 400 500
100 150 200 250 300100 150 200 250 300100 150 200 250 300
39
Crz1 - Simplified Diagram
Crz1wt
Crz1High
Schematic
Time
Loca
lizat
ion
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
14The screen versions of these slides have full details of copyright and acknowledgements
40
Mutation: Calcineurin Binding Affinity
w303a Crz1-cherry, Crz1High-GFP; 100mM extracellular calcium; plasmid provided by Martha Cyert
Crz1High=*PVIAVN
41Hanson et al., Neuron, 1994
Hints from Other Systems
• Frequency decoding of calcium waves in mammals
• Amplitude-modulated calcium waves in fish
• Fertilization in starfish
• Frequency-modulated bursts such as in S. cerevisiaeare probably present in other organisms as well
42Crablab.stanford.edu
NFAT Signaling in Development and Disease
There are several other transcriptional regulators that exhibit similar behavior to Crz1-bursting
Dynamic Signal Encodingin the S. cerevisiae Calcium Response
Chiraj K. Dalal
15The screen versions of these slides have full details of copyright and acknowledgements
43
Acknowledgements
• Long Cai
• Kasra Rahbar
• Michael Elowitz
Martha Cyert, Stanford
Kyle Cunningham, Johns Hopkins
Jochen Stadler, University of Vienna
Chin-Lin Guo, Caltech
Sharad Ramanathan, Harvard
Kurt Thorn, UCSF
Tsui-Fen Chou, Caltech
Lauren LeBon, Caltech
Elowitz Lab and Friends
NIHPackard Foundation
44
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