dynamic signal encoding in the s. cerevisiae calcium ... · dynamic signal encoding in the s....

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


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


Chiraj K. Dalal


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


Chiraj K. Dalal


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


Chiraj K. Dalal


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


Chiraj K. Dalal


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


Chiraj K. Dalal


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


Chiraj K. Dalal


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+]



Chiraj K. Dalal


22

[Crz1]nuc

Crz1

A BBA

Pro

mot

er A

ctiv

ity(T

rans

crip

tion)

[Ca2+]

Nor

mal

ized

Exp

ress

ion


Nor

mal

ized

Exp

ress

ion

[Ca2+]

AM

FM

FM, but not AM, Causes Proportional Expression


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


Chiraj K. Dalal


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


Chiraj K. Dalal


28

‘Failed’ Natural Genes

Nor

mal

ized

Exp

ress

ion


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


Chiraj K. Dalal


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



Chiraj K. Dalal


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


Chiraj K. Dalal


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


Chiraj K. Dalal


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


Chiraj K. Dalal


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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dynamic signal encoding in the s. cerevisiae calcium ... · dynamic signal encoding in the s....

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