Review of Cell Press Article titled: "Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module." Jared Toettcher, Orion Weiner, Wendell Lim Cell. 2013 Dec 5;155(6):1422-34. doi: 10.1016/j.cell.2013.11.004.

1. Farhoud Faraji Kent Hunter, PhD
November 4th, 2014

2. Agenda
1. Background
2. System Overview
3. System Validation
4. Characterization of Ras/Erk module properties
5. System application

3. Ras/Erk Signaling “Module”
• Activated by many
extracellular signals
• Can induce diverse
consequences
– Proliferation
– Differentiation
– Cell cycle arrest
– Etc.

4. When a shared internal node is activated,
how does a cell know which response to initiate?
Two different external stimuli activating same internal node…
1.Also induce other stimulus–specific pathways (digital bypass)
2.With different temporal patterns of activation (analog sensing)
– decoded by downstream modules to yield distinct
responses.
Current pathway dissection tools (small molecules, RNAi) do a poor
job of assessing signaling network dynamics.
•Low temporal resolution
•Not easily tunable

5. Optogenetics
Definition
•Genetic encoding of light-sensitive proteins that
activate signaling pathways in response to light
First application
•Optical manipulation of neuronal membrane potential
with channel rhodopsins
– Induce/suppress action potentials

6. Beyond Channel Rhodopsins: The Optogenetic Toolbox
High Spatial Resolution
•only limited by resolution
of light source
High Temporal Resolution
•can turn on/off within
seconds
Light
Light
Light
Control the activity of signaling systems in living organisms in real
time.

7. Question
Can optogenetics be used to deconvolute complex
and branched intracellular signaling systems?
Use light to directly manipulate a signaling sub-network and reveal its dynamic properties.

8. Agenda
1. Background
2. System Overview
3. System Validation
4. Characterization of Ras/Erk module properties
5. System application

9. Engineering Optogenetic Control of Ras
Addition of CAAX
prenylation motif to
Phy ensures membrane
localization
650nm – PIF-Phy associate
750nm – PIF-Phy dissociate
INPUT Readout
YFP-Opto-SOS enables
tracking membrane
localization
Ras activation by SOScat is highly
dependent on membrane recruitment
BFP-Erk
enables
tracking
nuclear
localization
OUTPUT:
Nuclear Erk

10. Agenda
1. Background
2. System Overview
3. System Validation
4. Characterization of Ras/Erk module properties
5. System application

11. Light stimulation induces membrane
localization of Erk
NIH3T3 cells
750nm reversed Erk nuclear
localization within minutes
Opto-SOS induces
similar magnitude of
nuclear Erk as PDGF
Activation is dependent on PhyB chromophore Phycocyanobilin.

12. Opto-SOS activation Induces Hallmark
Ras-mediated Biochemical Responses
PC12 cells NIH3T3 ells
NIH3T3 ells
Response is Reversible
PC12 cells
Response is MAPK pathway specific
Suggests Ras activation is not sufficient to activate PI3K.

13. Opto-SOS activation induces Hallmark
Ras-mediated Physiological Responses
Serum Starved

14. Kinetics and Levels of Opto-SOS and nuclear Erk
do not depend on prior stimuli

15. Agenda
1. Background
2. System Overview
3. System Validation
4. Characterization of Ras/Erk module properties
5. System application

16. Previous Studies of Cell Signaling Systems…
• Suggest cell-signaling is noisy
– Limited analog sensitivity - cannot alter output in
response to graded changes in input stimulus
• Measured bulk sample (population averages)
– Could not separate noise from intrinsic cell-to-cell
variability
Major advantage of Opto-SOS
system
Ability to quantitatively
analyze input/output
relationships in the
isolated Ras/Erk module
on a single cell scale!

17. How precisely can steady-state signals be
transmitted through the Ras/Erk pathway?
Experimental Design:
•Applied light doses in random order.
•Returned to each dose multiple times.

18. How precisely can steady-state signals be
transmitted through the Ras/Erk pathway?
Pooling data from just 25 cells
shows high variability
Single cells demonstrate high dose-response precision

19. How precisely can steady-state signals be
transmitted through the Ras/Erk pathway?
Cell-cell variability due to “variability in
the expression level of various relevant
[endogenous] molecular components.”
Intra-population variability persists within clonally derived lines
•Variability not a result of optogenetic component genomic integration site
or expression level.

20. Observed Single Cell Dose-Response Precision Enables
Quantitative analysis of Ras/Erk signal transmission
Frequency response analysis: the quantitative measure of
the output spectrum of a system in response to a stimulus
‒ Characterizes the dynamic response of the system:
Gain – ratio of output to input amplitude
Phase – delay in output oscillation relative to input

21. Types of Dynamic Filters
• Band-pass filter – responds best to a specific input
frequency or pulse length
• Low-pass filter – transmits low frequency signals
– Suppress noisy signals above a cutoff frequency
• All pass filter – faithfully transmits all inputs
Selectivity

22. Instead of stimulating cells with one frequency at a
time, applied a fluctuating input that simultaneously
contains information at many frequencies
SOS-to-ERK Gain (%)
Ras/Erk Demonstrates High Bandwidth Signal Transmission
2 hr to 4 min – frequency-response curve is flat (~100% gain)
EGF – single or periodic 15 min pulses
PDGF – >1 hr sustained Erk activation
< 4 min – Erk transmission efficiency drops dramatically
Low pass feature prevents responses to stochastic events

23. Experimental observations are consistent with
outcomes of a model of a second-order linear low-pass
filter with cut-off frequency of 2mHz

24. Ras/Erk demonstrates Low Pass Filtering
Gain – begins to drop off at 4 min
Phase – 3 min from opto-SOS activation to nuclear Erk
A system capable of rejecting inputs shorter than a cutoff time must:
(1) delay its response at least as long as the cutoff time
(2) dissipate inputs of this timescale so as not to initiate a
response

25. The Ras/Erk Module is a High-Bandwidth,
Low-Pass Filter
Hypothesis:
High bandwidth signal
transmission implicates
existence of
downstream decoding
mechanisms of dynamic
signals
Gain

26. Agenda
1. Background
2. System Overview
3. System Validation
4. Characterization of Ras/Erk module properties
5. System application

27. Does Decoding of Dynamic Signals Occur
Downstream of the Ras/Erk Module?
180 antibody
probes represented
27/180 proteins
assayed showed
response to PDGF
or optogenetic Ras
activation.

28. Class 1: Responsive to PDGF but not Ras
RTKs:
EGFR, HER2 (Abs may cross-react
with PDGFR)
AKT, JNK, SRC, YAP

29. Class 2: Responsive to PDGF AND transient AND
Long and Short
sustained Ras
MAPK members:
ERK1/2, MEK1, P90RSK
mTOR signaling (upstream)
mTOR, GSK3
Surprising: PKCβ (Ras-PLC crosstalk?)

30. Class 3: Responsive to PDGF AND sustained Ras
Long
mTORC1/2 signaling (downstream)
Rictor, p70S6K, S6, NDRG1
SNAIL
STAT3 – only after 1 hr

31. Pursuit of a Particularly Puzzling Pathway
pSTAT3 only observed under sustained Ras activation
•Known to be activated by
– IL-6 via JAK signaling
– Src - Activated only upon PDGF, but not light,
stimulation
Hypothesis:
Prolonged Ras activation induces
autocrine/paracrine release of
IL-6 family cytokines to activate
STAT3

32. Light induces pSTAT3 specifically in wt3T3 and
only when they are cocultured with opto-SOS 3T3
Surprisingly, no autocrine signaling was observed
•Opto-SOS induced pSTAT3 upon IL-6 but not light
stimulation

33. Paracrine Model

34. Paracrine signaling requires translation and is pErk
Dependent
Translational inhibition prevents pSTAT3 induction
MEK1 inhibition prevents pSTAT3 induction

35. Direct Demonstration of Paracrine Signaling by Media
Transfer from Light-Stimulated Cells

36. Dissecting the components of the Erk-STAT3 Circuit
Neutralizing antibody
against IL6R (GP130)
blocks light-activated
pSTAT3 in wt3T3.
Neutralizing antibody
against IL6 shows no
effect.

37. Mechanism of the Erk-STAT3 Circuit:
A Paracrine loop via GP130 receptor and LIF ligand
Conditioned Media:

38. Erk-STAT3 Circuit is a Persistence Detector: 2-
hour sustained pulse is necessary to induce
pSTAT3

39. Summary
Optogenetic activation of Ras via opto-SOS
provided precise characterization of Ras/Erk
module signaling properties
1.Individual cells display precise analog
sensitivity
– Can tune pathway amplitude in response to
varying input amplitudes

40. Summary
The Ras/Erk signaling module:
2.Is a High bandwidth channel
– Capable of responding across a large range of
timescales, transducing dynamic information
about a broad range of response programs
2.Is a Low pass filter
– Suppresses signals shorter
than 4 minutes in duration
4.First direct evidence
supporting dynamic filtering
of signal transduction.

41. Summary
Optogenetic activation of Ras via opto-SOS
efficiently identified previously characterized and
novel Ras effector programs.

42. Thank you!
Questions?