November 4 th , 2014 Farhoud Faraji Kent Hunter, PhD
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November 4th, 2014
Farhoud Faraji Kent Hunter, PhD
Agenda
1. Background2. System Overview3. System Validation4. Characterization of Ras/Erk module properties5. System application
Ras/Erk Signaling “Module”
• Activated by many extracellular signals
• Can induce diverse consequences– Proliferation– Differentiation– Cell cycle arrest– Etc.
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
Definition•Genetic encoding of light-sensitive proteins that activate signaling pathways in response to lightFirst application •Optical manipulation of neuronal membrane potential with channel rhodopsins
– Induce/suppress action potentials
Optogenetics
Beyond Channel Rhodopsins: The Optogenetic Toolbox
Control the activity of signaling systems in living organisms in real time.
High Spatial Resolution •only limited by resolution of light source
High Temporal Resolution •can turn on/off within seconds
Light
Light
Light
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.
Agenda
1. Background2. System Overview3. System Validation4. Characterization of Ras/Erk module properties5. System application
Engineering Optogenetic Control of Ras
Addition of CAAX prenylation motif to Phy ensures membrane localization
650nm – PIF-Phy associate750nm – PIF-Phy dissociate
INPUT ReadoutYFP-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
Agenda
1. Background2. System Overview3. System Validation4. Characterization of Ras/Erk module properties5. System application
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.
Opto-SOS activation Induces Hallmark Ras-mediated Biochemical Responses
PC
12 c
ells
NIH
3T3
ells
Response is MAPK pathway specific
Suggests Ras activation is not sufficient to activate PI3K.
NIH
3T3
ells
Response is Reversible
PC
12 c
ells
Opto-SOS activation induces Hallmark Ras-mediated Physiological Responses
Serum Starved
Kinetics and Levels of Opto-SOS and nuclear Erk do not depend on prior stimuli
Agenda
1. Background2. System Overview3. System Validation4. Characterization of Ras/Erk module properties5. System application
• 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
Previous Studies of Cell Signaling Systems…
Major advantage of Opto-SOS systemAbility to quantitatively analyze input/output relationships in the isolated Ras/Erk moduleon a single cell scale!
Experimental Design:•Applied light doses in random order. •Returned to each dose multiple times.
How precisely can steady-state signals be transmitted through the Ras/Erk pathway?
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
Cell-cell variability due to “variability in the expression level of various relevant [endogenous] molecular components.”
How precisely can steady-state signals be transmitted through the Ras/Erk pathway?
Intra-population variability persists within clonally derived lines•Variability not a result of optogenetic component genomic integration site or expression level.
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 amplitudePhase – delay in output oscillation relative to input
• 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
Types of Dynamic Filters
Selectivity
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 dramaticallyLow pass feature prevents responses to stochastic events
Instead of stimulating cells with one frequency at a time, applied a fluctuating input that simultaneously contains information at many frequencies
SOS-
to-E
RK G
ain
(%)
Experimental observations are consistent with outcomes of a model of a second-order linear low-
pass filter with cut-off frequency of 2mHz
Ras/Erk demonstrates Low Pass FilteringGain – begins to drop off at 4 minPhase – 3 min from opto-SOS activation to nuclear ErkA 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
Hypothesis: High bandwidth signal transmission implicates existence of downstream decoding mechanisms of dynamic signals
The Ras/Erk Module is a High-Bandwidth, Low-Pass Filter
Gai
n
Agenda
1. Background2. System Overview3. System Validation4. Characterization of Ras/Erk module properties5. System application
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.
Class 1: Responsive to PDGF but not Ras
RTKs: EGFR, HER2 (Abs may cross-
react with PDGFR) AKT, JNK, SRC, YAP
Long and Short
MAPK members: ERK1/2, MEK1, P90RSK
mTOR signaling (upstream) mTOR, GSK3Surprising: PKCβ (Ras-PLC crosstalk?)
Class 2: Responsive to PDGF AND transient AND sustained Ras
LongClass 3: Responsive to PDGF AND sustained Ras
mTORC1/2 signaling (downstream)Rictor, p70S6K, S6, NDRG1
SNAILSTAT3 – only after 1 hr
Pursuit of a Particularly Puzzling PathwaypSTAT3 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
Surprisingly, no autocrine signaling was observed •Opto-SOS induced pSTAT3 upon IL-6 but not light stimulation
Light induces pSTAT3 specifically in wt3T3 and only when they are cocultured with opto-SOS 3T3
Paracrine Model
Paracrine signaling requires translation and is pErk Dependent
Translational inhibition prevents pSTAT3 induction
MEK1 inhibition prevents pSTAT3 induction
Direct Demonstration of Paracrine Signaling by Media Transfer from Light-Stimulated Cells
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.
Mechanism of the Erk-STAT3 Circuit: A Paracrine loop via GP130 receptor and LIF ligand
Conditioned Media:
Erk-STAT3 Circuit is a Persistence Detector: 2-hour sustained pulse is necessary to induce
pSTAT3
SummaryOptogenetic activation of Ras via opto-SOS provided precise characterization of Ras/Erk module signaling properties1.Individual cells display precise analog sensitivity
– Can tune pathway amplitude in response to varying input amplitudes
SummaryThe 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
3.Is a Low pass filter– Suppresses signals shorter
than 4 minutes in duration
4.First direct evidence supporting dynamic filtering of signal transduction.
SummaryOptogenetic activation of Ras via opto-SOS efficiently identified previously characterized and novel Ras effector programs.
Thank you!
Questions?
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