11.02.14 - LCBG Journal Club

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


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


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


• 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?


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.”


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


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?

11.02.14 - LCBG Journal Club

Technology

Transcript of 11.02.14 - LCBG Journal Club