Modeling signal transductionEye as an evolutionary challengeEyes are present in most metazoan phyla: how did they evolve? The grand challenge:More broadly:Modeling molecular mechanisms of photo-transductionVertebrate Rods and ConesVertebrate photoreceptor cellIntracellular Voltage in Rods and ConesMeasuring currentsPatch clampSingle Photon ResponsePatch clamp recordingsElectrophysiology of RodsLight detector protein: Rhodopsincis/trans –retinal transitionLight-induced conformational transitionRhodopsin is a G-Protein coupled receptorRhodopsin is a G-Protein coupled receptorG- proteinsSteps in activation“Effector”Where did cGMP come from?G Protein Effectors IncludeShut-offRhodopsin inactivationTransducin is inactivated by the a intrinsic GTPase activity which hydrolyzes GTP to GDPRecovery to the resting state requires resynthesis of the cGMP that had been lost to hydrolysis. Ca- the second “2nd messenger”Negative feedback Signaling cascadePhototransduction Cascade as an enzymatic amplifier Olfaction:Olfactory receptor cascade Invertebrate Phototransduction Cascade Enzymatic amplifier modulesMore examples of amp modulesGeneral “push-pull” amplifier circuitAmplifier gain and time constantLinear analysis of vertebrate phototransductionLinear analysis of the cascade“Hard” and “Soft” parametersGeneral behaviorCase of feedbackConsequences of negative feedbackSignal and Noise: How much gain is enough?Reaction shot noiseChannel opening noisecGMP fluctuationsLocality of single photon responseSingle photon response variabilityWhat is the largest source of response variability?Multistep deactivation of Rh*Meta-Rhodopsin shut-off:Summary:AcknowledgementsPhototransduction from frogs to fliesModeling signal transductionEye as an evolutionary challenge“To suppose that the eye, with all its inimitable contrivances …, could have been formed by natural selection, seems, I freely confess, absurd in the highest possible degree. Yet reason tells me, … “C. Darwin, The Origin of Species:Darwin goes on to describe how eye may have evolvedthrough accumulation of gradual improvementsA number of different designs exist: e.g. vertebrate, molluskan or jellyfish “camera” eyes or insect “compound eye”The eye may have evolved independently 20 times!!(read “Cells, Embryos and Evolution” by Gerhart&Kirschner)Copepod's 2 lens telescopeTrilobite fossil 500 million yearsBlack antHouse FlyScallopOctopusCuttlefishSee Gerhartand Kirschener, “Cells, Embryos and Evolution”Eyes are present in most metazoan phyla: how did they evolve?The grand challenge:What can we learn from the similarities and differencesin the architecture/anatomy, development and molecular machinery of eyes (or light sensitive cells) indifferent species?To understand the evolutionary processes that underlie the appearance of a complex organ (an eye).To what extent is the similarity between different eyes (e.g. vertebrate and mollusk) is due to common ancestry or is the result of evolutionary convergence due to physical constraints?BUT before we can address the question of evolutionwe need first to learn how it works…More broadly:Molecular pathway(s) of phototransductionare similar to many other signaling pathways:olfaction, taste, etc“Comparative Systems Biology”Modeling molecular mechanisms of photo-transductionCase studies: 1) vertebrate and 2) insectPhototransduction as a model signaling systemVertebrate Rods and ConesVertebrate photoreceptor cellhvLight reduces the“dark current” into the cellNa+Na+, Ca2+100μmRodOuterSegment(2000Discs)DiscsRhodopsinTurtle ConeTurtle Rod: a flash response family shown on different time scalesIntracellular Voltage in Rods and ConesMeasuring currentsPatch clampSingle Photon ResponsePatch clamp recordingsKKNa, CaNa, CaKKKKNaNaNaNacGMPCa,KCa,KKKcGMPKIn the dark a standing current circulates through the rod.Light shuts off the influx of current into the outer segment, while K continues to exit the innersegment causing the cellto hyperpolarize.Na,Ca channels of outer segment need cGMP to stay openElectrophysiology of RodsATP drivenNa/K pumpRhodopsinundergoes light-induced conformational changeopsin, a membrane proteinwith 7 transmembranesegmentschromophore, 11-cis retinal+Light detector protein: Rhodopsincis/trans –retinal transitioncytoplasmic sideLight-induced conformational transitionRhodopsin is a G-Protein coupled receptorβ2adrenergic receptorRhodopsincytoplasmic endSeven-HelixG-protein-CoupledReceptorsform a large class(5% C. elegansGenome !)RhodopsinlightRh*TαβγGDPTα∗+ TβγGTPabout 109rhodopsinmolecules and about108 G-protein moleculesper rod outer segment (ROS)photoactivated rhodopsin(Rh*) forms in about 1 msand serially activates 100 to1000 Transducin moleculesper secondTransducin is a heterotrimericG proteinspecific to visionRhodopsin is a G-Protein coupled receptorγβαguanine nucleotide binding site onα subunitα subunit: 35-45 kDβ subunit: 35-40 kDγ subunit: 6-12 kDAt least 15 different genes for α subunit and several differentgenes for β and γ subunitsG- proteinsextracellularcell membraneγβαGDPG protein coupledreceptor (GPCR)Resting state:(G protein off)γβαGDPreceptor activated by action of external signalBinding of activated receptor to G proteinopens guanine nucleotidebinding pocket onα subunit 1.Steps in activationThe α and βγ subunits dissociate from activatedGPCRβγGDPαGTPγβα*GTP2.GDP -- GTP exchange at nucleotidebinding site activates the G proteinγβα*GTP3.Activated α subunitstimulates effectorproteins: enzymes or ion channelsRhodopsinlightRh*Tαβγ-GDP Tα∗-GTP + TβγPDE PDE*cGMP GMPCNG Channels: OPEN CLOSED“Effector”Guanylate CyclaseGTP cGMP + PPi GMPPhosphodiesteraseWhere did cGMP come from?Enzymes:Phosphodiesterase (PDE)Adenylyl cyclasePhospholipase Chydrolyzes cyclic nucleotide; e.g. cGMP GMP synthesizes cAMPfrom ATPhydrolyzes PIP2to produceIP3and diacylglycerol (DAG)Ion channels:e.g. K, Ca and Na channelsG Protein Effectors IncludeRhodopsinlightRh*Tαβγ-GDP Tα∗-GTP + TβγPDE PDE*cGMP GMPCNG Channels: OPEN CLOSEDNext question:How are the activated intermediates shut off?Shut-offRhodopsin inactivationγβα*GTPγβαGDP Pi+In TimeγβαGDPTα and βγre-associateTα-GDPdissociates from PDE*which re-inhibits PDE*Transducin is inactivated by the α intrinsic GTPaseactivity which hydrolyzes GTP to GDPAccelerated by PDE*γβα*GTPThe intrinsic GTPase