Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Cells Decide: Where to GoCell Motility and Actin PolymerizationHow Is Polymerization Controlled in Space and Time?The Control of Actin Polymerization at the Leading EdgeSignaling and PolymerizationComputing the Activity of the SwitchTuning Tethers to Alter SignalingBE/APh161 – Physical Biology of the CellBE/APh161 – Physical Biology of the CellRob PhillipsApplied Physics and BioengineeringCalifornia Institute of TechnologyTwo-Component Signal Transduction• Next few slides are courtesy of Michael Laub (MIT) and Mark Goulian (Upenn) – experts in the quantitative dissection of signaling networks.• This figure shows the generic features of the two-component signal transduction systems.SprEOmpREnvZPhoBPhoRCusRCusSDcuRDcuSNarLNarXNarPNarQKdpEKdpDQseBQseCYedWYedVYfJRYfhKYehTYehURcsBRcsCOmpRArcBRstARstBUhpCUhpBTorRTorSBasRBasSPhoPPhoQCpxRCpxACheYCheAAtoCAtoSUvrYBarAHydGHydDBaeRBaeSCheBRcsDNtrCNtrBFimZCreBCreCDpiBDpiAanimation by Mark GouilanYpdBYpdAEvgAEvgSCoordinating multiple signaling systems in a single cellPhosphotransfer profiling(use complete set of purified RRs)HK + ATP* HK~P* + ADP-RR1 RR2 RR44.........RR3incubate,separate bySDS-PAGEHK~P* + RR HK + RR~P*HK~PRR~PPhosphotransfer profiling(see Skerker, Laub et al..)Assessing Specificity: Phosphotransfer Profiling+PhoBC. crescentus PhoR profile – 60 min phosphotransfer reactions histidine kinases exhibit a strong kinetic preference in vitro for their in vivo cognate substrate specificity based on molecular recognitionC. crescentus PhoR profile – 5 min phosphotransfer reactions+PhoBSignal integrationSignal integration• Once we finish with our concrete example of chemotaxis, we will turn to the way in which cells decide where to put new actin filament and that will make us face this question of signal integration.• Cell as a computer: not just amplification, but also logical operations such as AND, OR, etc.Cellular decisions and swimmingCellular decisions and swimming• We have already seen the strategy of neutrophils for chasing down cellular offenders.• Bacteria also exhibit motile strategies based upon environmental cues.Cellular decisions and swimmingCellular decisions and swimming• We have already seen the strategy of neutrophils for chasing down cellular offenders.• Bacteria also exhibit motile strategies based upon environmental cues.Cellular decisions and swimmingCellular decisions and swimming(see Turner, Ryu, Berg – J. Bacteriol. 2000 .)• See the movies from Howard Berg website.A general view of bacterial chemotaxisA general view of bacterial chemotaxis• We will return to this figure several times in order to clarify various aspects of the bacterial chemotactic response.An amazing molecular machineAn amazing molecular machine• The flagellar motor uses a proton gradient to rotate at roughly the angular speed of a jet engine. • This motor is a darling of the creationists as an example of something that “couldn’t have evolved” because of its supposed “irreducible complexity”.Bacterial chemotaxis circuitBacterial chemotaxis circuit• The circuit for bacterial chemotaxis involves precisely the kind of two-component signal transduction system that we discussed earlier.Cellular decisions and swimmingCellular decisions and swimming(see Cluzel, Surrette and Leibler, Science.)• Concept of experiment: tether cell to surface and watch the rotation of the motor. They use a second color to watch the CheY-P in the cell. The result is a plot that shows the frequency of motor tumble rate as a function of CheY-P.Cellular decisions and swimmingCellular decisions and swimming(see Cluzel, Surrette and Leibler, Science.)• Measure the response of the motor as a function of the concentration of CheY-P.• Clockwise bias (CW) is obtained by taking fraction of time spent in clockwise state divided by the total time.The fret techniqueThe fret technique• Fluorescence resonance energy transfer is an amazing molecular ruler.• Proximity of acceptor and donor is readout by the efficiency of the transfer. https://netfiles.uiuc.edu/tjha/www/images/FRET_concept.jpgFret and chemotaxisFret and chemotaxis• FRET between CheY-P and CheZ.• This is from the paper of Sourjik and Berg, PNAS, 2002. They have two papers in PNAS that year, this is the first one.Changes in interactions between labeled proteins observed by FRET on chemotactic stimulation of E. coli cellsSourjik V, Berg H C PNAS 2002;99:12669-12674©2002 by The National Academy of Sciences• The circuit for bacterial chemotaxis involves precisely the kind of two-component signal transduction system that we discussed earlier.Receptor activity and chemoattractant concentrationReceptor activity and chemoattractant concentration• Measure the FRET activity for different chemoattractant concentrations.• Different mutants have different adaptive responses.Motor Binding and BiasMotor Binding and BiasBuilding a statistical mechanics modelBuilding a statistical mechanics model• Our goal is to find the probability that the receptor will be in the on state as a function of the chemoattractant concentration.States and weights States and weights • Each of the states has a corresponding statistical weight.Changes in FliM occupancy after flash-release of caged chemoeffectorsSourjik V, Berg H C PNAS 2002;99:12669-12674©2002 by The National Academy of Sciences• After addition of a chemoattractant, the FRET measurements permit direct determination of the kinetics of the process.Adaptation of the circuitAdaptation of the circuit• Methylation provides a scheme for tuning the energy difference between the on and off states of the receptor complex.• Quantitative measurements leave us with many interesting questions: mutants and their responses, sharpness of response, adaptation, motor binding vs switching, etc…Parkinson lab - UtahHigh resolution imaging of chemotaxis proteinsHigh resolution imaging of chemotaxis proteins• PALM is a high-resolution technique that permits beating the diffraction limit.•In this case, they are looking at chemotaxis proteins in E. coli.(Greenfield, Liphardt et al, PLoS Biology, 2009.)Nature of the receptorsNature of the receptors• Receptors assemble into trimers of dimers.• Receptor
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