BE/APh161 – Physical Biology of the Cell!Rob Phillips Applied Physics and Bioengineering California 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.SprE OmpR EnvZ PhoB PhoR CusR CusS QseB QseC YedW YedV YfJR YfhK BasR BasS PhoP PhoQ CpxR CpxA CheY UvrY BarA HydG HydD BaeR BaeS FimZ animation by Mark Gouilan Coordinating multiple signaling systems in a single cell!Phosphotransfer profiling!(use complete set of purified RRs) HK + ATP* HK~P* + ADP - RR1 RR2 RR44 ......... RR3 incubate, separate by SDS-PAGE HK~P* + RR HK + RR~P* HK~P RR~PPhosphotransfer profiling!(see Skerker, Laub et al..) Figure 4. Phosphotransfer Profiling Method(A) Phosphotransfer profile experiments involve three separate reactions: (1) autophosphorylation of the histidine kinase (HK) by radiolabeled ATP, (2)phosphotransfer to a response regulator (RR), and (3) dephosphorylation of the response regulator.(B) Schematic of the phosphotransfer profiling technique. A single preparation of purified, autophosphorylated kinase (HK;32P) is mixed with eachresponse regulator from a given organism and analyzed for phosphotransfer by SDS-PAGE and autoradiography. The first lane shows a single bandcorresponding to the autophosphorylated histidine kinase and is used as a comparison for every other lane. Lanes 2–4 illustrate the three possibleoutcomes of a phosphotransfer reaction. In lane 2, phosphotransfer from HK to RR1 leads to the appearance of a band corresponding to RR1. In lane 3,phosphotransfer from HK to RR2 also occurs, but owing to high phosphatase activity (either autophosphatase or catalyzed by a bifunctional HK), thenet result is production of inorganic phosphate (Pi) and the depletion of radiolabel from both the HK and RR2. In lane 4, no phosphotransfer occurs, andthe lane is indistinguishable from lane 1.(C–H) Phosphotransfer profiling was performed for three E. coli kinases (EnvZ, CheA, and CpxA) against all 32 purified E. coli response regulators, withphosphotransfer incubation times of either 1 h (C, E, and G) or 10 s (D, F, and H). For these three histidine kinases, a comparison of the short and longtime point profiles indicates a kinetic preference for only their in vivo cognate regulators: OmpR (C and D), CheY and CheB (E and F), and CpxR (G andH). After being examined for phosphotransfer, all gels are stained with Coomassie to verify equal loading of histidine kinase and response regulator ineach lane (data not shown). For each kinase profiled, we purified only its soluble, cytoplasmic domain, either as a thioredoxin-His6or a His6-MBP fusion,using standard metal affinity chromatography (see Materials and Methods). When necessary, we made successive N-terminal truncations until weidentified a construct that produced active kinase in vitro, always preserving the H-box and ATP binding domain (details on constructs used are in TableS3). All response regulators were purified as full-length fusions to a thioredoxin-His6tag. Purity was assessed by Coomassie staining, with each purifiedkinase domain and response regulator, except for E. coli FimZ, yielding an intense band of the correct approximate molecular weight (see Figure S5;Table S3).DOI: 10.1371/journal.pbio.0030334.g004PLoS Biology | www.plosbiology.org October 2005 | Volume 3 | Issue 10 | e3341777Systematic Analysis of Two-Component SignalingAssessing Specificity: Phosphotransfer Profiling!+PhoB C. 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 recognition C. crescentus PhoR profile – 5 min phosphotransfer reactions +PhoBSignal 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 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 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 swimming!(see Turner, Ryu, Berg – J. Bacteriol. 2000 .) • See the movies from Howard Berg website.A 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 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 circuit!• The circuit for bacterial chemotaxis involves precisely the kind of two-component signal transduction system that we discussed earlier.Cellular 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. ABFig. 1. (A) Schematic view of the experimental appa-ratus. We modified an inverted Zeiss microscope toperform FCS measurements on individual cells. The cellwas specifically attached by its flagella onto a micro-scope slide. A 0.5-!m latex bead (Polyscience), at-tached to a flagellum with rabbit antibodies to flagellin,is used as a marker to visualize a free rotating flagellum. The CW bias was computed as the ratio of the time spent in CW to the total time duration.The FCS technique allowed us to measure GFP-tagged protein concentration in the same bacterium. The fluctuations of the total fluorescence intensitywere processed in real time by a correlator (ALV-5000/E) that provided an autocorrelation function (14). CCD,
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