haploid saccharomyces cerevisiae yeast cells use a prototypic , g - protein coupled - receptor / map kinase cascade signaling system , the pheromone response system 1 , to sense and transmit information about the concentration of mating pheromone secreted by cells of the opposite mating type ( fig .
1 ) . the more information about pheromone concentration the system can transmit , the better a cell can distinguish between different pheromone concentrations , an essential ability for proper partner choice and mating . for example , a yeast cell ringed by potential mating partners strongly prefers to mate with partners producing the most pheromone 2 .
first , a cell grows up the pheromone concentration gradient 3 , a process that likely depends on measurement of precise differences in pheromone concentration at different points on the cell surface .
second , after contacting its partner and forming a prezygote , a cell preferentially completes fusion and forms a diploid with a partner that produces high amounts of pheromone 4 .
these experiments indicate it is important for cells to distinguish among different pheromone concentrations at multiple steps during the mating process .
prior work suggested that optimal transmission of information about pheromone concentration depends on both distinguishable receptor occupancies and distinguishable downstream system responses .
differences in receptor occupancy are clearly important for mating partner choice and discrimination ; for example , in the presence of exogenous pheromone at a concentration that saturates the receptor , cells lose the ability to discriminate high pheromone - secreting partners from low pheromone - secreting partners 2
. however , distinguishable receptor occupancies are not sufficient for partner discrimination , since hypersensitive cells , in the presence of exogenous pheromone at a concentration that does not saturate the receptor but does saturate downstream responses , also lose the ability to discriminate between partners secreting different levels of pheromone 2 . in complementary studies of orientation of mating projections in spatial gradients of pheromone ,
segall 3 showed that hypersensitive cells did not orient their mating projections as precisely as wild - type cells and suggested that this might result from saturation of downstream responses at most points in the gradient .
however , after reducing the gradient pheromone concentrations 100 fold to concentrations at which downstream responses are not predicted to be saturated , hypersensitive cells oriented their mating projections less precisely than wild - type cells orient in gradients of higher pheromone concentrations 3 .
these observations suggest that hypersensitive cells are inherently less able to respond distinguishably to different pheromone concentrations ( i.e. , transmit less information about pheromone concentration ) , even when they are responding to pheromone concentrations that saturate neither receptor nor downstream responses .
one characteristic of wild - type cells that we 5 and others 6 have previously found is that , despite the large number of intermediate signaling events in the system , the dose - response curve of receptor occupancy closely aligns with dose - responses curves of downstream system responses .
( here called dora ) between receptor occupancy and the amount of pheromone - activated ste12 ( pathway subsystem output p , which is reporter gene expression corrected for inherent cell - to - cell differences in the ability to express proteins 5 ) ( fig .
interestingly , dose - response alignment is commonly observed in many mammalian cell signaling systems , including the insulin 7 , acetylcholine 8 , thyroid stimulating hormone 9 , angiotensin ii 10 , and epidermal growth factor 11,12 response systems .
researchers in the past have often regarded alignment of curves for ligand binding by a candidate receptor and downstream response as evidence that the putative receptor was in fact the molecule that bound ligand and caused the cellular responses1315 . however , to our knowledge , researchers have investigated neither the implications of dose - response alignment for yeast pheromone response nor its general consequences for the function of cell signaling systems .
first , dora describes a linear relationship between receptor occupancy and downstream response ; consequently , the entire range of receptor occupancies evenly corresponds to the entire range of possible responses ( fig .
by contrast , even a modest dose - response misalignment , such as a 20-fold shift in the ec50 of downstream response ( fig .
2c ) , compresses the downstream responses corresponding to a wide range of receptor occupancies into a narrow range ( fig .
previous analysis of noise propagation in a synthetic gene circuit revealed analogous amplification of upstream noise in a system with misaligned dose - responses16 .
this reasoning suggested to us that cell signaling systems with misaligned dose - responses inherently transmit information with lower fidelity , even if downstream responses are not saturated , an idea consistent segall s observations that hypersensitive cells oriented mating projections less precisely in gradients than wild - type cells even at concentrations that did not saturate downstream responses 3 .
we hypothesized that dose - response alignment might indicate a system that can transmit large amounts of information , and therefore we sought to better understand the underlying molecular mechanisms required for dora and the linear relationship between upstream and downstream response that it defines .
proportional  negative feedback in electrical circuits , where a constant fraction of the output is subtracted from the input , can bring about a linear input - output voltage relationship17 .
biologists have also shown that negative feedback can make input - output relationships more linear in biological systems ; for example , bhalla et al . showed that , in a mapk / pkc - mediated signaling system , increasing the amount of a mapk - activated phosphatase that inactivates the mapk made the average output response more linearly related to ( i.e. , proportional to ) the input18 . research in both biology and engineering 17,1922 has also suggested or shown that negative feedback can increase the signal - to - noise ratio in system output and decrease the sensitivity of output to variation in properties of system components ( see supplementary information 8 for further discussion ) .
these observations suggested to us that negative feedback might mediate dose - response alignment and improve information transmission in the yeast pheromone response system .
prior work showed that the pheromone response system quickly establishes dose - response alignment ; the accumulation of reporter gene expression in cells increased linearly from 15 minutes to three hours after stimulation ( see fig . 2 in 5 ) , and at all times the normalized dose - response of downstream output aligned with the receptor - ligand binding curve .
these facts suggested that the molecular mechanisms that bring about and stabilize dora occurs in the first 15 minutes of pheromone stimulation .
however , no quantitative measurements of system activities in this time frame existed to indicate the action of negative feedback to align dose - responses .
we therefore developed tools to measure the early dynamics of molecular events that the system uses to operate before , during , and after establishment of dora .
we developed reporters and methods to measure real - time signal transmission in single cells , at the membrane , and in the nucleus , and supplemented these data with biochemical measurements .
we then measured system outputs ( i.e. , system activities at different stages in the signaling pathway , see fig .
1 ) after stimulating cells with 100 nm pheromone , a concentration that produces maximal downstream transcription reporter response ( fig .
two membrane - proximal system outputs , g - protein activation and ste5 recruitment to the membrane , peaked and declined rapidly . to follow g - protein activation in single cells over time , we measured loss of fluorescence resonance energy transfer ( fret ) between cyan fluorescent protein ( cfp)-tagged gpa1 and yellow fluorescent protein ( yfp)-tagged ste18 by image cytometry 23 ( supplementary information 2 ) in a derivative of a strain developed by yi and coworkers6 .
loss of g - protein fret rapidly peaked in the first minute and declined ( fig .
s2 ) , consistent with lower time resolution , single time point population measurements in an earlier study 6 .
we then measured , also in single cells and at sub - minute intervals , a subsequent membrane - proximal signaling event , the recruitment of ste5 to the membrane . to do this , we measured the redistribution of yfp - ste5 from the nucleus and cytosol to the membrane ( supplementary information 2 ) .
membrane recruitment of ste5 was rapid ( fig 3b ) . within 5 seconds of stimulation with high pheromone
, individual cells showed an increase in yellow fluorescence at the cell membrane , and a corresponding depletion of fluorescence from the cell interior ; no change in fluorescence was observed in unstimulated cells or cells with unlabeled ste5 ( fig .
average membrane recruitment reached near - maximal values within seconds and peaked by 20 seconds , before declining toward a plateau in later minutes , similar to the dynamics of g - protein loss of fret .
we then assessed intermediate system output further downstream by measuring the dynamics of mapk activation . using quantitative immunoblotting , we measured phosphorylation of fus3 residues thr180 and tyr182 , which is required for fus3 activity and pheromone response 24 .
the amount of phosphorylated fus3 relative to total fus3 increased rapidly , reaching a maximum in 2.5 min before dropping to a plateau level in approximately 57 min ( fig . 3c and fig .
we then measured nuclear mapk activity dynamics in single cells over time . to do this
, we developed a fret reporter to measure pheromone - induced changes in the association between the transcription factor ste12 and one of its inhibitors dig1 25 .
we deleted native ste12 and dig1 genes and chromosomally integrated versions of these proteins fused to cfp and yfp , respectively ( see supplementary information 5 ) .
we then measured changes in fret between cfp and yfp 26 in the nucleus by image cytometry 23 ( see fig .
s7b ) , but did require both ste5 ( fig s7c ) and map kinase activity ( fig s7d ) , consistent with the interpretation that loss of fret directly reported pheromone - induced , map kinase - mediated derepression of ste12 .
3d ) , and the overall signal dynamics were very similar to those of fus3 phosphorylation ( compare fig .
this fast signal transfer from fus3 activation to ste12 derepression is consistent with the idea that fus3 moves quickly in and out of the nucleus , as shown in studies of changes in fus3 localization by fluorescence recovery after photobleaching ( frap)27 .
we confirmed that the timing of ste12 de - repression measured by loss of ste12-dig1 fret was consistent with the dynamics of pheromone - induced mrna transcription . using ribonuclease protection assays
mrna levels peaked at 5 minutes following pheromone stimulation before declining ( fig . 3e and fig .
the maximum rate of increase in mrna occurred between 3 and 5 minutes , consistent with the time of maximum loss of ste12-dig1 fret .
all measurements of signal - relaying events showed a consistent pattern of rapid peak - and - decline toward a plateau after pheromone stimulation ( fig .
3f ) , which suggested the action of one or more fast - acting negative feedbacks that might modulate the dose - dependence of the signal to achieve dora .
a number of previous works suggested that the mapks fus3 and kss1 might mediate rapid negative feedback .
our previous study of regulated cell - to - cell variation in system output revealed a fus3-dependent reduction in variation , suggesting an autoregulatory negative feedback mediated by fus3 5 .
gartner et al . showed that levels of phosphorylated fus3 were higher in cells bearing a kinase - dead mutant version of fus324 .
showed that ste5 t287a mutant cells , in which the ste5 carries a lesion in a site of threonine phoshorylated by fus3 on peptides in vitro exhibited increased reporter expression 28 , albeit with no change in the ec50 of the dose - response . finally , phosphoproteomic studies of pheromone response system proteins29 have uncovered numerous sites of phosphorylation on pheromone response system proteins , whose levels change upon pheromone stimulation , many of which lie in consensus map kinase target sequences ( r. maxwell and o. resnekov , personal communication ) .
we therefore hypothesized that the signal decline at different measurement points depends on non - translational , fast - acting negative feedbacks mediated by fus3 or kss1 . to test
if fus3 or kss1 were sources of negative feedback on system activity , we compared the baseline system response , at system points up to and including fus3 phosphorylation , with system response after selective inhibition of either fus3 or kss1 kinase activity . to do this , we first modified reporter strains by replacing either fus3 or kss1 with the corresponding purine analog - sensitive allele 30 .
we did this by changing the  gatekeeper  residue in each kinase s atp binding pocket ( q93 in fus3 , n94 in kss1 ) to an alanine .
the mutant fus3-as2 and kss1-as2 kinases were active , as measured by fluorescent protein reporter gene output ( fig .
s9a ) , and10 m 1-nm - pp1 , a cell - permeable adenosine analogue , inhibited the activity of mutant kinases without inhibiting wild - type kinases ( fig .
we then quantified fus3 phosphorylation by quantitative immunoblotting after stimulation with pheromone , either with or without simultaneous inhibition with 1-nm - pp1 ( fig .
fus3 phosphorylation levels did not peak and decline to a plateau when we inhibited fus3-as2 , but , rather , remained high , near peak levels .
by contrast , when we inhibited kss1-as2 , fus3 phosphorylation levels were unaffected ( fig .
these results indicated that fus3 kinase activity mediated one or more negative feedbacks in this system .
we then studied where in the system the fus3-dependent feedback acted to diminish signal amplitude .
yi et al . showed that the decrease in g - protein fret within 30 seconds of stimulation depended on sst2 6 .
this finding suggested that the fus3-dependent negative feedback might upregulate the gtpase - activating protein ( gap ) function of sst2 , which would increase g protein reassociation and decrease downstream signal .
we tested if fus3-as2 inhibition affected the observed decline in both g - protein dissociation and ste5 recruitment .
4c ) , but surprisingly had no effect on the decline in g - protein dissociation in a g - protein fret reporter strain carrying fus3-as2 ( fig . 4d and fig .
these results indicated that fus3-mediated negative feedback acted downstream of mechanisms regulating g - protein association . to confirm that fus3 acted downstream of g - protein activation , we measured ste5 recruitment after deleting sst2 .
we expected deletion of sst2 to have no effect on fus3-mediated signal decline , since sst2 is required for efficient g - protein inactivation and , as we showed above , fus3-mediated negative feedback does not reduce g - protein dissociation levels .
unexpectedly , when we deleted of sst2 , we completely disrupted fus3-mediated signal decline ; unlike sst2 + cells , inhibition of fus3 did not cause an increase in ste5 recruitment ( fig .
furthermore , the ste5 recruitment ( with or without fus3-mediated feedback ) peaked and declined , similar to the baseline response of sst2 + cells ( compare squares and circles in fig .
this finding showed that signal peak - and - decline is the default behavior in the absence of sst2 . since a sustained non - declining signal is only evident in sst2 + cells in the presence of fus3-as2 inhibitor
, these results also indicate that sst2 promotes ste5 membrane recruitment , a hitherto unknown function of the rgs protein family , and that fus3 negatively regulates this novel signal - promoting function ( fig .
we then investigated which portions of the sst2 protein might be involved in promoting ste5 membrane recruitment . during analysis of sst2 point mutants
, we found that ste5 recruitment in a fus3-as2 strain that carried sst2-t134a instead of wild - type sst2 peaked - and - declined in the presence and absence of fus3 inhibitor ( fig .
the pheromone - induced growth - inhibition of sst2-t134a cells reported by halo assays was close to wild - type ( fig .
s11a ) , and the average number of sst2-t134a protein molecules per cell was similar to sst2 abundance in the parent strain , ( fig .
s11b ) , suggesting that the t134a mutation disrupted a significant fraction of the fus3-dependent , signal - promoting function of sst2 without disrupting the bulk of its signal - reducing gap activity .
t134 lies within the n - terminal dep domains of sst2 , which are required for localization of sst2 to the membrane by binding the cytosolic tail of ste2 31 .
these results indicate that the dep domains in sst2 might aid ste5 membrane recruitment , perhaps by providing additional membrane - proximal interaction surfaces , and suggest that mechanisms that regulate localization of sst2 to the membrane , such as disruption of sst2-ste2 interactions by yck1/2-mediated phosphorylation after longer periods of pheromone stimulation 31 , might consequently regulate ste5 membrane recruitment .
finally , we tested if dose - response alignment between receptor - pheromone binding and downstream activities required fus3 activity . in principle , fus3-mediated negative feedback might scale system activity by a dose - independent factor , and therefore cause no shift in the normalized dose - response curve .
for example , the ste5 t287a mutation increases the magnitude of system output relative to wild - type cells without changing the pheromone concentration yielding half - maximal response ( see fig . 5 in 28 ) .
we measured dose - responses of fus3 phosphorylation in a fus3-as2 strain with and without inhibitor 15 minutes after pheromone stimulation , the time when the amount of fus3 phosphorylation had declined to a steady - state level ( fig .
3c ) . inhibiting fus3 kinase activity shifted the dose - response of fus3 activation , lowering the pheromone concentration needed for half maximal response by 20-fold ( fig .
moreover , inhibiting fus3 kinase activity doubled the dynamic range of the output ( fig .
these results showed that fus3-mediated negative feedback was required for dose - response alignment in the yeast pheromone response system .
we found that mapk fus3 mediates rapid negative feedback that aligns the dose - responses of upstream and downstream system activities in the pheromone response system . we propose that dose - response alignment improves information transmission through this and other signaling systems .
furthermore , we found that fus3 negatively regulates a novel signal - promoting function of the rgs protein sst2 .
our results demonstrate that rgs proteins , present in many eukaryotic signaling systems ( the human rgs family , for example , contains more than 35 members 32 ) , can function in signal transduction systems by increasing signal in addition to accelerating g - protein inactivation , possibly ( as in the case of pheromone response ) by facilitating recruitment of mapk scaffolds to sites of activity .
the idea that dose - response alignment increases the amount of transmitted information has practical implications for drug discovery and design .
for example , consider a drug that increased sensitivity of cells to a naturally occurring antagonist of cell proliferation , analogous to the downstream dose - response shift we observed upon fus3 inhibition in the pheromone response system ( fig .
, the dose - response misalignment could reduce the amount of transmitted information about the signal . the decrease in transmitted information could increase cell - to - cell variation in response , causing a larger number of cells fall below a threshold in antagonist response and continue proliferation .
it is possible some existing drugs that allosterically modify gpcr signaling systems downstream of ligand binding ( see 33 , fig .
3 ) and those that target mid - system signaling molecules such as pkc 34 and akt 35 may decrease dose - response alignment and increase response variation , whereas drugs that specifically affect the affinity of receptor - ligand binding ( see 33 , fig .
we propose here that the fidelity with which a cell responds to different input concentrations of a ligand depends on a 
systems - level  quantitative behavior , dose - response alignment , found in many other cell signaling systems . for biological systems , a deeper understanding of key quantitative behaviors will likely depend on articulating appropriate analytical frameworks and metrics .
information theory36 defines a framework for quantifying the relationship between system input and output ( see supplementary information 9 for further discussion ) , and has enabled researchers to quantify , for example , the amount of information that an axon of a single sensory neuron can transmit 37 and the amount of information about morphogen gradient that a transcription factor can transmit to a downstream effector 38,39 .
much as concepts from classical electromagnetism provide rigorous means to describe and understand the determinants of behaviors of electrical circuits , we expect that concepts from information theory will enable more rigorous and quantitative understanding of how genes ( and the proteins they encode ) of more complicated signaling systems interact to sense and transmit information into the cell .
we constructed yeast strains and plasmids by standard methods 40,41 essentially as described ( 5 and supplementary information 1 ) . by doctrine
, we expressed all reporter constructs from native promoters integrated into the chromosome , and verified that the level of expressed protein was similar to the native level . with the exception of strains used for g - protein fret experiments , we constructed all strains were from otherwise- isogenic bar1- w303a reference parent strain , acl 379 5 , by the steps described .
we stimulated exponentially - growing cells with the indicated concentration of pheromone and/or other reagents ( such as the inhibitor 1-nm - pp1 ) in one of two ways .
for image cytometry , we affixed the cells to the bottom of wells in a glass - bottom 96-well plate , as described in 5 and in supplementary information 2.1 . using custom fluidic hardware , we evacuated medium from the well , injected fresh medium containing the indicated concentration of pheromone and/or inhibitor , and proceeded to record images over time . for mapk phosphorylation , fus1 mrna , and flow cytometry experiments , we stimulated cells by using a micropipette to mix a small volume of pheromone and/or inhibitor into the cell suspension to the final concentration ( as indicated , typically 100 nm pheromone and 10 m 1-nm - pp1 ) .
we performed image acquisition essentially as described in 5,23 , with modifications as detailed in supplementary information . for image cytometry , we extracted values for parameters of interest from images using cell - id 1.0 23 .
we analyzed image and flow cytometric data using physics analysis workstation ( paw ; see 42 ) and custom scripts , depending on the type of image , described in the text and in supplementary information .
supplementary information contains further details on plasmids , strains , construction methods , materials , and experimental methods .
 haploid saccharomyces cerevisiae yeast cells use a prototypic cell signaling system to transmit information about the extracellular concentration of mating pheromone secreted by potential mating partners . 
 the ability for cells to respond distinguishably to different pheromone concentrations depends on how much information about pheromone concentration the system can transmit . 
 here we show that the mapk fus3 mediates fast - acting negative feedback that adjusts the dose - response of downstream system response to match that of receptor - ligand binding . 
 this  dose - response alignment  , defined by a linear relationship between receptor occupancy and downstream response , can improve the fidelity of information transmission by making downstream responses corresponding to different receptor occupancies more distinguishable and reducing amplification of stochastic noise during signal transmission . 
 we also show that one target of the feedback is a novel signal - promoting function of the rgs protein sst2 . 
 our work suggests that negative feedback is a general mechanism used in signaling systems to align dose - responses and thereby increase the fidelity of information transmission . 