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28 February 2018
Push-pull signal control operates deeper within the yeast PRS

In the yeast pheromone response system, a year ago, Colman-Lerner and coworkers (Bush et al. 2016) published an elegant set of experiments showing that the physical interaction between the unliganded Ste2 receptor and the Sst2 RGS protein constituted the pull element of a push-pull mechanism (with the interaction between the receptor and the G protein the push element).  This mechanism presumably operates in other GPCR signaling systems.  The consequence of this mechanism is that the fraction of occupied G protein mirrors the fraction of occupied receptor, rendering G protein activation robust to cell-cell variation in receptor abundance and allowing dose-response alignment at the level of the G protein.

Last month, in beautiful work, Winters and Pryciak (2018) demonstrated a push-pull mechanism operating deeper in the signaling chain, at the level of the system MAPKs, Fus3 and Kss1.  In cells with both MAPKs, activated Fus3 stimulates PRS-induced gene expression while inactivated Kss1 inhibits it.  We imagine that push-pull should make fractional induction robust to correlated cell-cell variation in the amount of Fus3 and Kss1 and thus help bring about dose-response alignment in single cells and cell populations.

Their work strengthens the case that our previous assumption that alignment likely resulted from the closed-loop feedback control that would naturally have been employed by human designers (Yu et al. 2008) may have been wrong.

It is easy to see how, by allowing more precise and coherent responses in cell populations, both robustness to cell-cell variation in protein levels and dose response alignment should confer selective advantage during evolution.  In retrospect however, it would not be surprising if evolved signaling systems manifested coherent responses due to control mechanisms distinctly inferior to those that would be built by sentient engineers.

12 December 2016
Lab joins 21st century, posts work on bioRxiv

We've posted two major articles by Gustavo Pesce (et al.) on the bioRxiv preprint server. These describe how Gus screened more than 1000 yeast strains deficient in different nonessential genes for those that affected cell-cell variation in signal transmitted through the pheromone response system, how different data suggest that the origin of the noise they introduce is due to the ends of cytoplasmic microtubules interacting with the site on inside of the cell membrane at which signal is generated, and how that noise degrades the accuracy of a cell fate decision. We will describe the papers further when formally published. These papers have now been under editorial review at Molecular Systems Biology for 2 months. They represent revised versions of a single manuscript we originally submitted to that journal in July 2015. We are also cross-publishing and doi-ing these Pesce et al. papers via our FHCRC archives.

C. Gustavo Pesce, William Peria, Stefan Zdraljevic, Dan Rockwell, Richard C. Yu, Alejandro Colman-Lerner, Roger Brent (2016). Cell-to-cell variability in the yeast pheromone response: high throughput screen identifies genes with different effects on transmitted signal and response. bioRxiv 093187; doi: 

C. Gustavo Pesce, Stefan Zdraljevic, Alan Bush, Victoria Repetto, William Peria, Richard C. Yu, Alejandro Colman-Lerner, Roger Brent (2016). Cell-to-cell variability in the yeast pheromone response: Cytoplasmic microtubule function stabilizes signal generation and promotes accurate fate choice. bioRxiv 093195; doi

23 November 2016
Commentary on Andrews et al. by Weiße, Mannan, Oyarzún.

Description/ recapitulation and commentary on Andrews et al. Push-Pull paper in this issue of Cell Systems by Diego Oyarzún and coworkers at Imperial College London. Authors argue for the importance of thinking about many kinds of systems in terms of input-output relationships. They also make a point that illustrates how critical precise definition will be for systems biology to progress beyond its current state, sometimes characterized by slogans and buzzwords, to generate important new scientific knowledge. The authors note that because definitions of output differ among studies, the phrase "linear input-output relationship" covers a great deal of ground, and that serious attempts to infer general truths about "design principles" from studies of different systems will need to take such differences into account.

Weiße, A. Y, Mannan, A. A., and Oyarzún, D. A. (2016) Signaling tug-of-war delivers the whole message. Cell Systems 3, 415-416

27 October 2016 (epub) and 23 November 2016 (pub)
Andrews et al. Push-Pull now out/ up/ published in Cell Systems

In this paper, we asked how cell signaling systems, including the yeast pheromone response system, might exhibit ‘‘Dose-Response Alignment’’ (DoRA), in which output of one or more downstream steps closely matches the fraction of occupied receptors, and preserve this relationship throughout changes in the number of protein components. Systems with DoRA transmit information optimally. We found a non-feedback based, non-closed loop mechanism, Push-Pull, in which the nominally inactive form of upstream components actually reduces downstream activity, can generate DoRA. Although key aspects of the open-loop control enabled by Push-Pull are inferior to the closed-loop feedback control that would have been used by human (or other sentient) engineers, Push-Pull seems to be widespread throughout evolved eukaryotic signaling systems (even plants). Our work suggests means to detect when Push-Pull and closed-loop feedback control might be operating, and means by which synthetic closed-loop feedback control in signaling systems might be engineered.

Andrews, S., Peria, W., Yu. R,. C., Colman-Lerner, A., and Brent, R. (2016). Feedback and push-pull mechanisms can align signaling system outputs with inputs. Cell Systems 3 (5): 444-455.e2

22 September 2016
More from the vaults

We've published and doi-ed via FHCRC archives a number of previously unavailable papers from the lab.  The first (Ptashne et al. 2003) is a review on modular transcription activators from ergito.com, a now-defunct web textbook.  The second (Finley et al. 2003) is our original paper on a Drosophila gene and protein we called Cdi4, which interacted with Cdk2 and Cyclin E, and now called decapo.  The reasons we were unable to publish that work contemporaneously have to do with the occasional failure of peer review when the reviewers are untenured faculty members who view their work as competitive. 

Ptashne, M., Gill, G. and Brent, R.  (2003).   Modularity of eukaryotic transcription activators. doi: 10.6076/J7RN35SF

Finley, R. L. Jr., Cohen, B. and Brent, R. (2003) Drosophila Cdi4 is a p21/p27/p57-like cyclin-dependent kinase inhibitor with specificity for cyclin E complexes.  doi:10.6076/J7WD3XHS12

June 2016
ISAC/ CTYO 2016 meeting in Seattle

At this meeting Brent gave a "State of the art lecture" titled "Quantitative physiology of living single-cells: subtle pleasures and future promise".  The hope was to give the listeners a heads up on how one can use single cell measurements to define some non-obvious physiological states (including having high gene expression power or G) in yeast and worm cells, and the consequences of those states.

Colman-Lerner, A., Gordon, A., Serra, E., Chin, T., Resnekov, O., Endy, D., Pesce, G. and Brent, R. (2005) Regulated cell-to-cell variation in a cell fate decision system. Nature. 437, 699-706

15 July 2016
New controller design from Khammash lab

We know from our unpublished work that consideration of early 20th century control systems (Mindell 2002) has been helpful in understanding how evolved cell signaling systems like the yeast PRS operate.  We are now testing a related idea, that collaboration with living 21st century control engineers at ETHZ may help us learn more about evolved systems and build better artificial ones.  Work from our collaborator Mustafa Khammash illustrates the promise of such engagement.  In a just published paper (Briat et al. 2016a), Khammash and his coworkers show a new and simple design for an integral controller that gives robust "perfect adaptation", but at the price of sensitivity to stochastic intracellular events.  This result stands in contrast with a second recently articulated design from the Khammash lab (Briat et al. 2016b), whose workings are robust to intracellular stochastic events, and our lab is seeking to understand this difference in natural language terms.

Mindell, D.A. (2002). Between Human and Machine (Baltimore: Johns Hopkins University Press).

Briat, C., Zechner, C. and Khammash, M (2016a).  Design of a Synthetic Integral Feedback Circuit: Dynamic Analysis and DNA Implementation.  ACS Synth Biol. Article ASAP 2016 Jul 8. PMID: 27345033 DOI: 10.1021/acssynbio.6b00014

Briat, C., Gupta, A., and Khammash, M. (2016b). Antithetic Integral Feedback Ensures Robust Perfect Adaptation in Noisy Biomolecular Networks.  Cell Systems 2(1): 15-26. doi: 10.1016/j.cels.2016.01.004.

6 June 2016
Second phasor plotting paper published

Our second paper with the Houston lab at NMSU, on use of phasors and pseudophasors in flow cytometry, is now out.   In time resolved flow cytometry, we expect these methods will be as powerful as they are in microscopy. 

Cao R, Jenkins P, Peria W, Sands B, Naivar M, Brent R, Houston JP.  Opt Express. 2016 Jun 27;24(13):14596-607. doi: 10.1364/OE.24.014596. PMID: 27410612

31 March 2016
Mendenhall starts own lab at UW

Alex Mendenhall starts a tenure track faculty appointment in the Department of Pathology at UW School of Medicine.  The department is distinguished by the depth of its expertise on aging, making it something of a dream job for him. Will link to his lab website as soon as its up.  Mendenhall did very well here, and he will be deeply missed.

15 February 2016
Single cell physiological states and aging

Alex Mendenhall, Monica Driscoll, and Brent have a closely argued piece out in Aging Cell about how scientists can use single-cell measures of physiological state can contribute to an understanding of organismic aging (and response to perturbations) in adult organisms.  A major reason for this article was to put forward, particularly for aging researchers, but also for researchers seeking to understanding organismic physiology, logically consistent and eventually computable definitions of concepts such as "physiological state"  We come down pretty strongly on the viewpoint that to view aging as the execution of a "genetic program" in the same sense that embryonic development clearly is the working out of genetic program. We hope to provoke discussions about and lay groundwork for measurement of single cell physiological variables through the entire developmental and adult trajectory of organisms.  We also hope to give the aging community some new ideas to accept or criticize.

Mendenhall A, Driscoll M, Brent R.  Using measures of single-cell physiology and physiological state to understand organismic aging.  Aging Cell. 2016 Feb;15(1):4-13. doi: 10.1111/acel.12424. Epub 2015 Nov 29. Review. PMID: 26616110

4 January 2016
post-Cohen-Boyer DNA assembly now out

Overview of post Cohen-Boyer methods for single segment cloning and for multisegment DNA assembly.  Sands B, Brent R.  Curr Protoc Mol Biol. 2016 Jan;113:3.26.1-3.26.20. Epub 2016 Jan 4. PMID: 27152131

With the overview now published, the plan is to publish Bryan's latest assembly methods as a separate paper later in 2016

11 September 2015
21st century DNA assembly methods.

Much of the research at the Hutch and elsewhere relies on new DNA constructions.   Since the early 1970s, researchers have continued to use "classical" means to construct DNA molecules, but they have also developed additional powerful means to make complex molecules of desired sequence.  In many cases, researchers rely on kits made by commercial manufacturers that use methods the researchers do not understand. A forthcoming paper in Current Protocols in Molecular Biology by Bryan Sands and Roger Brent describes and explains the most important methods, including the improved assembly methods based on recombination in yeast developed here.

20 July 2015
Derek Britain to graduate school in Biophysics at UCSF

Derek M. Britain, the most junior lab member, will be graduating from the lab to go enter graduate school in Biophysics at UCSF. Derek joined the lab as an undergraduate in the UW bioengineering program and then stayed on as a full time researcher. Derek's work here was on the response of single yeast cells to mating pheromone. He demonstrated effects of particular allelic variants of microtubule plus end binding proteins on cell-to-cell variation in signal transmitted through the system. The work may be important because these variants occur in homologs of these proteins found in members of the human population, and so may contribute to human disease.

1 July 2015
M. Allen Northrup joins lab

Allen Northrup, a distinguished microfluidics engineer and successful biotech executive, has joined the lab as a visiting scientist. Allen (see bio) is one of the pioneers of microfluidics and among other things is responsible for the first devices that carried out PCR in micromachined silicon. He plans work that may improve the ability of researchers and diagnosticians to detect and quantify rare molecules in dirty samples.

24 - 28 June 2015
C. elegans international meeting

At UCLA, Alexander Mendenhall, now out of stealth mode, presented his results on single cell quantification at the annual worm meeting.

Results included studies of effects of configuration of different reporter gene elements total measured fluorescence. This work is important because it provides the necessary foundation for the lab's ongoing studies of variation and physiological state.

7 May 2015
Mendenhall et al. paper now out/ live animal single-cell quantification for all

A paper by Alexander Mendenhall and other lab members is now published in PLOS ONE. The paper tells researchers how to quantify with great accuracy reporter gene expression in living single cells in adult Caenorhabditis elegans. Lab researchers view development of these quantification methods as the foundation on which much future work worldwide will depend. The work opens the way to understanding the origin of non-genetic variation in living animals-- the "neither" in the triad sometimes termed "nature, nurture, or neither". It will also allow study of now-mysterious physiological states that can affect lifespan and other outcomes.

March 2015
Phasors work for discriminating populations in flow cytometry too.

In work with the lab, Roufan Cao and Jessica Perea Houston at NMSU have shown that phasor plots can distinguish populations of cells in flow cytometry based on fluorescent lifetime. Experiments continue.

21 March 2015
yeastpheromonemodel.org wiki passes 150,000+ accesses, 50 full downloads.

More than 10 years ago, Ty Thomson, then a graduate student with collaborator Drew Endy, was working with the lab on the Alpha project, an effort to make a comprehensive predictive model of the yeast pheromone response system. For his PhD thesis (http://dspace.mit.edu/handle/1721.1/45206), Ty carried out a full review of the literature, extracted key facts and parameters from the literature, and documented this in a wiki. Significantly, each fact in the wiki was synonymously encoded in a particular language, BioNetGen, which facilitated its automatic incorporation into an SBML coded model on demand. By this means, changes in the prose wiki automatically rippled through to changes in the model.

Today, eight years after launch, the model site continues to be near the top on searches for quantitative information about the pheromone response system and supply starting materials for researchers modeling cell signaling systems.

14 February 2015
Negative feedback is not enough

Work from the lab in 2008 (Yu et al. 2008) refocused our interest on the fact that some signaling systems turn on downstream events in proportion to the number receptors activated at the cell surface. This is "Dose-Response Alignment", or DoRA. Ongoing work theoretical work by Steve Andrews and other lab members now shows that simple negative feedback is not able to maintain DoRA but that augmented negative feedback and a second control mechanism can achieve it. Experimental work to establish the existence of the second mechanism continues.

19 January 2015
Brent, Houston labs sort on lifetimes with pseudophasors

In collaboration with Jessica Perea Houston and her coworkers at New Mexico State University in Las Cruces, Bryan Sands, Bill Peria and other lab members have sorted engineered yeast cells engineered to express fluorescent proteins with different lifetimes http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109940

The work required Sands to build proteins with different lifetimes and Peria to find and deploy a relatively obscure algorithmic method (reference)

Goertzel, G. (1958) An Algorithm for the Evaluation of Finite Trigonometric Series. American Mathematical Monthly 65 (1): 34–35, doi:10.2307/2310304

to program the sorter's Field Programmable Gate Array (FGPA) to compute lifetimes quickly enough to allow sorting. One consequence of this work will be to separate populations of cells in different physiological states defined by differences in cell signaling.
25 July 2014. Collaborating PI Jessica P. Houston receives a basic research award from The NCI’s Center to Reduce Cancer Health Disparities (CRCHD) at an investigators workshop for "Partnerships to Advance Cancer Health Equity" (PACHE) for collaborative work with Brent lab. Seehttp://crchd.cancer.gov/news/spotlights/program-spotlights_PACHE_2014.html

1 August 2014. Alexandra Ventura et al.
" Utilization of extracellular information before equilibrium receptor binding expands and shifts the input dynamic range", accepted for PNAS. Work is in collaboration with the lab of Alejandro Colman-Lerner lab at the University of Buenos Aires. The mechanism, PRESS, is important for the operation of the large number of cell signaling systems that share certain dynamic properties.

5 September 2014. Bryan Sands et al,
"Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes" is accepted in PLOS ONE. Work is in collaboration with the lab of Jessica P. Houston at New Mexico State University. Going forward, we hope that altered-lifetime fluorescent proteins and appropriate equipment will enable us to quantify signaling events involving very small (<10) numbers of molecules.

23 September 2014. Eddie Altszyler et al.
"Impact of upstream and downstream constraints on a signaling module’s ultrasensitivity " is accepted in Physical Biology. Work is from collaborating Colman-Lerner lab. If certain "modules" in cell signaling systems generate steep (or "switchlike") response curves, and interface with certain kinds of upstream or downstream reactions, the "modules" can give response curves that are even steeper.

1 April 2014.  Roger Brent and Gaymon Bennett recently joined a AAAS working group
on understanding big data in biology and its impacts on national security (http://www.aaas.org/event/big-data-life-sciences-and-national-security). This working group provides the Bennett and Brent with an opportunity to inform themselves about the science surrounding big data in 2014 and to define national security in ways they believe might be most conducive to democratic governance and human flourishing.

Center for Biological Futures: Roger Brent and Senior Research Fellow Gaymon Bennett were recently at the National Academy of Sciences Workshop on the H5NI controversy. See the CBF timelines on how these events have unfolded in scrolling or text form, or download a pdf.