2. “Secreting and Sensing the Same Molecule
Allows Cells to Achieve Versatile Social
Behaviors.”
• Aims
– To build a mathematical model which explains the
versatility of the secrete-and-sense motif.
– To explain the recurrence of the secrete-and-sense
self-signaling motif across species.
“The advantages of using secrete-and-sense circuits have
been unclear in many situations. For example, if a cell’s
primary purpose is self-communication, then it is unclear
why the cell secretes molecules instead of relying entirely
on intracellular signaling.” (1)
Science 343 (2014), H. Youk, W. A. Lim
3. Definitions
• Synthetic biology: an interdisciplinary branch of
biology, designing and constructing biological
devices and systems for useful (?) purposes.
• System biology: an interdisciplinary field of study
that focuses on complex interactions within
biological systems using a holistic approach and
engineering principles to biological and
biomedical research.
• Quorum sensing: a system of stimulus and
response correlated to population density.
7. Assay 1: Secretion Rate and Receptor
Abundance
No proof was delivered for
the localization of the α
factor – receptor reaction
on the extracellular side of
the membrane.
14. Their Conclusions
• Self-communication competes with neighbor
communication because they both use the same
molecule and receptor.
• The differences between local high concentration
of alpha factor and global concentration of alpha
factor determines the behavior of secrete-and-
sense circuit motif.
• Population and individual behavior can be
defined by models of reaction in unison, bimodal
activation, and positive feedback versus signal
degradation dynamics.
15. My Discussion Points
• The problem of translating a mathematical
model or simulation from a synthetic
approach to a biological one.
• A holistic approach should be accompanied by
an attention to the specifics (e.g. localization
of receptor signal, diffusion rate and
distances, and the question of evolution).
• They did not explain why the chose these
concentrations and these time frames.
Authors, Title, Date: Feb. 2014
Their Aims
2nd aim: They pose a research question throughout the article which I want us to remember and address later on.
My aims & way I’ll present: take apart their article to five assays, give their conclusions and offer my critiques (hopefully you may have some comments)
Synthetic biology (as a method): is an interdisciplinary branch of biology combining disciplines such as biotechnology, evolutionary bio. and molecular biology, system biology, and biophysics. It is in many ways related to genetic engineering.
System biology (as a model): is a biology based interdisciplinary field of study that focuses on complex interactions within biological systems using a holistic approach (which is good, but they missed important aspects of the proof) to biological and biomedical research.
Quorum sensing is a system of stimulus and response correlated to population density.
The term SnS they use refers to an action of a cell secreting a signal outside of its membrane which affects itself (self-signaling) or other cells (quorum sensing) by sensing the same signal.
As we’ve already discussed, however, and this is the key question: why do we need this kind of self communication?
B. SnS motif can be found in: EXAMPLES
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Synthetic secrete-and-sense circuit motif in yeast.(A) Cells that secrete a signaling molecule without sensing (top), cells that sense a molecule without secreting (middle), and cells that secrete and sense the same signaling molecule (bottom). (B) Examples of “secrete-and-sense” cells in nature: bacteria secrete and sense an autoinducer for sensing a quorum, human pancreatic β cells secrete and sense insulin, human T cells secrete and sense the cytokine IL-2 to control their proliferation, and the vulva precursor cells in C. elegans secrete and sense the diffusible Delta for specifying their cell fates. (C) Schematic of self-communication and neighbor communication between two identical secrete-and-sense cells. (D) Schematic of synthetic secrete-and-sense system: haploid budding yeast (yellow box) engineered to secrete and sense α-factor (orange circle). GFP fluorescence is a readout of the concentration of α-factor sensed by the cell.
In trying to answer the question we raised above, those guys engineered a budding yeast mating pathway as a secrete-and-sense circuit motif and built a model system which they could modify, to determine what features affect the self-communication versus the quorum sensing.
A Haploid Budding Yeast was engineered to secrete and sense the same mating pheromone alpha factor, meaning it can secrete the alpha factor and sense the alpha factor, through the Ste2 Receptor. The gene responsible for the secreting is the MFalpha1; and as a reporter they used a GFP which is a readout of the concentration of the alpha factor.
In order to quantify the GFP they used a flow-cytometer. The flow-cytometer uses a laser beam to count cells and measure their fluorescence activity.
I want to stop for a moment and pose several primary questions that rise from their research which we can keep in mind as we continue the presentation.
The intracellular problem – which they attempt to address
In addition, I see two main gaps in their discussion:
2. The evolutionary problem and the ‘purpose’ of self-signalling
3. The signal after the secretion-life : what happens to a signal after it was secreted – environs (environment modifications)?
But first things first: lets address the assays as they were introduced in the article.
In the first assay, they built two strains of yeast:
Strain A can secrete and sense the α factor
Strain B can only sense the α factor.
- For both of the strains the gene for the alpha factor has a PTET promoter which is induced by doxycycline concentration: the more doxycycline the more alpha factor expressed. And also for both of them, they engineered a PFUS promoter to express GFP: the more sense activity the more GFP expressed.
For both basic strains they used a matching Pvaried promoter, in order to get the same Ste2 abundance.
For the sense-only strain, they engineered a constitutive expressed mCherry gene (in order to distinguish it from the sense-and-secrete strain.)
So they measured two situations of heterogeneous cultures: high and low cell densities. In these, they measured two sub-criteria: low and high secretion rates.
We can see that when we have a low cell density we can see a high-degree of self-communication which is presented by the heat map.
Fig: C & E: explain the graph […High Secretion Rate: we do see a gradual rise in the GFP expression in strain B by the end of the six hours of the experiment. I see a big problem here, but I’ll get to that in a minute.]
Fig. D & F: explain the graph briefly [Quorum sensing/ Social behavior]
Fig. G: When we take the numbers of the Strain A cells and subtract the Strain B cells, we are left with the secrete-only activity.
CONCLUSION: Here they defined in what range of secretion rate and receptor abundance we get an asocial behavior or a social behavior.
I see two problems in this assay which were not answered in the article:
How do they know that the self-signalling is not in the cell? Why do they assume that it is extra-cellular? MARTIN: I’m not the yeast expert – you tell me, is there any probability that the Ste2 protein and the alpha factor are being edited or transferred in the same vascile near the membrane from the intracellular side of the membrane – so what we see here as an outside self-signalling is actually inside self-signalling.
The assay only measures six hours of culturing. We can see in figure C that the sense-only strain starts to have a rise in its GFP expression. Maybe, if they had continued the culturing for another several hours the GFP expression would get to the same rate (or close enough) as the expression for strain A which would call their calculations into question.
They built a different type of SnS Strain which had a positive feedback in its secrete-and-sense circuit motif. Here, they connected the GFP and alpha factor expression to the alpha factor receptor reaction. We have a positive feedback mechanism because we have linked two inducive pathways to increase the alpha factor. The first one is a closed circuit in which the alpha factor itself regulates positively the FUS1 promoter, which in its way increases the expression of the alpha factor. The second one is a tunable expression of the alpha factor, again, as before, by doxycycline.
What they tried to prove in this assay is whether activation properties were primarily due to self-communication or neighbor communication.
By activation, I mean a high concentration of GFP in relation to the cell count percentage. Activation has an ‘on’ and ‘off’ state. An ‘off’ state is when the cells secrete the alpha factor at a low basal rate (indicated by its low basal GFP fluorescence)
An ‘on’ state is the opposite: when the cells secrete the alpha factor at a high rate (indicated by its high GFP fluorescence)
In this assay they used both homogenous secrete-and-sense culture and heterogeneous cultures with both the secrete-and-sense and the sense-only strain. They did this in order to distinguish between self-communication and neighbour communication (information on this appears only in the supplementary information, and is based on assay #1).
Similar to their conclusion in assay 1, here too, they determined that in low cell density there is no significant quorum sensing activity. Thus, as we can see, in Fig. B, when the Low Cell Density was checked, a weak positive feedback had no activation, on both the secrete-and-sense and the sense-only strains. A strong positive feedback, on the other hand, turned the secrete-and-sense strain into an ‘on’ state, while the sense-only strain remained non-activated – hence Self-Activation.
In the case of High Cell Density, we see that at each positive feedback the GFP expression of the sense-only strain reached its maximum even before the secrete-and-sense strain, indicating both neighbor and self activation.
with a weak positive feedback there was a matching GFP expression, increasing at the same time and same rate with both secrete-and-sense and sense-only strains, thus indicating neighbor communication.
To summarise, self-activation can occur without any neighbor communication whereas neighbor activation can occur in regimes where self communication is insufficient for self-activation
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Effects of self-communication and neighbor communication on positive feedback linking secretion with sensing.(A) Basic secrete-and-sense circuit modified by a positive feedback link (highlighted in blue). (B and C) Representative histograms showing the single-cell GFP fluorescence level of the basic secrete-and-sense strain with the positive feedback link obtained by a flow cytometer. This strain was cultured by itself at two different initial cell densities [(B) low cell density (OD = 0.001) and (C) high cell density (OD = 0.1)] and in two representative concentrations of doxycycline {[doxycycline] = 3 μg/ml (weak positive feedback) and 40 μg/ml (strong positive feedback)}. Blue histograms, beginning of the time course (0 hour); red histograms, 8 hours into the time course (full data sets in figs. S11 and S12). Under each panel, the corresponding type of activation behavior is mentioned. a.u., arbitrary units. (D) Main population-level behavior: activation of all cells in near unison.
In their third assay, to the positive feedback circuit they added a signal degradation by constructing a Pvaried promoter attached to Bar1 gene. This gene expressed the Bar1 protease which then degrades alpha factor in the periplasmic space.
Again, we see that the assay is divided into two experiments: low and high cell density. We can see in the Low Cell density that a constitutive expression of Bar1 created a No Activation in the weak positive feedback and a Bimodal Activation in the strong positive feedback. This behavior of the culture population responds more or less the same when we talk about high cell density. Only this time, in a weak positive feedback we get a Slow neighbor activation, instead of no activation at all.
CONCLUSION: From this assay they concluded that a Bimodal Activation implies a division to two main populations which helps them to hedge their bets. This phenomenon is a complex addition to the population behavior in addition to the behavior we saw in earlier assays of self activation or quorum sensing activation.
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Effects of self-communication and neighbor communication on positive feedback with signal degradation.(A) Basic secrete-and-sense circuit with positive feedback link (blue highlight) and the Bar1 protease (gray). Six different strains of this type were constructed, each with a different constitutive promoter Pvaried controlling expression of BAR1. (B and C) An example strain (with pCYC1-BAR1) cultured by itself at two different initial cell densities [(E) low cell density (OD = 0.001) and (F) high cell density (OD = 0.1)] and in two representative doxycycline concentrations {[doxycycline] = 6 μg/ml (weak positive feedback) and 20 μg/ml (strong positive feedback)}. Representative histograms showing the single-cell GFP fluorescence levels of this strain are plotted at two different time points (blue and red histograms). Under each panel, the corresponding type of activation behavior is mentioned (figs. S14 and S15). (D) Phase diagrams from analyzing each time course for the seven secrete-and-sense strains, each with different amounts of Bar1 (including none, Fig. 3) and positive feedback strengths at low (OD = 0.001) and high (OD = 0.1) cell density cultures (summarizes fig. S11, S12, S14, and S15). (E) Main population-level behavior: bifurcation of an isogenic population into subpopulations of transiently quiescent and maximally secreting cells.
