Pavithran Ravindran has been a part of the preLights team for two years, and has highlighted many interesting preprints in synthetic biology and beyond (for a full list click here). In fact, in 2019 one of his highlights became our most-read post of the year. Pav was recently first author on a preprint so we asked him to tell us more about their findings and why they shared the work on bioRxiv.
A synthetic gene circuit for imaging-free detection of dynamic cell signaling
, , (bioRxiv, 2021) https://doi.org/10.1101/2021.01.06.425615
Could you explain the main finding of your new preprint?
Cells employ signaling pathways to communicate with one another and to learn about their environment. In more recent years, it has become clear that mammalian signaling pathways do not solely turn on or off in a constant manner but rather display ornate time-varying patterns. It has also become clear that pathways contain oscillations or pulses of activity and these may determine specific cell fates. Even more recently, with advances in optogenetics and microfluidics, researchers have found that cells interpret different dynamic patterns of signaling pathways to make cell fate decisions. However, measuring these dynamics requires the use of sub-cellular resolution imaging and watching cells go through phases of off, on, and then back to off. This limits the field to study imaging-viable models and prevents the use of large-scale screens to understand the networks that lead to such dynamics. In this preprint, we aimed to develop a method to selectively label cells that are experiencing pulsatile signaling by engineering a synthetic gene that ignores constant signaling.
To begin to address this problem, we decided to do a computational study of network structures to identify one that selectively responds to pulses. After finding a particular incoherent feed forward loop (a structure where the input activates an intermediate node that attempts to make the output but the original input is simultaneously inhibiting this output node) that works, we were able to design a system that implements the design for the Erk signaling pathway. We accomplish this by driving the expression of a synthetic transcription factor using an Erk-responsive promoter and a GFP downstream of the transcription factor; the trick however is that an Erk-kinase-translocation reporter (KTR) is attached to the transcription factor. This KTR exits the nucleus upon phosphorylation by Erk, and returns to the nucleus when Erk is off. Therefore, when Erk is constantly on, the transcription factor is made, but is out of the nucleus, unable to make GFP. In the case of a pulse, the transcription factor is made, goes out of the nucleus while the pathway is on and then when Erk is off again, the transcription factor returns to the nucleus, making GFP. Using this system, we show that we can robustly detect pulses of Erk signaling in fixed cells. For some cool videos of the system in action, check out this twitter thread from my advisor: https://twitter.com/toettch/status/1347221267267657729. Overall, we believe this tool will open doors for studying signaling dynamics by enabling large scale screens and in vivo situations where high-resolution imaging may not be tractable.
Movie S1 from the preprint. Time-lapse imaging of NIH3T3 READer cells in response to different dynamic stimuli.
Why did you decide to post your work on bioRxiv?
Even though this is not my first, and definitely not my last, time posting a manuscript on bioRxiv, there were a few concrete reasons that we decided to post this work as a preprint. First and foremost, from a philosophical point, I think that the time between finishing a project and getting it published, which can be dragged on for many months, is wasted time. That time could instead be used by the authors and others to further the work through collaborations based on the preprint. Furthermore, a whole community of researchers now can comment on the work to point out any fatal flaws that we may have overlooked. From a more practical standpoint, preprinting this paper allows me to strengthen my CV as I look for MD/PhD programs and for my advisor to use it for grant-writing.