Deciphering a Prototypical MAP Kinase Signaling Network at the Single-Cell Level Using a Genetically-Encodable Optogenetic Circuit
Receptor tyrosine kinases (RTK) are key elements that convert growth factors into intracellular signaling patterns such as the mitogen activated protein kinase (MAPK) pathway. Even though the importance of this pathway in cell fate has been assessed, the mechanisms that regulate its molecular players still need to be investigated.
Our lab has recently uncovered new insights into this network. By delivering growth factor pulses to PC-12 cells using microfluidics and recording ERK activation dynamics at the single-cell level with a genetically encoded biosensor, they gained key novel insights into how the MAPK signaling network is wired.
The next question is to identify the molecular players that mediate different feedback and feedforward structures. This will be done using perturbation approaches. Because microfluidic technology allows limited dynamic stimulation patterns, we propose to build a circuit with an optogenetic FGF receptor (INPUT) and an ERK biosensor (OUTPUT). This will allow us to activate the MAPK network using dynamic light pulses and record the ERK signaling in hundred of single cells.
The first step of this project is to establish a stable cell line expressing an opto-FGFR and an ERK biosensor. The behavior of the system subject to different dynamical patterns of FGF receptor activation will then be established. In a second time, siRNA perturbations of a small number of candidates will be performed to identify the implication of these molecular players in the MAPK network. These observations will be implemented in mathematical modeling to build a refined model of the MAPK signaling Network.
Keywords: MAPK, signaling network, single-cell analysis, opto-genetic, siRNA, modelingback