Our main goal is to understand how signaling network dynamics controls cell fate. Mitogen Activated Protein Kinases, as well as the entire metabolism signaling network (i.e. AKT, AMPK, mTORC1), are clinically relevant signaling molecules that orchestrate cellular responses to a diverse array of stimuli. There are three major MAPK signaling cascades (ERK, p38 and JNK) that are highly interconnected with the metabolism signaling network and control critical cellular decisions such as survival/apoptosis or proliferation/senescence. Our current understanding of how signaling controls cell fate is incomplete because: (i) a lack of integrated methods to quantify the dynamics of the network as a whole and (ii) the use of cell population assays that average unsynchronized single cell behaviors. To address this need, my laboratory has pioneered a new generation of biosensors that allow simultaneous quantification of multiple kinase activities in thousands of live single cells. These biosensors convert phosphorylation into a nucleocytoplasmic shuttling event that can be easily measured by fluorescent microscopy. Eukaryotic cells constantly adjust metabolic fluxes in response to environmental demands via the mammalian Target of Rapamycin (mTOR). Over the past 20 years, the topology of the mTOR signaling network has been dissected using classic biochemical and genetic approaches. However, recent advances in single cell analysis have demonstrated that cell populations often behave qualitatively different than individual cells. Thus, we recently generated a single cell biosensor to measure mTOR complex 1 activity in live single cells (mTORC1- KTR). Surprisingly, our preliminary data shows striking oscillations of mTORC1 activity occurring out-of-phase between neighboring cells of epithelial monolayers. This finding is in agreement with a recent preprint suggesting that individual cells undergo autonomous cycles in energetic balance under normal conditions. However, the cause and the consequence of these oscillatory patterns are not understood. Our lab has previously shown that oscillatory or sustained MAPK activity differentially regulate cell fate highlighting the role of temporal dynamics in regulating biological functions. Taken together, we hypothesize that the temporal patterns of mTOR activity differentially regulate metabolism to fine tune energetic demands. To address this hypothesis we divided the proposal in three aims:(i) Aim 1 will address the molecular mechanisms responsible for mTORC1 oscillatory dynamics, (ii) Aim 2 will implement a new optogenetic tool to modulate mTORC1 activity using light, and (iii) Aim 3 will measure the physiological consequences of mTORC1 dynamics at the transcriptome and metabolome levels.
Mitogen Activated Protein Kinases (MAPKs) are clinically relevant signaling molecules that orchestrate cellular responses to a diverse array of stimuli. Our current understanding of this highly interconnected signaling network is incomplete due to the lack of integrated single cell approaches. The work we propose here overcomes these limitations and seeks to define how MAPK and mTORC signaling dynamics regulates single cell fates (i.e. proliferation, quiescence, senescence, apoptosis) during tissue homeostasis.
