Dynamics and function of stimulus-dependent SMAD signaling in proliferating versus quiescent epithelial cells.

• Applicants: Professor Dr. Stefan Legewie; Professor Dr. Alexander Loewer
• Subject Area: Cell Biology ; General Genetics and Functional Genome Biology ; Bioinformatics and Theoretical Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 468927954

SMAD-mediated signaling regulates embryonic development, adult tissue homeostasis, and regeneration. Alterations in the response to ligands of the TGFβ family are involved in severe human diseases including cancer. To understand how cells process these inputs, we recently combined live-cell imaging, computer-aided analysis and mathematical modelling to investigate pathway activity at the single-cell level. Our combined experimental and theoretical study revealed that the response to a given dose of TGFβ is determined cell-specifically by the levels of defined signaling proteins, and placed the dynamics of SMAD signaling as an important determinant of the variegated cell fates elicited by TGFβ. We are now challenged to understand how SMAD dynamics in individual cells depend on specific ligands, how they are affected by cellular states, and how they are translated into gene expression changes. Preliminary experiments indicated that ligand sensitivity is adjusted to the proliferation state: while TGFβ -induced signaling is amplified in proliferating and attenuated in quiescent cells, we observed the reversed response to ligand such as GDF 8 and GDF11. Genome-wide expression analysis indicates that cell-specific signaling dynamics determine the transcriptional response. However, the underlying molecular mechanisms and the consequences for the physiological response of the cell remain elusive. Here we propose to use an experimental and theoretical approach to characterize state- and stimulus-specific dynamics of SMAD signaling in individual cells, to determine the key processes involved in re-wiring the signaling network in proliferating and quiescent cells and to investigate how stimulus-specific dynamics are translated into distinct gene-expression patterns. We will use time-varying stimulation patterns, microscopy based–measurements as well as pharmacological perturbations to systematically characterize SMAD signaling. A modeling -based approach will allow us to identify key processes in the SMAD network that are altered in proliferating and quiescent cells and we will validate the resulting model predictions using Cas9-mediated genome engineering. Finally, we will combine genome-wide expression analysis with time-resolved measurements in living cells to generate mathematical models of stimulus-specific target gene regulation and determine to which extent SMAD dynamics are sufficient to predict cell- and stimulus-specific responses or whether they require ligand-specific alternative inputs. Taken together, our study will delineate how and why cells modulate ligand sensitivity depending on their proliferation state and how this affect their physiological response. This may provide insights into the oncogenic potential and tumor suppressive capacity of TGFβ family ligands and help to develop new therapeutic concepts.

• DFG Programme: Research Grants