Bohn, Stefan Jürgen (2021)
The Interplay of Signaling Dynamics and Cell Cycle Regulation in Single Cells.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00019077
Dissertation, Erstveröffentlichung, Verlagsversion

Kurzbeschreibung (Abstract)

Signaling pathways that control cellular responses such as proliferation, quiescence, migration and apoptosis are crucial for embryonic development, tissue homeostasis and regeneration. Dysregulation of these signaling processes can result in severe human diseases including cancer. Therefore, to maintain a balance between the above-mentioned cell fates and to prevent pathological events, an interplay between different signaling pathways is indispensable. For instance, mitogenic signaling induced by the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT networks is required for cells to enter the cell cycle and divide. However, to prevent uncontrolled proliferation, anti-mitogenic signals such as those transmitted by mothers against decapentaplegic homologue (SMAD) proteins are essential. To gain a deeper understanding of these pathway interactions, I employed quantitative time-resolved measurements of fluorescent reporters and computer-aided data analysis. In the first part of the present study, I examined how the MAPK and PI3K/AKT pathways synergize to regulate cell cycle entry and progression. Although these networks have been well characterized in earlier studies, their relative contribution, especially at later cell cycle stages, remains largely unexplored. In order to investigate the response of cells outside of an active cell cycle to acute mitogenic signals, untransformed human breast epithelial cells were first arrested in a quiescent state by growth factor deprivation. Afterwards, epidermal growth factor (EGF)-induced signal processing in individual cells was quantified over time, which revealed that both pathways were necessary for initial cell cycle entry, whereas only PI3K/AKT affected the duration of S-phase at later stages of mitogenic signaling. My results provide evidence that the high metabolic demands of replication are unmet in the absence of AKT signaling, which results in a strongly prolonged S-phase of the cell cycle. In the second part, I investigated how the cell cycle and mitogenic signals influence the SMAD signaling pathway. I uncovered that ligands of the transforming growth factor beta (TGFb) superfamily signaled very differently in quiescent versus proliferating cells. While TGFb mediated a stronger SMAD2 response in proliferating cells compared to quiescent cells, the opposite was observed upon growth differentiation factor 11 (GDF11) stimulation. I was able to show that MAPK activity was responsible for the switch in ligand sensitivity, most likely through regulation of target genes. Therefore, the question arose whether a single key player or a complete pathway-rewiring were accountable. As RNA sequencing revealed considerable changes in the expression of multiple SMAD associated genes and single perturbations of SMAD signaling regulators could not explain the observed phenomenon, I hypothesized that a more wide-ranging rewiring of the network is necessary to shift the sensitivity to different ligands of the TGFb superfamily. However, further studies using genome-scale knockout or activation screenings need to be carried out to validate this idea and recreate the pathway-rewiring in proliferating cells. Besides the switch in ligand sensitivity, I observed different SMAD-mediated cell fates in proliferating and quiescent cells. While apoptosis was only induced in quiescent cells, epithelial to mesenchymal transition (EMT) and cytostasis were found in dividing cells. Interestingly, cellular responses correlated well with the dynamics of SMAD signaling, which suggested that ligands mediate diverse cellular outcomes through different dynamical patterns of SMAD2 nuclear accumulation. This quantitative nature of the pathway was later validated by real-time quantitative PCR (RT-qPCR) and RNA sequencing.

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