Miniature organ-like tissues that can be grown in the lab, called organoids, proved to be a valuable model to investigate the process of cell extrusion. To test genes and signaling pathways for their effect on cell extrusion we developed an intestinal organoid-based culture assay that allowed us to quantitatively analyse the number of cells that extrude as live or dead cells using flow cytometry. We performed a drug screen to perturb different signaling pathways and measured if their inactivation or activation affects extrusion. We identified both, pathways that increased and that reduced cell extrusion. We found that the increased cell extrusion rates triggered by one pathway can be compensated by the activity of the others suggesting that the integration of multiple signaling pathway inputs controls cell extrusion in the intestine. We leveraged the culture model to profile extruded cells that have just left the epithelium at the transcriptomic level using bulk RNA sequencing. The results revealed a specific transcriptomic signature of extruded cells and provided a set of differentially regulated candidate genes that are currently being functionally characterized by loss of function mutants. Altogether, a regulatory signaling network underlying the control of live cell extrusion was identified which is highly dependent on the balance between major homeostatic signaling pathways of the intestine.
We furthermore tested whether also mechanical signals play a role in extrusion regulation. We employed quantitative live imaging-based analyses of all single cell movements in intestinal organoids over several days and found that live cell extrusion is not regulated by a timer set at cell division, nor by tissue crowding. To characterize the cytoskeletal rearrangements and mechanical forces that underly cell extrusion, we investigated the activity of the molecular force generator Myosin generated using CRISPR/Cas9 technology. We grew Myosin reporter organoids on synthetic substrates resembling the intestine’s anatomy, which allowed the targeted observation of cell extrusion with high spatiotemporal resolution. We observed that extruding cells show lower levels of Myosin in the hours preceding extrusion. Our idea was that these cells have lost mechanical stability and are therefore extruded in a regulated way. We tested this idea by intentionally damaging the base of the cells with a laser and found that these cells indeed extrude. When more then only one cells were damaged, all damaged cells extruded unless we block the activity of the molecular motor Myosin. These findings show that the molecular mechanism to probe mechanical cell stability relies on force generation by Myosin. We then created a tool that allows us to control the activity of Myosin with light by artificially combining an activator of Myosin with proteins from plants. Consistently, when Myosin was activated with light, we increased the tension in the tissue and thereby accelerated the tug of war between neighbouring cells which resulted in an increase in the rate of cell extrusion. This part of the work demonstrates that tissue-scale mechanical forces play a critical role in the regulation of cell extrusion, and therefore in maintaining tissue homeostasis.
Together, the results mapped the process of cell extrusion within the complex signaling landscape of the intestine and identified pathways that specifically regulate this important fate decision. At the same time, local tissue mechanics were identified to have a regulative function as the mechanical stability of cells in the epithelium is continuously probed and weakened or damaged cells are being removed through the activity of Myosin. The obtained transcriptional signature of extruding cells suggests strong interactions and mutual feedback of both, cell signaling pathways and cellular mechanics through their molecular cytoskeletal regulators. These interactions will be interesting subjects for further research.
The two parts of this work will be published individually by scientific publications and all new reagents, protocols and data sets shared with the research community. The results will be communicated via social media outlets of the host institution and the host lab.
