Periodic Reporting for period 1 - MCExtrusion (Mechanisms of Cell Extrusion controlling Tissue Homeostasis in the Intestine)
Période du rapport: 2021-06-01 au 2023-05-31
The small intestine is an integral component of the gastrointestinal tract and responsible for the absorption of nutrients from ingested food within the gut lumen. Its distinct anatomy supports this role: The intestinal epithelium is folded into long tissue protrusions containing specialized cells that for example absorb nutrients and cells that secrete mucus. At the same time the intestinal epithelium functions as a protective barrier shielding the underlying tissue from the harsh environment of the gut lumen containing a high pathogenic load. It is therefore exposed to environmental and internal stresses to which it responds by dynamic cell remodelling. The intestine is among the tissues with the fastest turnover of cells revealing a precise orchestration of cell production and removal. Unbalancing of this equilibrium results in severe pathologies, as excessive proliferation causes tissue overgrowth and excessive cell removal compromises the epithelium’s barrier function. Many human pathogens distort this equilibrium either by preventing the removal of infected intestinal cells to allow for bacterial replication or by inducing removal of healthy cells to enable the penetration of the intestinal barrier. Excessive removal of cells from the intestine affects the anatomy of the intestine and compromises its digestive function. This is also hallmark of certain chronic diseases such as inflammatory bowel disease highlighting the importance of cell extrusion for intact tissue homeostasis. Despite the importance of controlled cell extrusion for maintaining the structure and function of the intestine, remarkably little is known about its regulation. With this research I investigate biological mechanisms underlying the regulation of cell extrusion in the intestine. Cells might either commit to extrusion through specific genetic programs or local tissue mechanical constraints might force the extrusion of otherwise indistinguishable cells. I aimed to characterize cell extrusion and identify gene networks that regulate it. At the same time, I investigated the role of mechanical forces for cell extrusion using precision methods including optogenetics and laser microsurgery.
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.
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.
Cell extrusion represents a critical aspect of intestinal homeostasis that was not well understood. Its deregulation is implicated in human diseases caused by pathogen infection or chronic diseases. Thus, the results of this study are of interest for a broad scientific community as they have general implications for epithelial morphogenesis and maintenance and resolve a critical aspect of intestinal biology that remained so far elusive. The research might have implications with respect to human diseases and might lead to the identification of new therapeutic targets in the future. The results are therefore of relevance for a bigger public audience. The findings furthermore open new avenues for future research such as investigating the connection between mechanical and signaling pathways in the regulation of extrusion and other biological processes. The reporters and tools developed with this work will foster research with a diverse set of potential applications.