During morphogenesis, epithelial tissues undergo dramatic changes in shape, transitioning from flat sheets to three-dimensional folded structures. This remarkable transformation relies on dynamic changes in mechanical tension at both their apical and basal surfaces. While it is well-established that the generation of mechanical tension at the apical side is driven by the actomyosin network, research on this process has often overlooked the generation of mechanical tension at the basal surface. Moreover, the mechanical response to stress, encompassing both elastic (spring-like) and viscous (fluid-like) properties, is important for epithelial transformations, yet this mechanical response is poorly understood for the basal cell surface. In this thesis, we investigated how basal tension is influenced by the basement membrane - an extracellular matrix layer which has been widely regarded as a passive scaffold for cells. We probed the material mechanical response of the basement membrane and directly measured and analyzed basal tension in the wing imaginal disc epithelium of Drosophila.
To study the mechanical response, I used long-term confocal imaging and fluorescence recovery after photobleaching (FRAP) to analyze the turnover and mobility of Collagen IV, a component of the basement membrane. The low Collagen IV mobility and turnover (≈ 40 hours) suggest a solid-like behavior of the basement membrane at the time scale of hours. Moreover, Atomic Force Microscopy (AFM) force-indentation curves reveal low hysteresis and an elastic solid-like response.
To measure basal mechanical tension, I probed the basement membrane with an AFM. Interpreting the results of AFM shallow indentations on the basal side of explanted wing discs as indenting into a fixed, elastic, stretched thin film, I investigated in control conditions and after molecular perturbations basal mechanical tension. Mechanical tension was ≈ 0.4 mN/m. The removal of collagen IV by collagenase significantly reduced basal tension while increasing basal cell surface area. In addition, inhibition of actomyosin activity through different reagents reduces basal tension while decreasing basal cell surface area. These results indicate that basal tension depends on both the ECM and actomyosin activity. They also indicate that the basement membrane is under expansile stress.
Finally, to further investigate the mechanisms underlying the generation of stretch in the basement membrane, I analyzed the influence of hydrostatic pressure and actomyosin contractility along the lateral cell surfaces. These mechanisms exert mechanical forces that increase basal cell area, inducing a stretch in the basement membrane. Mild hypo- or hyperosmotic shocks resulted in increased or decreased basal cell area and basal tension, respectively. Moreover, optogenetic activation of actomyosin at lateral cell surfaces resulted in an increase in both basal cell area and basal tension.
In summary, our research quantifies basal tension and unveils that the basement membrane is an elastic material (at time scale of hours). Furthermore, our data suggest that the basement membrane is under elastic stretch generated by hydrostatic pressure and actomyosin contractility. Thus, rather than being a passive scaffold for cells, the elastic properties of the basement membrane contribute to basal tension and thereby the shaping of cells and tissues.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:90785 |
| Date | 10 April 2024 |
| Creators | Guerra Santillán, Karla Yanín |
| Contributors | Fischer-Friedrich, Elisabeth, Dahmann, Christian, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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