Making waves: active microrheology of complex systems

In this thesis, we developed a toolkit to quantify viscoelastic properties of cellular structures, from the DNA itself to isolated nuclei and cells. This toolkit comprises two single-molecule techniques - Acoustic Force Spectroscopy (AFS) and optical tweezers - which possess distinct advantages: the former offers high throughput while retaining single-cell characterization, while the latter offers a highly precise manipulation geometry and the combination with fluorescence imaging. By using this toolkit, we not only characterized the viscoelastic properties of single cells and biopolymers, but also explored the role of individual cellular structures, such as chromatin, intermediate filaments and lipid membranes as contributors to the overall resilience of cells. Chapter 2 introduced Acoustic Force Microrheology (AFMR). Combining active microrheology and acoustic forces, AFMR serves as a microrheology tool capable of applying a large range of forces and frequencies. In this Chapter, AFMR was used to quantify the viscoelastic properties of a diverse range of biological samples: collagen gels, red blood cells, and motile fibroblasts. Furthermore, using AFMR’s ability to provide single-bead information, we investigated heterogeneity in collagen gels and cell-to-cell variance. We then zoom into the mechanics of individual cellular components with Chapter 3, which focuses on the nucleus, the most important cell organelle and vital mechanical element. Here, we used a combination of optical tweezers with fluorescence imaging to manipulate isolated nuclei in solution while mapping the deformation of chromatin and lamina under force. By investigating their force response, we revealed the nonhomogeneous response of nuclei, which we could explain via a hierarchical chain model. In parallel, fluorescence imaging exposed a high euchromatin compliance compared to the lamina. Finally, the high manipulation precision allowed us to pull nuclear envelope tethers. By investigating the rigidity and composition of the nuclear membrane, we shed light on a rarely studied aspect of nuclear mechanics. Focusing further into the mechanics of the genome, Chapter 4 investigates DNA-intercalator interactions, where mechanics and chemistry converge. By using optical tweezers to perform active microrheology, and interpreting the data via Eigen's chemical relaxation theory, we were able to not only characterize the mechanics of a DNA-protein complex, but also to determine its binding kinetics via the same mechanical measurements. Finally, in Chapter 5 the toolkit developed in Chapter 3 finds its role within the context of synthetic cell development. This chapter not only underscores the toolkit's adaptability to study bottom-up assembled GUVs, but also its significance in assessing mechanical properties across different stages of synthetic cell evolution. As synthetic cells gradually evolve, the toolkit would also offer a help in deciphering the individual components' mechanical roles. To demonstrate this, we characterised the mechanical properties of a simple membrane system (GUV) and investigated how changes of chromatin state in nuclei impact nuclear stiffness.