Making waves
Authors:   , Bergamaschi Giulia 
Published: April 2024

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.