The relationship between intracellular forces and cellular stiffness investigated by atomic force microscopy

The characterization of the mechanical behavior of cells has always captured the interest of scientists and, in the last decades, has been facilitated by the development of techniques capable of measuring a cell’s deformability. However, if on one hand, cells are active, living materials that regulate their physiology by generating and transmitting forces throughout their volume, common mechanical characterizations of cells involve material science approaches, which mostly address them as inert materials. As a consequence, although mechanical characterizations of cells have so far provided a wealth of correlations between stiffness and physio-pathological states, they have rarely provided insights into biological function and regulation. In this thesis, a cell nanomechanical platform is presented, whose resolution allows the isolation of the mechanical contribution of load-bearing cellular components. We first demonstrated that tensional forces - rather than the passive viscoelastic properties of the cytoplasm - govern the stiffness of cells at the nanoscale. We then quantitatively characterized the relationship between intracellular forces and the µm-scale patterns of stiffness across the cell surface. This analysis allowed us to calculate multiple physiologically-relevant quantities, such as membrane tension, cortex tension, actin bundle tension, tension-free elastic modulus, and mechanical coupling distances, all from single high-resolution cell stiffness images, providing an unprecedented connection between distinct mechanobiology fields

In vitro assembly of a human DNA pre-replication complex

An in vitro assembly of a human DNA pre-replication complex was obtained. Biotynilated DNA containing known origins of replication was incubated with HeLa nuclear extract in the presence of 100-fold excess of competitor DNA. The resulting complex was analyzed with western blotting, border mapping and mass spectrometry

Large and reversible myosin-dependent forces in rigidity sensing

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