Sculpting Rupture-Free Nuclear Shapes in Fibrous Environments.

Cytoskeleton-mediated force transmission regulates nucleus morphology. How nuclei shaping occurs in fibrous in vivo environments remains poorly understood. Here suspended nanofiber networks of precisely tunable (nm-µm) diameters are used to quantify nucleus plasticity in fibrous environments mimicking the natural extracellular matrix. Contrary to the apical cap over the nucleus in cells on 2-dimensional surfaces, the cytoskeleton of cells on fibers displays a uniform actin network caging the nucleus. The role of contractility-driven caging in sculpting nuclear shapes is investigated as cells spread on aligned single fibers, doublets, and multiple fibers of varying diameters. Cell contractility increases with fiber diameter due to increased focal adhesion clustering and density of actin stress fibers, which correlates with increased mechanosensitive transcription factor Yes-associated protein (YAP) translocation to the nucleus. Unexpectedly, large- and small-diameter fiber combinations lead to teardrop-shaped nuclei due to stress fiber anisotropy across the cell. As cells spread on fibers, diameter-dependent nuclear envelope invaginations that run the nucleus's length are formed at fiber contact sites. The sharpest invaginations enriched with heterochromatin clustering and sites of DNA repair are insufficient to trigger nucleus rupture. Overall, the authors quantitate the previously unknown sculpting and adaptability of nuclei to fibrous environments with pathophysiological implications.

Aligned and suspended fiber force probes for drug testing at single cell resolution.

The role of physical forces in disease onset and progression is widely accepted and this knowledge presents an alternative route to investigating disease models. Recently, numerous force measurement techniques have been developed to probe single and multi-cell behavior. While these methods have yielded fundamental insights, they are yet unable to capture the fibrous extra-cellular matrix biophysical interactions, involving parameters of curvature, structural stiffness (N m(-1)), alignment and hierarchy, which have been shown to play key roles in disease and developmental biology. Using a highly aggressive glioma model (DBTRG-05MG), we present a platform technology to quantify single cell force modulation (both inside-out and outside-in) with and without the presence of a cytoskeleton altering drug (cytochalasin D) using suspended and aligned fiber networks (nanonets) beginning to represent the aligned glioma environment. The nanonets fused in crisscross patterns were manufactured using the non-electrospinning spinneret based tunable engineering parameters technique. We demonstrate the ability to measure contractile single cell forces exerted by glioma cells attached to and migrating along the fiber axis (inside-out). This is followed by a study of force response of glioma cells attached to two parallel fibers using a probe deflecting the leading fiber (outside-in). The forces are calculated using beam deflection within the elastic limit. Our data shows that cytochalasin D compromises the spreading area of single glioma cells, eventually decreasing their 'inside-out' contractile forces, and 'outside-in' force response to external strain. Most notably, for the first time, we demonstrate the feasibility of using physiologically relevant aligned fiber networks as ultra-sensitive force (∼nanoNewtons) probes for investigating drug response and efficacy in disease models at the single cell resolution.