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Abstract 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. 

Abstract Cell fragments devoid of the nucleus play an essential role in intercellular communication. Mostly studied on flat 2D substrates, their origins and behavior in native fibrous environments remain unknown. Here, cytoplasmic fragments’ spontaneous formation and behavior in suspended extracellular matrices mimicking fiber architectures (parallel, crosshatch, and hexagonal) are described. After cleaving from the parent cell body, the fragments of diverse shapes on fibers migrate faster compared to 2D. Furthermore, while fragments in 2D are mostly circular, a higher number of rectangular and blob‐like shapes are formed on fibers, and, interestingly, each shape is capable of forming protrusive structures. Absent in 2D, fibers’ fragments display oscillatory migratory behavior with dramatic shape changes, sometimes remarkably sustained over long durations (>20 h). Immunostaining reveals paxillin distribution along fragment body‐fiber length, while Forster Resonance Energy Transfer imaging of vinculin reveals mechanical loading of fragment adhesions comparable to whole cell adhesions. Using nanonet force microscopy, the forces exerted by fragments are estimated, and peculiarly small area fragments can exert forces similar to larger fragments in a Rho‐associated kinase dependent manner. Overall, fragment dynamics on 2D substrates are insufficient to describe the mechanosensitivity of fragments to fibers, and the architecture of fiber networks can generate entirely new behaviors.