Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration, Nucleus Shape, and Focal-Adhesion-Cluster Length

Abstract

It has been shown that cellular migration, persistence, and associated cytoskeletal arrangement are highly dependent on substrate stiffness (modulus: N/m(2) and independent of geometry), but little is known on how cells respond to subtle changes in local geometry and structural stiffness (N/m). Here, using fibers of varying diameter (400, 700, and 1200 nm) and length (1 and 2 mm) deposited over hollow substrates, we demonstrate that single mouse C2C12 cells attached to single suspended fibers form spindle morphologies that are sensitive to fiber mechanical properties. Over a wide range of increasing structural stiffness (2 to 100+ mN/m), cells exhibited decreases in migration speed and average nucleus shape index of ∼57% (from 58 to 25 μm/h) and ∼26% (from 0.78 to 0.58), respectively, whereas the average paxillin focal-adhesion-cluster (FAC, formed at poles) length increased by ∼38% (from 8 to 11 μm). Furthermore, the increase in structural stiffness directly correlates with cellular persistence, with 60% of cells moving in the direction of increasing structural stiffness. At similar average structural stiffness (25 ± 5 mN/m), cells put out longer FAC lengths on smaller diameters, suggesting a conservation of FAC area, and also exhibited higher nucleus shape index and migration speeds on larger-diameter fibers. Interestingly, cells were observed to deform fibers locally or globally through forces applied through the FAC sites and cells undergoing mitosis were found to be attached to the FAC sites by single filamentous tethers. These varied reactions have implications in developmental and disease biology models as they describe a strong dependence of cellular behavior on the cell's immediate mechanistic environment arising from alignment and geometry of fibers.

Crosshatch nanofiber networks of tunable interfiber spacing induce plasticity in cell migration and cytoskeletal response

“…Although paxillin adhesions were distributed throughout the cells on the 2D flat substrata, on fibers, they were observed to be concentrated in FACs (Fig. 1C), consistent with our previous observations (61,62). The observed migratory phenotype was not unique to Hras1 because other commonly used cell lines with migratory potential [NIH 3T3 fibroblasts, human smooth muscle cells, human bone mesenchymal stem cells, and human breast adenocarcinoma MDA-MB-231 cells ( Fig.…”

“…7A). Contrastingly, cells having elongated shapes similar to dense networks but cultured on multiple parallel fibers or on single parallel fibers in spindle shapes move faster at lower stiffness (43,62). Thus, we emphasize the need to conjointly examine geometric arrangement of fibers and their stiffness for a comprehensive description of the migratory phenotype.…”

Helical nanofiber yarn enabling highly stretchable engineered microtissue

“…4 G-I). This behavior of nuclear elongation and organization in a fibrous system is consistent with previous studies (52)(53)(54). Moreover, the idea of nuclear deformation regulating gene expression and leading to varied cell functions is also in line with previous observations (55 -61).…”

Extracellular Matrix Elasticity Regulates Osteocyte Gap Junction Elongation: Involvement of Paxillin in Intracellular Signal Transduction

“…Paxillin is a signaling transduction adaptor that activates cytoplasmic signals such as Wnts [34] and can cause changes in downstream targets [35]. Moreover, paxillin can directly alter the cytoskeleton via the reorganization and recombination of microfilaments and microtubules that alter cell responses [36]. In this study, we detected changes in paxillin expression and distribution in response to different substrate stiffnesses and thus inferred the activation of cytoplasmic signaling and the reorganization of the cytoskeleton.…”