Cell Fragment Formation, Migration, and Force Exertion on Extracellular Mimicking Fiber Nanonets

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.

Fiber Diameter and Architecture Direct Three-Dimensional Assembly of Pericytes into Spheroids

“…The two layers of the fibers were fused at the contact points resulting in fixed-fixed boundary conditions. 28,30 Cells applied contractile forces as visualized by the deflection of the fibers, resulting in the assembly of spheroids. To visualize spheroid 3D organization on fiber networks using confocal microscopy, pericytes were seeded on fibers coated with Rhodamine-conjugated fibronectin.…”

“…Since pericyte spheroid assembly caused the fiber networks to deflect, we measured the forces exerted during this process. Using nanonet force microscopy 27,30,32,33 , we found the spheroid force exertion to be higher (P=.003) than individual pericyte forces. Individual cells had an average force of 41.55 ± 18.78 nN (N=10), while spheroids had a wide range of forces depending on their size (1383 ± 1787 nN ((N=49), Sup.…”

“…Aligned fiber networks were fabricated by depositing ∼2 µm thick ‘ base’ fibers spaced ∼350 µm apart, over which orthogonal layers of ∼200/500/800 nm ‘aligned’ fibers with ∼10 µm spacing were deposited. 26–28 Crosshatch fiber networks involved two densely spaced (∼10 µm) orthogonally deposited layers of fibers having the same diameter (∼200/500/800 nm 29,30 ). The intersections were fused using Tetrahydrofuran vapors to preserve their architecture (Sup.…”

“…Cell and spheroid forces were calculated using our established nanonet force microscopy technology. 27,30,32,33 This technique involves the use of aligned fiber networks to calculate cellular forces. The thinner aligned fibers fused to the stiff base fibers on both sides are modeled as fixed-fixed beams.…”

Cellular tango: How extracellular matrix adhesion choreographs Rac-Rho signaling and cell movement

“…This is interpreted as a transect across the diameter of the cell, with ends at opposite cell edges. This 1D model can also be interpreted as the geometry of a cell that is confined to a narrow channel as in [60]or moving along thin "ECM" fibers or nano-wires, as in [61,62,63]. (b) A static 2D circular domain, as illustrated in Figure 1c.…”