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Abstract Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and tilting of the metaphase plate (MP). To understand the effect of confinement on mitosis by fibers (ECM confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesions on fibers. We discover that the extent of confinement influences RF organization in 3D, forming triangular and band-like patterns on the cell cortex under low and high confinement, respectively. Our mechanistic analysis reveals that the patterning of RFs on the cell cortex is the primary driver of the MP rotation. A stochastic Monte Carlo simulation of the centrosome, chromosome, membrane interactions, and 3D arrangement of RFs recovers MP tilting trends observed experimentally. Under high ECM confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM confinement leads to low and high MP tilts, which we mechanistically show to depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in ECM-confining microenvironments in vivo. 

A high porosity (88%) and ultrathin (<3 μm) fibrous basement membrane mimic using (A) suspended nanofiber networks for a (B) brain endothelial–pericyte co-culture model. (C) Our approach achieved low cell membrane and nuclei separations. 

Abstract Protrusions at the leading-edge of a cell play an important role in sensing the extracellular cues during cellular spreading and motility. Recent studies provided indications that these protrusions wrap (coil) around the extracellular fibers. However, the physics of this coiling process, and the mechanisms that drive it, are not well understood. We present a combined theoretical and experimental study of the coiling of cellular protrusions on fibers of different geometry. Our theoretical model describes membrane protrusions that are produced by curved membrane proteins that recruit the protrusive forces of actin polymerization, and identifies the role of bending and adhesion energies in orienting the leading-edges of the protrusions along the azimuthal (coiling) direction. Our model predicts that the cell’s leading-edge coils on fibers with circular cross-section (above some critical radius), but the coiling ceases for flattened fibers of highly elliptical cross-section. These predictions are verified by 3D visualization and quantitation of coiling on suspended fibers using Dual-View light-sheet microscopy (diSPIM). Overall, we provide a theoretical framework, supported by experiments, which explains the physical origin of the coiling phenomenon. 

During mitosis, cells round up and utilize the interphase adhesion sites within the fibrous extracellular matrix (ECM) as guidance cues to orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we explore mitotic outcomes and error distribution for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo significant 3-dimensional (3D) displacement while being held by retraction fibers (RFs). Increasing the number of parallel fibers increases FACs and retraction fiber-driven stability, leading to reduced 3D cell body movement, metaphase plate rotations, increased interkinetochore distances, and significantly faster division times. Interestingly, interphase kite shapes on a crosshatch pattern of four fibers undergo mitosis resembling single-fiber outcomes due to rounded bodies being primarily held in position by RFs from two perpendicular suspended fibers. We develop a cortex–astral microtubule analytical model to capture the retraction fiber dependence of the metaphase plate rotations. We observe that reduced orientational stability, on single fibers, results in increased monopolar mitotic defects, while multipolar defects become dominant as the number of adhered fibers increases. We use a stochastic Monte Carlo simulation of centrosome, chromosome, and membrane interactions to explain the relationship between the observed propensity of monopolar and multipolar defects and the geometry of RFs. Overall, we establish that while bipolar mitosis is robust in fibrous environments, the nature of division errors in fibrous microenvironments is governed by interphase cell shapes and adhesion geometries. 

Tunneling nanotubes (TNTs) comprise a unique class of actin-rich nanoscale membranous protrusions. They enable long-distance intercellular communication and may play an integral role in tumor formation, progression, and drug resistance. TNTs are three-dimensional, but nearly all studies have investigated them using two-dimensional cell culture models. Here, we applied a unique 3D culture platform consisting of crosshatched and aligned fibers to fabricate synthetic suspended scaffolds that mimic the native fibrillar architecture of tumoral extracellular matrix (ECM) to characterize TNT formation and function in its native state. TNTs are upregulated in malignant mesothelioma; we used this model to analyze the biophysical properties of TNTs in this 3D setting, including cell migration in relation to TNT dynamics, rate of TNT-mediated intercellular transport of cargo, and conformation of TNT-forming cells. We found that highly migratory elongated cells on aligned fibers formed significantly longer but fewer TNTs than uniformly spread cells on crossing fibers. We developed new quantitative metrics for the classification of TNT morphologies based on shape and cytoskeletal content using confocal microscopy. In sum, our strategy for culturing cells in ECM-mimicking bioengineered scaffolds provides a new approach for accurate biophysical and biologic assessment of TNT formation and structure in native fibrous microenvironments. 

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous submesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here we fabricated extracellular matrix mimicking suspended fiber networks: cross-hatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying interfiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease while a force-disease biphasic relationship exhibited F-actin stress fiber network dependence. However, unique to suspended fibers, coiling occurring at the tips of protrusions and not the length or breadth of protrusions displayed the strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on a timescale of hours. 

Contact inhibition of locomotion (CIL), in which cells repolarize and move away from contact, is now established as a fundamental driving force in development, repair, and disease biology. Much of what we know of CIL stems from studies on two-dimensional (2D) substrates that do not provide an essential biophysical cue—the curvature of extracellular matrix fibers. We discover rules controlling outcomes of cell–cell collisions on suspended nanofibers and show them to be profoundly different from the stereotyped CIL behavior on 2D substrates. Two approaching cells attached to a single fiber do not repolarize upon contact but rather usually migrate past one another. Fiber geometry modulates this behavior; when cells attach to two fibers, reducing their freedom to reorient, only one cell repolarizes on contact, leading to the cell pair migrating as a single unit. CIL outcomes also change when one cell has recently divided and moves with high speed—cells more frequently walk past each other. Our computational model of CIL in fiber geometries reproduces the core qualitative results of the experiments robustly to model parameters. Our model shows that the increased speed of postdivision cells may be sufficient to explain their increased walk-past rate. We also identify cell–cell adhesion as a key mediator of collision outcomes. Our results suggest that characterizing cell–cell interactions on flat substrates, channels, or micropatterns is not sufficient to predict interactions in a matrix—the geometry of the fiber can generate entirely new behaviors.