Search for: All records

Abstract The cell migration cycle, well‐established in 2D, proceeds with forming new protrusive structures at the cell membrane and subsequent redistribution of contractile machinery. Three‐dimensional (3D) environments are complex and composed of 1D fibers, and 1D fibers are shown to recapitulate essential features of 3D migration. However, the establishment of protrusive activity at the cell membrane and contractility in 1D fibrous environments remains partially understood. Here the role of membrane curvature regulator IRSp53 is examined as a coupler between actin filaments and plasma membrane during cell migration on single, suspended 1D fibers. IRSp53 depletion reduced cell‐length spanning actin stress fibers that originate from the cell periphery, protrusive activity, and contractility, leading to uncoupling of the nucleus from cellular movements. A theoretical model capable of predicting the observed transition of IRSp53‐depleted cells from rapid stick‐slip migration to smooth and slower migration due to reduced actin polymerization at the cell edges is developed, which is verified by direct measurements of retrograde actin flow using speckle microscopy. Overall, it is found that IRSp53 mediates actin recruitment at the cellular tips leading to the establishment of cell‐length spanning fibers, thus demonstrating a unique role of IRSp53 in controlling cell migration in 3D. 

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. 

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. 

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. 

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. 

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. 

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.