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.
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Rules of contact inhibition of locomotion for cells on suspended nanofibers
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.
more » « less- NSF-PAR ID:
- 10217946
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 118
- Issue:
- 12
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- Article No. e2011815118
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Collagen fibers in the 3D tumor microenvironment (TME) exhibit complex alignment landscapes that are critical in directing cell migration through a process called contact guidance. Previous in vitro work studying this phenomenon has focused on quantifying cell responses in uniformly aligned environments. However, the TME also features short‐range gradients in fiber alignment that result from cell‐induced traction forces. Although the influence of graded biophysical taxis cues is well established, cell responses to physiological alignment gradients remain largely unexplored. In this work, fiber alignment gradients in biopsy samples are characterized and recreated using a new microfluidic biofabrication technique to achieve tunable sub‐millimeter to millimeter scale gradients. This study represents the first successful engineering of continuous alignment gradients in soft, natural biomaterials. Migration experiments on graded alignment show that human umbilical vein endothelial cells (HUVECs) exhibit increased directionality, persistence, and speed compared to uniform and unaligned fiber architectures. Similarly, patterned MDA‐MB‐231 breast cancer cell aggregates exhibit biased migration toward increasing fiber alignment, suggesting a role for alignment gradients as a taxis cue. This user‐friendly approach, requiring no specialized equipment, is anticipated to offer new insights into the biophysical cues that cells interpret as they traverse the extracellular matrix (ECM), with broad applicability in healthy and diseased tissue environments.
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In vertebrate brains, virtually all neural circuits operate inside a dense matrix of axons (fibers) that have a strongly stochastic character. These fibers originate in the brainstem raphe region, produce highly tortuous trajectories, and release serotonin (5-hydroxytryptamine, 5-HT), with other neurotransmitters. They can robustly regenerate in the adult mammalian brain and appear to support neuroplasticity [1], with implications for mental disorders [2] and artificial neural networks [3]. The self-organization of this “serotonergic” matrix remains poorly understood. In our previous study, we have shown that serotonergic fibers can be modeled as paths of fractional Brownian motion (FBM), a continuous-time stochastic process. FBM is parametrized by the Hurst index, which defines three distinctive regimes: subdiffusion (H < 0.5), normal diffusion (H = 0.5), and superdiffusion (H > 0.5). In two-dimensional (2D) shapes based on the adult mouse brain, simulated FBM-fibers (with H = 0.8) have produced regional distributions similar to those of the actual serotonergic fibers [4]. However, increments of superdiffusive FBM trajectories have long-range positive correlations, which implies that a fiber path in one 2D-section depends on its history in other sections. In a major extension of this study, we used a supercomputing simulation to generate 960 fibers in a complex, three-dimensional shape based on the late-embryonic mouse brain (at embryonic day 17.5). The fibers were modeled as paths of reflected FBM with H = 0.8. The reflection was caused by natural neuroanatomical borders such as the pia and ventricles. The resultant regional densities were compared to the actual fiber densities in the corresponding neuroanatomically-defined regions, based on a recently published comprehensive map [5]. The relative simulated densities showed strong similarities to the actual densities in the telencephalon, diencephalon, and mesencephalon. The current simulation does not include tissue heterogeneities, but it can be further improved with novel models of multifractional FBM, such as the one introduced by our group [6]. The study demonstrates that serotonergic fiber densities can be strongly influenced by the geometry of the brain, with implications for neurodevelopment, neuroplasticity, and brain evolution. Acknowledgements: This research was funded by an NSF-BMBF CRCNS grant (NSF #2112862 to SJ & TV; BMBF #STAXS to RM). References: 1. Lesch KP, Waider J. Serotonin in the modulation of neural plasticity and networks: implications for neurodevelopmental disorders. Neuron. 2012, 76, 175-191. 2. Daws RE, Timmermann C, Giribaldi B, et al. Increased global integration in the brain after psilocybin therapy for depression. Nat. Med. 2022, 28, 844-851. 3. Lee C, Zhang Z, Janušonis S. Brain serotonergic fibers suggest anomalous diffusion-based dropout in artificial neural networks. Front. Neurosci. 2022, 16, 949934. 4. Janušonis S, Detering N, Metzler R, Vojta T. Serotonergic axons as fractional Brownian motion paths: Insights Into the self-organization of regional densities. Front. Comput. Neurosci. 2020, 14, 56. 5. Awasthi JR, Tamada K, Overton ETN, Takumi T. Comprehensive topographical map of the serotonergic fibers in the male mouse brain. J. Comp. Neurol. 2021, 529, 1391-1429. 6. Wang W, Balcerek M, Burnecki K, et al. Memory-multi-fractional Brownian motion with continuous correlation. arXiv. 2023, 2303.01551.more » « less
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Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple “cryptic leading edges” within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as “cell polarization barriers,” decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.
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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.
