Session M27: Mechanics of Cells and Tissues II

Active response of metabolically intact isolated nuclei to compression

The nucleus is generally considered to be the stiffest part of the cell with an apparent Young's modulus of ~10 kPa, but cells deform their nuclei as they move through much softer matrices and apply <kPa stresses. Metabolically active nuclei were produced from cells by a centrifugation process that enucleates the cell, leaving behind a cytoplast and producing a nucleus that is wrapped with a plasma membrane and a few hundred nanometers of cytosol (a karyoplast) but no discernible cytoskeleton, endoplasmic reticulum, ribosomes or other large organelles. Karyoplasts have a wrinkled nuclear lamina, as expected since the nuclei transform from flattened structure to spheres as they are centrifuged out of the cell spread on a substrate. If the nucleus is modeled as an elastic object, force-indentation relations lead to an apparent Young's modulus of 5-10 kPa when measured at low indentations that increases with larger indentations. However, new data show that most of the work of compressing the nucleus is dissipated rather than elastically stored, even though nuclei maintain volume and recover shape after large deformations. Dissipation is driven by active mechanisms since depleting ATP by inhibiting glycolysis eliminates most of the dissipation and increases nuclear stiffness. The ATPase motor BRG1 in the chromatin remodeling BAF complex appears to generate the active motions since its inhibition mimics global ATP depletion.   

Complex Rheology in Single Cells: compression stiffening but shear softening

Cells need to be able to withstand mechanical deformation.  Whether in the human body or any other organism, cells are subject to various physical stresses, such as stretching, compression, and shear forces.  This resilience is essential for activities including tissue regeneration, adaptation to changing microenvironment and preventing cell damage or even rupture.  Our aim is to understand the response of cells to external mechanical cues.  For this purpose, we have developed a novel single cell rheology setup that allows us for the first time to make direct comparisons between a living mammalian cell’s response to compression and shear strain.  In this work, I have identified the relative contribution of actin and vimentin intermediate filaments in uniaxial compression experiments on single fibroblasts.  Our findings reveal that individual fibroblasts undergo stiffening under physiologically relevant compressive strains, but the removal of vimentin reduces this stiffening effect.  Furthermore, we present, to our knowledge, the pioneering example of single-cell shear rheology experiments, where we discovered that cells soften when sheared, in stark contrast to their stiffening behaviour under compression.  Finally, we propose a minimal constitutive model to elucidate these phenomena and compare our results to semiflexible polymer models used to explain the mechanics of reconstituted cytoskeletal systems.   

Stoichiometric Model for the Microtubule-mediated dynamics of centrosome and nucleus

The Stoichiometric Model for the interaction of centrosomes with cortically anchored pulling motors, through their associated microtubules (MTs), has been applied to study key steps in the cell division such as spindle positioning and elongation. In this work we extend the original Stoichiometric Model to incorporate (1) overlap in the cortical motors, and (2) the dependence of velocity in the detachment rate of MTs from the cortical motors. We examine the effects of motor overlap and velocity-dependent detachment rate on the centrosome dynamics, such as the radial oscillation around the geometric center of the cell, the nonlinear nature (supercritical and subcritical Hopf bifurcation) of such oscillation, and the nonlinear orbital motions previously found for a centrosome. We explore biologically feasible parameter regimes where these effects may lead to significantly different centrosome/nucleus dynamics. Furthermore we use this extended Stoichiometric Model to study the migration of a nucleus being positioned by a centrosome. This is joint work with Justin Maramuthal, Libin Liu, Reza Farhadifar, Alex Barnet and Michael Shelley.   

In situ single-cell mechanics of mouse preimplantation embryos

The first lineage segregation and self-organization of mammalian embryos take place during the transition from zygote towards blastocyst, where large mechanical deformations can be observed. Yet the role of mechanics in this process is not clear, especially due to the lack of approaches to probe mechanical properties in situ. In this work, we use microrheology to monitor the evolution of mechanical properties of mouse preimplantation embryos at single-cell resolution.   

On the Topology and Dynamics of Breast Cancer Cell Morphologies

A prerequisite to understanding a dynamical system is to understand the underlying topology. In past research our lab has extracted a wide variety of morphological metrics for many in vitro spheroid cells. Since we are dealing with biological systems, these morphological realizations are governed by the expression of phenotypes, which will endow the phase space with a particular topology. To better understand this natural topology, we employed the use of an adversarial autoencoder, a neural network, to reduce the dimensionality and better understand the phase space's topology. In our work, we were able to reduce this morphological phase space to two dimensions while still being able to reconstruct the encoded vectors back to their corresponding morphological metrics. We then characterize the similar topologies and probe different questions about the space: What do individual cell trajectories look like, i.e. what do the dynamics look like in this phase space? Are there regions where the 4 main morphological phenotypes are characterized? How strongly are the embedded vectors bound to their neighbors? Do different priors significantly affect the topology?   

Cancer Cell Footprinting and Exploration in Micropatterned Fibrinogen Mazes

During metastasis, cancer cells utilize the surrounding Extracellular Matrix (ECM) to grow and move. Tumors actively remodel surrounding ECM to promote this migration. In this study, we placed MDA-MB-231 cancer cells onto 2-dimensional micropatterned fibrinogen coated coverslips with circular maze structure. The maze connectivity and cell density were both varied as a model for local cancer environments within ECM. Simulations were also completed to model the cells as active particles with random walk, tuning the properties of particle adhesion and maze structure to compare with cellular migration and test for self-guided motion. Results suggest that some cells are weakly interacting, however they tend to develop footprints quickly after adhering to fibrinogen. Cells then use each other's footprints as local bridges towards new locations. Other cells tend to be highly migratory with stochastic behavior which more closely matches simulations. Future directions of this work may include seeding cells in a centralized location on the fibrinogen maze to better model metastasis, as well as testing cancer cell interaction with immune cells on fibrinogen mazes.   

Deciphering Tumor Heterogeneity in Triple-Negative Breast Cancer: The Crucial Role of Dynamic Cell-Cell and Cell-Matrix Interactions

Epithelial tissue development, wound healing, and tumor progression are intricate processes influenced by the dynamic interplay between cells and the surrounding extracellular matrix. These physical cell-cell and cell-matrix interactions are especially complex in triple-negative breast cancer (TNBC), which displays dramatic spatiotemporal heterogeneity over the course of cancer progression. Consequently, TNBC is highly aggressive and associated with poorer patient prognosis compared to other breast cancer subtypes, posing a significant clinical challenge due to the limited availability of targeted therapies. To address this issue, we have established a scalable method that captures the spatial heterogeneity of multicellular cluster-induced matrix deformations. We find that induction of the epithelial-mesenchymal transition dramatically alters the 3D mechanophenotype of mammary cell clusters through a progressive loss of protrusive and circumferential tractions to more localized contractile tractions. To reveal how individual cell differences may contribute to overall tumor progression, we used a microfluidic model to strat­egically engineer tumors with precise composition using noninvasive and invasive clonal TNBC cell subpopulations exhibiting epithelial and mesenchymal characteristics, respectively. Our findings revealed that the physical presence of invasive clonal cells in multiclonal tumors could dramatically enhance overall tumor growth, invasion, and escape to a nearby vessel, which could be delayed by selectively targeting the invasive subpopulation. Moreover, we used a reverse-engineering approach to locally pattern regions of noninvasive and invasive cell subpopulations, revealing how spatial heterogeneity at the cellular level affects the dynamics of tumor progression. Overall, our work enables the systematic interrogation of breast tumor heterogeneity in the context of dynamic cell-cell and cell-matrix interactions, providing insight into the physical basis of TNBC progression. These findings shed light on how selective targeting of tumor subpopulations with distinct mechanophenotype and invasion signatures may offer a promising therapeutic strategy for managing heterogeneous tumors.   

Fluidization of jammed epithelia by noninvasive carcinoma in a heterogeneous tumor model.

Cellular jamming has been found useful in describing biological systems, including solid tumors. We use spheroids, tumor approximations, composed of isogenic cell lines to investigate jamming across the epithelial to mesenchymal transition. In particular, we investigate spheroid fusion, which can capture both 'fluid-like' and 'solid-like' dynamics – by investigating the macroscopic dynamics of the fusion, we are able to elucidate the importance of single cell properties as well as emergent behavior. Cell shapes are found to reflect and dictate fusion capabilities. In each cell line investigated we perturb various cell properties that may influence spheroid fluidity, including nuclear stiffness and cell proliferative capacity. Additionally, we investigate the roles of cell-cell adhesion and cell contractility on fusion. These studies reveal that cell deformability promotes fusion and that increased cell-cell adhesion, and conversely decreased cell contractility, aids in jamming a spheroid system. In addition to characterizing fusion in homotypic spheroids, we also study heterotypic spheroids from the same cell lines. In this system, fluid-like cells readily fluidize otherwise solid-like spheroids and thus are able to unjam the cellular collective.   

Modeling Nonlinear Dynamics of Cells in Confinement

During physiological processes like tissue development and metastasis, migrating cells navigate various constrictions that affect their behaviors. Recent work on the confined migration of cancerous MDA-MB-231 cells in two-state adhesive micropatterns suggests their motion can be described by a limit cycle, i.e., persistently hopping from side to side, while they remain stationary on average in rectangular confinements [1]. Moreover, cells in two-state geometry have a nonlinear acceleration-velocity relationship. By contrast, healthy MCF10A cells plated on two-state micropattern can be described as bistable, with only noise-induced hopping between the two states [1]. Can we explain these different behaviors with a single model of cell-extracellular matrix interaction? What cell properties determine whether cells limit-cycle or are bistable? To investigate these questions, we build a computational model within the phase field framework. Our model incorporates a simple cell-micropattern coupling where cells decrease their polarity when they leave the micropattern. We also include stochastic dynamics of cell protrusions. We can recapitulate, depending on cell parameters and the geometry of the micropattern, limit cycle, bistable, or simple linear behavior. Our model predicts that larger cells and cells with lower tension are more likely to develop a limit cycle, while smaller and stiffer cells are more likely to be bistable. Other key factors affecting cell migration are the time between protrusions and the level of noise in protrusion amplitude.

[1] David Bruckner, et. al, Nature Physics, 15, 595-601 (2019)   

Long-Wavelength Interface Fluctuations Between Different Cell Populations are Suppressed by Friction

The study of interfaces between different cell populations or between cells and their environment is of great importance due to its relevance to various biological processes, such as compartmentalization during embryonic development and interfacial instability during cancer metastasis. A common theoretical framework to understand these phenomena is treating different cell populations as immiscible fluids with interfaces controlled by effective surface tensions arising from the interactions between the cells. Capillary Wave Theory (CWT) is usually the theoretical basis for these studies. However, special features in cellular systems such as complex cell-cell interactions and non-equilibrium can result in non-regular interface phenomena different from the predictions of regular CWT. In these cellular systems, another general feature is the high dissipation due to friction between cells and their environment. In this study, we use vertex model to simulate the interface between two groups of deformable cells moving under Brownian Dynamics and show that the interface fluctuation spectrum for these friction-dominated systems is qualitatively different from the prediction of CWT, with fluctuations in the long-wavelength regime being suppressed. These results greatly change our understanding of interfacial phenomena in overdamped systems and point out better ways to estimate surface tension.   

Model of cell-cell adhesions as cytockeleton-to-cytoskeleton linkers

Adhesion molecules such as E-cadherin are known to contribute to the overall mechanics of multicellular systems by directly coupling cells through their cytoskeletons. Even though adhesion molecules interact directly with the cytoskeletons of the adherent cells, many models still describe them by an effective affinity between cell membranes rather than as a direct interaction between cytoskeletons. To understand how tissues behave mechanically and how forces are propagated between cells, we present a model of cell-cell coupling that takes into explicit account the dynamics of adhesion molecules and their interaction with the cytoskeleton. In our models, we describe cellular adhesions as a one-dimensional field that couples to fields describing the cytoskeleton of two adjacent cells. We capture forces exerted between adhesions and the cytoskeleton, turnover of both adhesions and cytoskeleton, cytoskeletal activity, and the dynamics of adhesion molecules binding and unbinding to actin. Importantly, we show that a population of adhesions connecting two cytoskeletal fields can compel the cytoskeletons to coordinate their dynamics.   

Microrheology of suspended cells using optical traps

Mammalian cells are highly complex, structured, active soft materials. The cytoskeleton is largely responsible for activity and mechanical response of whole cells. The cell nucleus typically occupies a substantial fraction of the cell volume and is itself an active material with a complex structure. We here report on measurements of viscoelastic properties as well as non-equilibrium fluctuations of both, suspended cells and isolated cell nuclei, using optical traps. Combining manipulation experiments with confocal microscopy, we also observe how drug interference affects mechanical response and activity.   

Combinatorial role of mechano-chemical cues in cell mechanics and state transitions

Cells typically reside in complex physiological milieus which exhibit spatiotemporally heterogeneous material properties and diverse signalling cues. Two such widely studied microenvironmental parameters are mechanical stimuli and chemical sensing, which are themselves modulated by cellular activity. For instance, complex physiological processes such as embryogenesis and tissue patterning feature dynamic ECM remodelling and chemotactic migration by cells. However, conventional approaches largely study these players in isolation, despite substantial experimental evidence hinting at extensive crosstalk and cross-regulation. Our present work will explore a combinatorial mechano-chemical phase space that influences the mechanical state of cells and regulates cell state transitions. Leveraging quantitative measurements and phenotypic assays at both the collective and single cell levels, we will characterise this conjoined axis.