Schedule for: 23w5120 - Mechanics of Cells and Polymer Networks: Bridging Theory, Simulation and Experiment

A buffet dinner is served daily between 5:30pm and 7:30pm in Vistas Dining Room, top floor of the Sally Borden Building.

Informal Gather in BIRS Lounge, located on the 2nd floor of PDC Building.

Breakfast is served daily between 7 and 9am in the Vistas Dining Room, the top floor of the Sally Borden Building.

A brief introduction to BIRS with important logistical information, technology instruction, and opportunity for participants to ask questions.

The cell cytoskeleton is a composite of protein filaments, including actin, microtubules and intermediate filaments, as well as their associated crosslinking proteins, that is pushed out-of-equilibrium by molecular motors to mediate wide-ranging processes from migration to morphogenesis. The cytoskeleton is, thus, paradigmatic active matter and its composite nature is one of its hallmarks. Yet, state-of-the-art active matter focuses on single force-generating components and substrates. Here, we engineer programmable composites of actin filaments and microtubules that can be versatilely crosslinked and driven by dual motors, kinesin and myosin, to contract, flow, and restructure into diverse morphologies, ranging from interpenetrating scaffolds to phase-separated clusters. We couple optical tweezers microrheology with differential dynamic microscopy and particle-image-velocimetry to demonstrate that composites exhibit emergent rheological properties that arise from cooperativity between actin and microtubules as well as competition between myosin and kinesin. Microtubules confer enhanced force resistance, elastic memory, and ordered dynamics to myosin-driven actin networks, while kinesin and myosin motors compete to delay composite restructuring and suppress de-mixing. Moreover, we find that the nonlinear force response of active composites exhibits emergent high-strength and multi-modal stiffening at intermediate kinesin concentrations and strain rates that vanish at the low and high limits. Our composite designs, along with our robust microscale measurements, offer a powerful platform for active matter interrogation and discovery, and may prove foundational for diverse materials applications from wound-healing to soft-robotics.

On small length-scales, the mechanics of soft materials may be dominated by their interfacial properties as opposed to their bulk properties. These effects are described by equilibrium models of elasto-capillarity and wetting. In these models, interfacial energies and bulk material properties are held constant. However, in biological materials, including living cells and tissues, these properties are not constant, but are ‘actively’ regulated and driven far from thermodynamic equilibrium. As a result, the constraints on work produced during the various physical behaviors of the cell are unknown. Here, by measurement of elasto-capillary effects during cell adhesion, growth and motion, we demonstrate that interfacial and bulk parameters violate equilibrium constraints and exhibit anomalous effects, which depend upon a distance from equilibrium. However, their anomalous properties are reciprocal, and thus in combination reliably define energetic constraints on the production of work arbitrarily far from equilibrium. These results provide basic principles that govern biological assembly and behavior.

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (vertical height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual vertical oscillations and the collective spatiotemporal wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for both the oscillatory growth of individual podosomes and the wave-like dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers within the cluster drives wave-like mesoscale coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments (targeting myosin activity, actin polymerization, and mechanosensitive Rho-ROCK pathway) and the impact of microenvironment stiffness on chemo-mechanical waves. Overall, our integrated theoretical and experimental approach reveals how collective wave dynamics arise from the coupling between chemo-mechanical signaling and actin diffusion, shedding light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Lunch is served daily between 11:30am and 1:30pm in the Vistas Dining Room, the top floor of the Sally Borden Building.

Meet in foyer of TCPL to participate in the BIRS group photo. The photograph will be taken outdoors, so dress appropriately for the weather. Please don't be late, or you might not be in the official group photo!

A buffet dinner is served daily between 5:30pm and 7:30pm in Vistas Dining Room, top floor of the Sally Borden Building.

In this talk, I will introduce our recent works in quantitatively studying how cells interact with their surrounding extracellular matrix. In particular, I will discuss the critical impact of the matrix nonlinear elasticity in regulating cell-ECM mechanical interactions. For example, the nonlinear stiffening nature of the ECM enables a significantly extended stress dissipation, and the creation of a stiff shell surrounding the cell. These effects have important role in regulating cell-cell communications, as well as mechanobiology of cell-ECM interactions. In addition, I will also show results revealing the critical role of interfacial curvature on cell migratory behaviors.

Breakfast is served daily between 7 and 9am in the Vistas Dining Room, the top floor of the Sally Borden Building.

Cell migration is a critical process underlying proper tissue maintenance. While a soft nucleus allows a cell to squeeze through small pores, the resulting physical stress can lead to nuclear damage and genomic variability. We have shown that the cytoskeletal intermediate filament protein vimentin protects against DNA damage during migration. Here, we present a mechanical model in which vimentin modulates force transmission in a substrate-stiffness dependent manner, helping fine-tune the cell’s response to different matrix stiffnesses. Further, we present new results on how vimentin mediates the microtubule network and mediates extracellular matrix remodeling to facilitate cell motility.

Integrin-based focal and fibrillar cell-extracellular matrix adhesions (FA and Fib) are critical for the matrix rigidity sensing and fibronectin fibrillogenesis, respectively. Besides the association with the actin cytoskeleton, they interact with microtubules through molecular complexes containing KANK family proteins. Here, we compare the mechanisms underlying the FA and Fib dynamics. Microtubule disassembly or uncoupling from the adhesions by disruption of KANK-mediated link results in augmentation of FA but elimination of Fib. Thus, microtubules negatively regulate FA and positively - Fib. The underlying mechanism in both cases is a release from adhesion-uncoupled microtubules of Rho activator GEF-H1, triggering Rho-ROCK-myosin-IIA signaling cascade. Consequent burst of actomyosin contractility promotes the growth of mechanosensory FA and the disassembly of Fib. The same mechanism is involved in the disruption of the individual FA upon microtubule targeting by local optogenetic activation of KANK1. Here, microtubule attachment (weakening FA) is succeeded by microtubule withdrawal and a local GEF-H1-dependent accumulation of myosin-II filaments at the proximal end of the FA. The myosin-dependent traction force triggers the adhesion detachment, centripetal sliding, and disassembly. While both FA and Fib can be formed by cells on planar substrate, Fib formation is specifically activated (in a myosin-II independent manner) by physiologically relevant micropatterns – decellularized extracellular matrix, electrospun nanofibers, or edges of microfabricated ridges. Consistently, the treatments increasing membrane/cortical tension disassemble fibrillar adhesions, but not focal adhesions. Thus, selective sensitivity of fibrillar adhesions to microtubule uncoupling and subsequent local myosin II activation can be explained by the myosin II-driven increase of membrane/cortical tension.

The mechanical properties of tissues can be studied either at the macroscopic scale of the tissue or at the mesoscoscopic scale of the cell. The macroscopic description must include the non conservation of the cell number due to cell division and cell death. It leads to a non-conventional hydrodynamic theory and to spontaneous flows due to the cell activity. At the mesoscopic scale, we use a so-called vertex model which starts from a geometrical description of an individual cell. We will show on a simple example how one can derive the macroscopic properties of the tissue by a coarse-graining of the mesoscopic vertex model. Our important result is an explicit calculation of the cell pressure in the homeostatic state of a confined growing tissue: it can be negative and depends on the existence of mechanical residual stresses. Within the coarse-grained model, we determine the linear rheological properties of the tissue as a function of frequency and the non-linear rheology at large shear rate.

Lunch is served daily between 11:30am and 1:30pm in the Vistas Dining Room, the top floor of the Sally Borden Building.

A buffet dinner is served daily between 5:30pm and 7:30pm in Vistas Dining Room, top floor of the Sally Borden Building.

The mechanical properties of soft materials can be probed on small length scales by microrheology, commonly done by tracking embedded micrometer-sized beads. We here introduce filament-based microrheology (FMR) using high-aspect-ratio semi-flexible filaments as probes. Such quasi-1D probes are much less invasive than beads due to their small cross sections. By imaging transverse bending modes, we can simultaneously probe multiple length scales. As a proof of principle, we use semiflexible single-walled carbon nanotubes (SWNTs) as probes that can be accurately and rapidly imaged based on their stable near-IR fluorescence. We find that the viscoelastic properties of sucrose, polyethylene oxide, and hyaluronic acid solutions measured in this way are in good agreement with those measured by conventional micro- and macrorheology. When probing cells, it can be advantageous to avoid introducing probe particles altogether. We show that one can directly use fluctuation analysis of native cytoskeletal filaments, in this case microtubules, to perform intracellular microrheology when filament properties are known. Alternatively, one can probe filament mechanics when the response properties of the embedding cytoplasm are known. The latter approach is useful because microtubule mechanics in living cells are believed to be regulated by post-translational modifications, but are extremely difficult to probe directly, while fluctuations are difficult to interpret because they are generated by active forces in a surrounding cytoplasm with poorly understood material properties. We discovered that polyglutamylation, a post-translational modification enriched on microtubule networks that need to withstand large mechanical forces such as those in axons or cilia, significantly increases microtubule stiffness in living cells.

"Actomyosin is a dynamic network of interacting proteins that reshapes and organizes in a variety of structures, including bundles, clusters, and contractile rings, depending on its function. Motivated by observations from the reproductive system of the roundworm C. elegans, we use an agent-based modeling framework to simulate interactions between actin filaments and myosin motor proteins inside cells. We develop techniques based on topological data analysis, network theory, and spatial statistics to understand time-series data extracted from these filament network interactions, as well as from fluorescence experiments. These measures allow us to compare the filament organization resulting from myosin motors with different properties. In particular, we study how different myosin motor properties may serve to regulate various actin organizations, as observed in vitro and in vivo. Recently, we have been interested in understanding actin organization driven by the force-sensitive unconventional myosin VI motor. Joint work with Adriana Dawes."

Breakfast is served daily between 7 and 9am in the Vistas Dining Room, the top floor of the Sally Borden Building.

Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the mechanics of the extracellular environment. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force. It is unclear how the cell manages the plasticity of the FA structure and the associated traction force for accurate mechanosensing. In synergy with experimental tests, we developed a PDE-based mathematical model of FA growth that integrates the contributions of the branched actin network and stress fibers (SFs). This model provides a unified framework that quantitatively explains two types of mechanosensing activities: i.e., durotaxis and viscotaxis that the cell prefers to migrate toward the stiffer ECM and the more viscous environment, respectively.

Two important mechanisms for cellular self-organization include liquid-liquid phase separation of protein species to form membraneless organelles and self-organization of cytoskeletal filaments into larger-scale arrangements such as the actin cortex and microtubule mitotic spindle. We demonstrate that microtubule-crosslinking proteins, MAP65 and PRC1, can self-assemble into liquid-like condensates that can catalyze spatially controlled nucleation and growth of microtubule asters with control over the aster organization. Microtubules grown from gel-like condensates can reverse the gelation, controlling the material properties. This interplay between protein condensates and the cytoskeleton could allow for dynamic reorganization of the cell interior in space and time.

Articular cartilage is a remarkable material able to sustain millions of loading cycles over decades of use outperforming any synthetic substitute. Crucially, how extracellular matrix constituents alter mechanical performance, particularly in shear, remains poorly understood. Here, I will describe experiments and theory in support of a rigidity percolation framework that quantitatively describes the structural origins of cartilage’s shear properties and how they arise from the mechanical interdependence of the collagen and aggrecan networks making up its extracellular matrix. This framework explains that near the cartilage surface, where the collagen network is sparse and close to the rigidity threshold, slight changes in either collagen or aggrecan concentrations, common in early stages of cartilage disease, create a marked weakening in modulus that can lead to tissue collapse. More broadly, this framework provides a map for understanding how changes in composition throughout the tissue alter its shear properties and ultimate in vivo function.

Lunch is served daily between 11:30am and 1:30pm in the Vistas Dining Room, the top floor of the Sally Borden Building.

A buffet dinner is served daily between 5:30pm and 7:30pm in Vistas Dining Room, top floor of the Sally Borden Building.

Spinal cord injury is a dynamic process that is difficult to recapitulate in controlled environments, both in vitro and ex vivo). In order to characterize the time-dependent breakdown in blood-spinal cord barrier permeability and subsequent progression of glial scarring that alters the mechanics of parenchymal tissue, our laboratory has created a perfused ex vivo spinal cord injury model. The model includes a bioreactor capable of measuring the dynamic mechanical properties of the extracellular matrix following injury. The new system overcomes the limitations of current slice models that are unable to incorporate blood flow and in vivo studies which are incapable of continuous measurement of blood flow and extracellular matrix mechanics. Damage to the blood-spinal cord barrier (BSCB) following SCI is a key mediator of the secondary phase of SCI which causes increased inflammatory levels and cell death that correspond to changes in mechanics, though currently the relationship between barrier permeability and extracellular matrix mechanics is unclear. This system will test our hypothesis that the altered blood flow in the spinal cord alters extracellular matrix mechanics during glial scar formation. Moreover, using magnetically active hydrogels, we describe a method to mimic the time-dependent changes in mechanics in an in vitro platform. These studies provide insight into the interplay between matrix mechanics and cell response that leads to glial scarring and subsequent inability to regenerate the injured spinal cord.

The forces which position and orient the spindle in human cells remain poorly understood due to a lack of mechanical measurements. I will describe our recent work using magnetic tweezers to measure the force on human mitotic spindles. Combining the spindle’s measured resistance to rotation, the speed it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to ~1 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindle’s resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and positioning, and demonstrates the central role of cortical dynein clustering.

Breakfast is served daily between 7 and 9am in the Vistas Dining Room, the top floor of the Sally Borden Building.

The organization and dynamics of chromatin is essential for the regulation of gene expression. Transcription factors (TFs) regulate gene expression by binding to specific consensus motifs within enhancers or promoter-proximal regions. The mechanism by which TFs bind to their cognate chromatin targets within a complex and heterogenous nuclear environment to assemble the transcriptional machinery at specific genomic loci remains elusive. Single-molecule tracking (SMT) has emerged as a powerful approach to explore chromatin and transcription factor dynamics and their interaction in living cells. Using single-molecule tracking, coupled with machine learning-based analysis, we show that chromatin displays two distinct low-mobility states. Our experimental observations are consistent with a minimal active copolymer model for interphase chromosomes. Remarkably, we find that a diverse set of transcription factors, transcriptional co-regulators, architectural proteins, and remodelers also exhibit two distinct low-mobility states, reflecting the mobility of chromatin. Ligand activation results in a marked increase in the propensity of steroid receptors to bind in the lowest-mobility state. Mutational analysis reveals that interactions with chromatin in the lowest-mobility state require an intact DNA binding domain and oligomerization domains. We further find that, in the case of the glucocorticoid receptor, an intrinsically disordered region is a key determinant of the second low mobility state. Interestingly we find that these states are not spatially separated as previously believed, but individual H2B and bound-TF molecules can dynamically switch between them on time scales of seconds. We then used single-molecule tracking to directly measure the interaction dynamics of a broad spectrum of transcription factors in live cells. We found that TFs follow power-law distributed binding times, with TF molecules of different mobilities exhibiting different dwell time distributions, suggesting that the mobility of TFs is intimately coupled with their binding dynamics. Together, our results elucidate how TF and chromatin mobility regulates transcriptional activation in mammalian cells.

Intermediate filaments are one of the components of the cytoskeleton; they are involved in cell mechanics, signaling and migration. The organization of intermediate filaments in networks is the major determinant of their functions in cells. Their spatio-temporal organization results from the interplay between assembly/disassembly processes and different types of transport. An overview of mathematical models used to investigate important mechanisms such as the filament elongation or intracellular transport of filaments will be presented. Dallon et al. Stochastic modeling reveals how motor protein and filament properties affect intermediate filament transport. Journal of Theoretical Biology 464: 132-148 (2019). Dallon et al. Using Fluorescence Recovery After Photobleaching data to uncover filament dynamics. PLoS Computational Biology 18 (2022). Mücke et al. A general mathematical model for the in vitro assembly dynamics of intermediate filament proteins. Biophysical Journal, 121: 1094-1104 (2022). Park et al. Models of vimentin organization under actin-driven transport. Physical Review E, 107:054408 (2023). Portet et al. Keratin dynamics: modeling the interplay between turnover and transport. PLoS ONE, 10: e121090 (2015). Portet et al. Deciphering the transport of elastic filaments by antagonistic motor proteins. Physical Review E, 99: 042414 (2019). Portet et al. Impact of noise on the regulation of intracellular transport of intermediate filaments. Journal of Theoretical Biology. 111183 (2022). Schween et al. Dual-wavelength stopped-flow analysis of the lateral and longitudinal assembly kinetics of vimentin. Biophysical Journal 121: 3850-3861 (2022).

Fiber networks are characterized by complex disordered structures as well as nonlinear elasticity and plasticity. Interestingly, a lot can be learnt about their mechanical properties by using simple discrete lattice models. In this talk, I will review how recent work based on diluted lattice models explained multiple aspects of fiber networks, in terms of their linear and nonlinear rigidity transitions, unusually fracturing processes, and new topologically protected floppy modes. I will also discuss perspectives on bridging these theories to experimental systems.

Lunch is served daily between 11:30am and 1:30pm in the Vistas Dining Room, the top floor of the Sally Borden Building.

A buffet dinner is served daily between 5:30pm and 7:30pm in Vistas Dining Room, top floor of the Sally Borden Building.

Nuclei are generally the stiffest organelle in the cell, and their large elastic modulus is thought to be due to a combination of polymer networks formed by chromatin and the 2D lamin network underlying the nuclear membrane. Direct measurement of nuclear stiffness within the cell is complicated by the surrounding cytoskeleton, and isolating nuclei by disrupting the cell results in diffusion of solutes through the nuclear pores, loss of ATP-dependent active processes, and osmotic dysregulation that might affect nuclear mechanics. Therefore, metabolically active isolated nuclei were produced from live cells by a centrifugation process that enucleates the cell. This process leaves behind a cytoplast and produces a nucleus that is wrapped by a plasma membrane and a thin layer of cytosol (a karyoplast), but no discernible cytoskeleton, endoplasmic reticulum, ribosomes or other large organelles. The metabolic activity within this membrane-wrapped nucleus remains intact for at least 12 hours after isolation. Force-indentation curves measured by atomic force microscopy show that the apparent Young's modulus of these nuclei is on the order of 5 to 8 kilopascal. This large apparent Young's modulus contrasts with the ability of cells to deform the nucleus with acto-myosin dependent stresses less than 200 Pa. The large Young’s modulus inferred from force-indentation curves assumes a purely elastic object, but comparison of the force-indentation and force-retraction curves from AFM studies shows that most of the work of nuclear indentation is dissipated. Repeated deformations show little or no weakening of the nucleus and very little rate dependence, suggesting that the material is not a passive viscoelastic body. In contrast, treatment with a glycolysis inhibitor nearly eliminates the dissipation, suggesting that it depends on active processes within the nucleus.

From foods to bio-inks to cement hydrates, soft particulate gels can be composed of various types of particulate matter (proteins, polymers, colloidal particles, or agglomerates of various origins) embedded in a continuous fluid phase. The solid components are self-assembled to form a porous matrix, providing rigidity and control of the mechanical response, even at low solid content. The rheological response and gel elasticity are direct functions of the particle volume fraction, however, the diverse range of different functional dependencies reported experimentally has challenged all efforts to identify general scaling laws. I will discuss a hidden hierarchical organization of fractal elements that controls the viscoelastic spectrum of these materials, and which is associated with the spatial heterogeneity of the solid matrix topology. The fractal elements form the foundations of a viscoelastic master curve, which we construct using large-scale 3D microscopic simulations of model gels, and can be described by a recursive rheological ladder model over a range of particle volume fractions and gelation rates. The hierarchy of the fractal elements provides the missing general framework required to predict the gel elasticity and the viscoelastic response of these ubiquitous complex materials.

Breakfast is served daily between 7 and 9am in the Vistas Dining Room, the top floor of the Sally Borden Building.

Collagen is the predominant structural protein in humans, representing over 25% of protein mass in our bodies. Its unique triple-helical structure has the ability to assemble into biomechanically important, higher-order structures, such as the extracellular matrix and connective tissues including tendon. How amino-acid sequence prescribes local mechanics of collagen is not known, yet is key for understanding mechanobiological signalling and remodelling of strained and damaged tissues. We are investigating this link between sequence and mechanics at the single-molecule level, using atomic force microscopy (AFM) imaging of collagens [1] and centrifuge force microscopy (CFM) [2] for high-throughput force measurements. In this talk, I’ll present our first mapping of sequence to mechanics, and share insight into how this might influence the properties driving self-assembly of collagens into higher-order structures. I’ll also highlight the importance of environmental conditions on collagen’s mechanics and stability at the single-molecule level. [1] A. Al-Shaer, A. Lyons et al. Biophysical Journal 120, 4013-4028 (2021) [2] M.W.H. Kirkness and N.R. Forde. Biophysical Journal 114, 570–576 (2018)

Nanostars are multi-armed DNA structures that can condense into viscoelastic liquid phases through interactions of single-stranded sticky ends that decorate each arm. These phases are analogous to biological condensates, but are more colloidal in that each nanostar in the dense phase solely occupies its pervaded volume, rather than entangling in a polymeric fashion with its neighbors. As a result, nanostar liquids are tenuous and highly programmable through control of the sticky-ends; have viscoelastic behaviors sensitive to particle valence and the semi-flexible nature of double-stranded DNA; and are functionalizable through sequence-specific interactions with solutes. I will discuss various aspects of these DNA liquids, including recent work exploiting particle valence and/or heterogeneous bonding schemes to control nanostar phase and mechanical behavior; advances in understanding and controlling the formation of nanostar liquid droplets; and means to active nanostar droplets through enzymatic interactions.

Osteoarthritis, the degradation of cartilage that cushions joints, is a major cause of disability, suffering, and lost productivity. A thorough understanding of how patient symptoms arise as a result of changes in the composition of cartilage is essential for diagnosis of symptoms’ root cause and for the design of more effective regenerative medicine and prosthetic tissue. While poroelasticity theory has been highly successful in accounting for the resistance of cartilage to volume change, cartilage shear mechanics has received comparatively little attention. Over the past few decades, rigidity percolation has emerged as a valuable tool for understanding the shear mechanics of semiflexible biopolymer networks, such as the type II collagen in cartilage. In my talk, I will discuss new modeling techniques to address the role of local concentration fluctuations in articular cartilage, and to describe the highly nonlinear elastic response of cartilage under large strain. I will begin by presenting recently published results demonstrating the ability of structural correlation in dilute lattice models of semiflexible biopolymer networks to non-monotonically vary the fraction of retained bonds in the network at which rigidity percolation occurs. I will argue for a micromechanical explanation of this phenomenon in terms of spatial correlations in non-affine rearrangements. Next, I will discuss a new modeling paradigm in which a discrete network, used to model the type II collagen scaffold in articular cartilage, is superposed with a finite element mesh, used to describe the mechanics of the polyelectrolyte gel in which the collagen is immersed. I will exhibit the ability of this model to mimic two important experimental phenomena: the initial drop in the storage modulus of articular cartilage upon large uniaxial compression, and the strain hardening of articular cartilage under large shear.

5-day workshop participants are welcome to use BIRS facilities (TCPL ) until 3 pm on Friday, although participants are still required to checkout of the guest rooms by 11AM.