Session M35: Emerging Trends in Soft Microscale Mechanics I

Dynamics and rheology of active biomaterials and gels quantified with optical microscopy tools

How can we quantify the microscale dynamics and mechanics of soft materials that are quickly restructuring in time, exhibit spatial and temporal heterogeneities, and move in all three dimensions? Here, I will discuss optical microscopy techniques that can capture such complex dynamics observed in, e.g., active soft materials. I will highlight the image analysis technique of differential dynamic microscopy and recently developed microscopy modalities and their applications to cytoskeleton networks driven by molecular motors and to suspensions and gels of colloidal rod-shaped particles. Through optical tweezers microrheology and bulk rheology used in combination with video microscopy, we robustly connect observed microscale dynamics with a material's mechanical properties. I will present new methods we are developing to extend differential dynamic microscopy to achieve finer temporal resolution and improved detection of 3D motion.   

Development of hydrogel-based platforms to investigate the mechanics and dynamics of active biological networks

This work focuses on exploring the potential of hydrogel-based materials and devices to control and study the feedback-driven responses of active cytoskeletal composites formed using actin, microtubules, motor proteins, and crosslinkers. We report our development of a measurement platform that uses monodisperse hydrogel microspheres created using microfluidic processing and photopolymerization. The spheres are elastic and are surface functionalized to enable adhesion to both glass substrates and proteins. When encased within an actively-contracting cytoskeletal composite material, the spheres can be deformed due to the stochastic application of contractile, compressive, and shear forces generated by kinesin and/or myosin motors. In this work, we develop protocols to tune the size, composition, and placement of the hydrogel spheres within a microfluidic channel. We show that it is possible to measure the sphere deformation using confocal microscopy and to quantitatively relate the sphere deformation to the applied stresses using a technique we call microsphere-based traction force microscopy, which can be implemented in a high throughput fashion.  This high degree of synthetic control enables us to investigate the relationships between hydrogel geometry and stiffness and the resultant cytoskeletal response to elucidate mechanobiological circuits that underpin cytoskeletal dynamics.   

Deformation of Hydrogel Inclusions by Tunable Motor-Driven Cytoskeletal Composites

The cytoskeleton is a dynamic and versatile network, composed of protein filaments such as semi-flexible actin and rigid microtubules, along with motor proteins, including kinesin and myosin. The forces generated throughout this composite network can range from highly heterogeneous to globally contractile or extensile depending on the concentrations and types of filaments, motors and crosslinkers. However, measuring these forces has proven challenging due to, in large part, the spatiotemporal heterogeneity of the force field. Here, we use synthetic hydrogel inclusions as reporters of motor-generated forces exerted by these composites by observing the extent to which the hydrogels flatten, shrink, or asymmetrically deform during motor activity. In turn, we characterize the impact of hydrogel inclusions on the structure and active dynamics of the composites, revealing increased resilience and connectivity. This unique ability to manipulate hydrogels through the controlled reconfiguration of cytoskeletal composites, and likewise improve the resilience of biopolymer networks using synthetic hydrogels, makes our bio-synthetic platform a promising route towards next-generation materials engineering and biomechanics.   

Curves and Tension Alter the Shapes of Crystals Growing in 2D Fluid Membranes

It is understood that the growth of 2D crystals can be dramatically affected by template curvature. Contrasting crystal growth on a curved substrate, we employ a membrane that integrates the growing crystal to impose out of plane stresses that impart curvature. We show that besides the geometry of the interface, membrane tension, which is dictated by thermal contraction and water permeation, can together control the crystal morphology within the closed, flexible fluid membrane of lipid vesicles. Due to the area-to-volume ratio difference for different size vesicles, which affects the permeation process and thus, tension, crystal shapes show a size dependence: Small vesicles exhibit compact domains while larger vesicles produce floret shapes.  The observations run counter to the trends expected based on colloidal crystals on rigid spherical substrates. Theoretical models, accounting for elasticity and line energy, suggest solid domains with zero-Gaussian curvature form petals within the floret-shaped domains to help release elastic energy under high inflation. Experimental techniques like micropipette aspiration and phase contrast image tracking during controlled cooling can implicate the tension history of real samples. This shape dependence of crystals on vesicle size is ubiquitous among different choices of lipid composition, cooling rate, fluorescent tracer, or even a different system like lipid/copolymer hybrid membrane. The findings here could provide potential insight in industrial and pharmaceutical applications like nanoparticle coatings, drug delivery or even virus assembly.   

The Effect of Anillin on Actomyosin Network Dynamics

The actin crosslinking protein, anillin, has been shown to autonomously constrict actin rings without the presence of motors. This contractililty is reminiscent of actomyosin contraction, which requires a careful balance of connectivity and force-generation. Here, we investigate the effect of anillin crosslinking on global contractility of actomyosin networks. We use fluorescence confocal microscopy to image actomyosin network dynamics and use differential dynamic microscopy and spatial image autocorrelation to quantify the rate of contraction and degree of restructuring. We show that increasing the concentration of anillin slows the rate of global network contraction while at the same time enhancing local bundling and restructuring.   

Cofilin concentration controls cofilactin gel stress response

F-actin is a scaffold protein that provides structural support for the cell and mediates its mechanical behaviors. Actin binding proteins (ABP), such as a-actinin and fascin, link F-actin into a polymer network gel. The increase in crosslinking leads to a general increase in rigidity. As a traditional-actin severing protein, cofilin has been shown to oligomerize at low pH and induce F-actin crosslinking through the formation of di-sulfide bonds. Given its known role in softening F-actin in bending, we sought to explore how F-actin crosslinking and F-actin softening contribute to the network-scale mechanical properties of cofilactin. We therefore perform rheology in the linear and nonlinear regimes of F-actin networks crosslinked by cofilin and compare them to well-known F-actin crosslinkers.  Unlike a-actinin, which is known to strain-stiffen, we find that cofilin crosslinked networks strain-stiffen at low concentration while strain-soften at saturating concentration, suggesting a novel role for cofilin in mediating the mechanics of the F-actin cytoskeleton.   

Using Simulations to Guide the Design of Biotic-Abiotic Machines

Wide availability of purified cytoskeletal proteins and advances in click chemistry have motivated interest in coupling structural and force bearing components of the cell cytoskeleton to inert structural elements to construct actuators powered by chemical potential gradients. The potential design space for such devices is vast, and computational models are called for to steer experiments. Here, we present a simulation framework that couples semiflexible polymer mechanics, molecular motor kinetics, and nonlinear gel elasticity to simulate biotic-abiotic machines. An agent-based description of cytoskeletal protein mechanics is coupled to a finite element mesh, used to simulate the elastodynamics of a passive hydrogel. In pursuit of high throughput simulations, we expedite calculations with thread parallelism and efficient modern algorithms for rapid spatial queries and finite element mesh topology optimization. We will discuss the potential of this model to anticipate emergent properties of a cytoskeletal protein actuator, such as traction forces exerted by the actomyosin gel on a boundary, and process dependence of material properties resulting from repeated cycles of activity.   

Optical tweezers map spatiotemporal force generation in active actin-microtubule composites

The cytoskeleton is a dynamic network of proteins, including actin, microtubules, and their associated motor proteins that enables essential cellular processes such as motility, division, and growth. Previous study has shown co-entangled actin-microtubule networks driven by myosin exhibit organized and controlled contraction dynamics. In contrast, similar actomyosin networks that lack microtubules undergo fast, multidirectional motion and network rupturing. However, the underlying motor-generated forces that give rise to these different behaviors is poorly understand. Here, we combine optical tweezers microrheology, fluorescence microscopy and photo-controlled myosin activity, to measure local contractile forces in active co-entangled actin-microtubule composites. By measuring the forces on beads that are held fixed at different positions throughout the network, with a particular focus on force generation at the network boundary, we determine the stress that the composites can self-generate, and how these stresses propagate through the network from the contractile edge. By simutaneously imaging the actively restructuring filaments in the composite we directly correlate structure and deformation fields of the network to force generation.   

Spatio-temporally reconfigurable photo-activable kinesin driven Active Cytoskeleton Composites

The cytoskeleton is a dynamic network of filaments of varying properties, such as stiff microtubules, tensile actin, and space-filling intermediate filaments working alongside associated proteins like motors and crosslinkers. Inspired by this robust biological system of proteins, we are trying to develop reconfigurable active cytoskeleton composites. In this work, we examine formulation of actin-microtubule composites with photo-activatable crosslinking between kinesin motors. This kinesin driven network has contractile properties which can be tuned by photoactivation and characterized through quantitative fluorescence imaging.   

Excess entropy, microstructure, and rheology in disordered solid suspensions under shear

How do disordered particulate systems yield under shear? The answer to this question is challenging, though we know that particle-scale microscopic interactions and configurations are important for understanding how local rearrangements influence bulk responses. To date, it has been difficult to construct a universal local structural descriptor that can be related to the heterogenous rearrangement dynamics. In supercooled liquids and glasses, recent progress on data-driven structural descriptors has alleviated this issue in some systems. Recent work [Galloway et al. Nat. Phy. 2022] uncovered a connection between the structure and rheology of sheared dense suspensions utilizing the quantity excess entropy. In this work we extend this relationship between structure and rheology to the microstructural-level using local excess entropy. We analyze both experimental and simulated dense suspensions under oscillatory shear and explore how the microscopic structure and rheology evolve as systems approach and surpass yield.   

Dynamics of nearly spherical, multicomponent vesicles in simple shear flow

In biology, cell membranes are often multi-component in nature, made up of multiple phospholipids and cholesterol mixtures that give rise to interesting thermodynamics and fluid mechanics.  Often, these cells are surrounded by fluids whose flows influence their shape and stability. This project deals with the analysis of shear flow around a multi-component vesicle. We consider a nearly spherical giant unilamellar vesicle (GUV) made up of a ternary mixture of a saturated phospholipid, an unsaturated phospholipid, and cholesterol. The bending energy of the vesicle is governed by the Helfrich model and the mixing energy is governed by a Landau-Ginzburg model with an order parameter that represents the phospholipid composition as one marches along a tie line of the ternary phase diagram. We use spherical harmonics basis sets to come up with reduced order equations that solve the Stokes equations inside and outside the vesicle as well as the phospholipid distribution on the membrane surface, in the limit of small deformations (small excess area). We observe a wide range of regimes including tank treading, phase treading, swinging, and tumbling depending on the characteristic dimensionless numbers governing the line tension, average bending stiffness, and flow variables. This talk discusses what gives rise to the behaviors seen.   

Using Mechanochemistry to Visualize the Microballistic Impact of Block Copolymer Films

Current measurement platforms for studying the high-strain-rate impact properties of materials remain limited as they cannot experimentally capture the in-situ deformation behavior of the material with the requisite temporal (ns scale) and spatial (sub-μm scale) resolution. In this work, we address this challenge by studying the microballistic response of an anthracene-based mechanophore-functionalized diblock copolymer material system. Upon rupture of the covalent bond due to a high-velocity impact, this mechanophore exhibits a fluorescence signature that scales with the impact velocity. To demonstrate the utility of this mechanophore for studying the high-strain-rate material response, we impact this material system with microprojectiles at impact velocities ranging from 100 to 500 m/s. AFM measurements reveal permanent deformation at the surface of the impacted sites, indicating that plastic deformation is one of the energy dissipation mechanisms. However, laser scanning confocal microscopy reveals fluorescence information well below the deformed surfaces. More importantly, this subsurface deformation volume resembles a Mach cone that is confirmed by simulations, thus suggesting the significant role of acoustic wave attenuation for energy dissipation in these materials. Finally, we demonstrate that this mechanophore system allows for the three-dimensional visualization of the deformation volume of the material that scales with the impact velocity, thus providing results that cannot be imaged otherwise and offering new insights into microballistic impact studies.