Session B10: Focus Session: Mechanics of Cells and Biological Networks II

Matrix mimicry and nuclear biophysics: biostructures are maintained where stresses are high

You are more collagen than any other protein. We will describe various collagenous assemblies as thin and thick matrices that help elucidate how important matrix physical properties are to cells. We start with tissues analyses that reveal power law scaling of collagen levels versus of the stiffness of real tissues conform to polymer physics. Our unbiased `omics methods also reveals similar scaling for a main structural protein of the nucleus, a filamentous coiled-coil protein akin to collagen but in the same family as keratin in your hair and nails. This nuclear protein called lamin-A protects DNA from stresses that differ between tissues as stiffness does. Ultimately, the data suggests lamin-A is mechanosensitive in ways similar to collagen: degradation and turnover is repressed by stress -- which is distinct from the conventional ``stress it and break it'' of dead materials.   

Response of microscale cell/matrix constructs to successive force application in a 3D environment

Mechanical dilation of arteries by pulsatile blood flow is directly opposed by coordinated contraction of a band of smooth muscle tissue that envelops the vessels. This mechanical adaptation of smooth muscle cells to external loading is a critical feature of normal blood vessel function.~While most previous studies on biomechanical systems have focused on single cells or large excised tissue, we utilize a device to apply forces to engineered smooth muscle microtissues. This device consists of arrayed pairs of elastomeric micro-cantilevers capable of magnetic actuation. Tissues are formed through self-assembly following the introduction of cell-infused collagen gel to the array. With this system, we are able to dynamically stretch and relax these sub-millimeter sized tissues. The timing and magnitude of the force application can be precisely controlled and thus can be used to mimic a wide range of physiological behavior. In particular, we will discuss results that show that the interval between successive force applications mediates the both the subsequent mechanical and active dynamics of the cell/matrix composite system. Understanding this process will lead to better understanding of the interplay between cell and extracellular matrix responses to mechanical stimulus at a novel length scale.   

High contrast single molecule tracking in the pericellular coat

The pericellular coat is a robust, hydrated, polymer brush-like structure that can extend several micrometers into the extracellular space around living cells. By controlling access to the cell surface, acting as a filter and storage reservoir for proteins, and actively controlling tissue-immune system interactions, the cell coat performs many important functions at scales ranging from the single cell to whole tissues. The cell coat consists of a malleable backbone - the large polysaccharide hyaluronic acid (HA) - with its structure, material properties, and ultimately its bio-functionality tuned by a diverse set of HA binding proteins. These proteins add charge, cross-links and growth factor-like ligands to the coat To probe the dynamic behavior of this soft biomaterial we have used high contrast single molecule imaging, based on highly inclined laser illumination, to observe individual fluorescently labeled HA binding proteins within the cell coat. Our work focuses on the cell coat of living chondrocyte (cartilage) cells, and in particular the effect of the large, highly charged, protein aggrecan on the properties of the coat. Through single molecule imaging we observe that aggrecan is tightly tethered to HA, and plays an important role in cell coat extension and stiffening.   

Dynamic non-linear response of cross-linked actin networks: an energy dissipation approach

Cross-linked bio-polymer networks that primarily maintain the shape and rigidity in eukaryotic cells show striking non-linear mechanical properties. Here, we study the steady-state energy dissipation ($E_{diss}$) over a complete sinusoidal shear strain cycle for a macroscopic assembly of reconstituted network of actin filaments cross-linked with Filamin A, over wide range of strain amplitude and frequency values. For small values of the applied strain amplitudes (linear regime) $E_{diss}$ increases monotonously with the increasing frequency over the entire frequency range studied but in the non-linear regime (larger applied strain amplitudes), a clear saturation in $E_{diss}$ is observed at higher frequencies. Also, the normalized dissipated energy distribution binned over the fixed strain intervals along the shear cycle show frequency dependence in the nonlinear regime but remains frequency independent in the linear regime. Remarkably, the monotonously increasing behavior of $E_{diss}$ with frequency is also observed in the non-linear regime when a more rigid cross-linker A-Actinin is used, suggesting the importance of flexibility of cross-linkers in controlling the non-linear mechanical response in this class of materials.   

Mechanics of composite actin networks: in vitro and cellular perspectives

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.   

Nonlinear Elasticity: From Single Chain to Networks and Gels

Biological and polymeric networks show highly nonlinear stress-strain behavior leading to material hardening with increasing deformation. Using a combination of theoretical analysis and molecular dynamics simulations we develop a model of network deformation that describes nonlinear mechanical properties of a broad variety of biological and polymeric networks and gels by relating their macroscopic strain-hardening behavior with molecular parameters of the network strands. The starting point of our approach is a nonlinear force/elongation relation for discrete chain model with varying bending rigidity. This theory provides a universal relationship between the strain-dependent network modulus and the network deformation as a function of the first invariant and chain elongation ratio that depends on a ratio of the unperturbed chain size to chain dimension in a fully extended conformation. The model predictions for the nonlinear shear modulus and differential shear modulus for uniaxial and shear deformations are in a very good agreement with both the results of molecular dynamics simulations of networks and with experimental data for biopolymer networks of actin, collagen, fibrin, vimentin, neurofilaments, and pectin.   

Composite fiber networks mechanics

Random fiber networks are present in many soft biological and engineering materials. In most cases, these networks are composite, in the sense that they are constructed from multiple fiber types. In this work we develop elements of a theoretical understanding of the elasticity of these structures. To this end, we consider systems made from a softer base and varying fractions of stiff fibers and investigate the effect of various system parameters on the overall behavior. The small strain elasticity depends strongly on the presence of a small concentration of stiff fibers for some types of base networks, but is essentially insensitive to these additions for other types. The way in which the stiff fibers are cross-linked to the soft fibers and to themselves is also important. These issues will be discussed within a framework general enough to make the conclusions relevant for diverse applications.   

Elasticity on the edge of stability: what Maxwell can teach us about biology

Life makes use of filamentous proteins for many structures, both in cells and tissues. In the cell, the cytoskeleton consists of networks of protein biopolymers for mechanical stability, organization and transport within the cell. Extracellular proteins such as collagen and fibrin form similar networks. One hundred and fifty years ago, Maxwell taught us about the minimal conditions for stability of simple spring-based networks [J. C. Maxwell, Philos. Mag. 27, 27 (1864).]. Interestingly, as a function of connectivity, such networks exhibit second-order rigidity transitions. We discuss recent theoretical and experimental progress in understanding the mechanics of such networks. We focus particularly on implications of the marginal state of networks near, and below Maxwell's isostatic connectivity. We show how fields such as stress, molecular motor activity and thermal fluctuations can stabilize networks. In the process, this can help us to understand long-standing problems in collagenous tissue mechanics.