Session T47: Focus Session: Mechanical Structure-Function Relations in Biological Matter II

Heritable adhesion geometries and mechanosensing of surfaces by biofilm-forming bacteria

Biofilms are dense, interacting communities of single-celled organisms that are bound to each other with a self-produced polymer matrix.~ Biofilms have devastating clinical impact as they increase resistance to antibiotics and the immune system as well as the production of virulence factors that damage the host.~ Here we examine effects very early in biofilm development, when the infection is still in a stage of a few cells not yet characterized by high biofilm densities. ~\textit{Pseudomonas aeruginosa}, an opportunistic human pathogen, produces multiple extracellular polysaccharides that form the biofilm's structuring matrix.~ We have recently shown that the two primary polysaccharides, Pel and Psl, have distinct roles in controlling the mechanics of single-cell adhesion to a surface -- Psl dominates adhesion to the surface, and Pel makes the bacterium lie down flat (Cooley \textit{et al}., 2013 Soft Matter). Here, we show that expressing Pel alters the symmetry of Psl's distribution on the surface of rod-shaped \textit{Pseudomonas}. We also show that expressing Pel decreases the work of detachment from the surface. It seems paradoxical that a biofilm-forming organism should pay the cost of maintaining and making a gene product that reduces the energy input required to detach it from a surface. Therefore, we probe the possibility that a flat-lying bacteria may better sense a solid surface and change its signaling state accordingly.   

How the velvet worm squirts slime

The rapid squirt of a proteinaceous slime jet endows the ancient velvet worms (Onychophora) with a unique mechanism for defense from predators and for capturing prey by entangling them in a disordered web that immobilizes their target. However, to date neither qualitative nor quantitative descriptions have been provided for this unique adaptation. Here we investigate the fast oscillatory motion of the oral papillae and the exiting liquid jet that oscillates with frequencies $f\sim 30-60$ Hz. Using anatomical images, high speed videography, theoretical analysis and a physical simulacrum we show that this fast oscillatory motion is the result of an elastohydrodynamic instability driven by the interplay between the elasticity of oral papillae and the fast unsteady flow during squirting. Our results demonstrate how passive strategies can be cleverly harnessed by organisms, while suggesting future oscillating micro-fluidic devices as well as novel ways for micro and nano fiber production using bioinspired strategies.   

Micromechanics of cellularized collagen I networks

Collagen gels are commonly used in experiments on cell mechanics because collagen is the most abundant protein in the mammalian extracellular matrix. Collagen gels are often approximated as homogeneous elastic materials; however, variations in the collagen fiber microstructure and cell adhesion forces cause the mechanical properties to be inhomogeneous at the cellular scale. We study the mechanics of type I collagen on the scale of tens to hundreds of microns by using holographic optical tweezers (HOT) to apply pN forces to micron-sized particles embedded in the collagen fiber network. We calculate the local compliance and elastic modulus of the collagen network and find that particle displacements are inhomogeneous, anisotropic, and often have components perpendicular to the direction of the applied force. Confocal reflection microscopy (CRM) is used to reveal the local fiber structure and a simulation treating fibers as rigid rods is used for comparison to the HOT measurements. Collagen samples prepared at 21$^\circ$C and 37$^\circ$C show that gels formed at lower temperature are more inhomogeneous, anisotropic, and compliant than those formed at high temperature, and cellularized samples allow us to characterize the effects of cell adhesion forces on the network mechanics.   

Cellular Contact Guidance Through Dynamic Sensing of Nanotopography

We evaluated the contact guidance of nanoscale ridges on the cellular motion and actin waves in HL60 neutrophil- like cells, a model system for studying cell migration. By analyzing the velocity of cell motion and actin waves, we found that the nanoridges exert bidirectional guidance on migrating cells and actin wave propagation. More cells migrate parallel to the nanoridges than any other direction. Nanoridges nucleate actin polymerization waves which then proceed preferentially along the ridges. Contact guidance efficiency depends on the spacing between adjacent ridges. The greatest guidance efficiency occurs on 1.5-$\mu $m-spaced ridges. A larger average actin wave speed is observed on 5-$\mu $m-spaced than 1.5-$\mu $m-spaced ridges, which may arise from a larger portion of random propagation on 5-$\mu $m-spaced ridges rather than confined directional propagation along the 1.5-$\mu $m-spaced ridges.   

Cardiac tissue as a mechanically and electrically active medium

The heart is an active solid in which energy is injected at the cell scale when cardiomyocytes contract. This energy is tranduced up to macroscopic scales, leading to a collective function--the pumping of the heart--in which a wavefront of contraction propagates across the heart from one end to the other. We will present results for a model that couples a traditional model for electrical signaling to an overdamped biphasic model for tissue mechanics to look at the competition between mechanical and electrical signaling in the contractile wavefront in the embryonic heart. We speculate on the ramifications of our results for the adult heart, which is conventionally described exclusively in terms of electrical signaling.   

The dynamics of body elongation in vertebrate morphogenesis

Vertebrate embryos have a body axis that grows by the addition of cells in a posterior growth zone in the embryo. Experiments show that these cells show a gradient in motility that decays towards the anterior of the embryo, consistent with a degradation of specific cellular signals (FgF) that control cellular motility. However, this motility is primarily diffusive in nature, and converted into an advective gradient by virtue of inhomogeneous confinement. We use these observations to build a minimal mechanochemical model for tissue extension as a function of FgF activity, cell motility and tissue rheology with results that allow us to explicitly test the model in a variety of in-vivo and ex-vivo situations, with implications for normal and pathological axis elongation.   

Quantitative analysis of actin monomer funneling: how capping protein enhances actin filament growth and nucleation on biomimetic beads

Capping protein (CP) caps the growing ends of actin filaments and thereby halts their polymerization. However, CP is required for actin-based motility, and experiments by Akin and Mullins [1] have shown that CP also enhances the rate of filament nucleation. Proposed explanations for these phenomena include the Actin Funneling Hypothesis (AFH) [2], in which the presence of CP increases the free-actin concentration, and structural changes of the actin networks induced by increasing CP [1]. In this article, we provide a quantitative analysis of the AFH based on rate equations including actin nucleation and branching, polymerization and capping, plus monomer depletion near the surface of the bead. With two adjustable parameters, our simulation results accurately match several aspects of the results of Akin and Mullins [1]. We find that CP increases the local monomer concentration at the bead surface, but has a much smaller effect on the global free-actin concentration. The increased local monomer concentration gives rise to an enhanced rate of branching events and thus a larger number of actin filaments. [1] O Akin and R. D. Mullins. Cell 133.5 (2008): 841-851. [2] M-F Carlier, and D. Pantaloni. Journal of molecular biology 269.4 (1997): 459-467.   

Measurement of DDR-Collagen interaction Forces with Atomic force Microscopy

Discoidin Domain Receptors (DDR) are membrane proteins of the tyrosine kinase receptor family. The binding of collagen to the extracellular domain of DDR stimulates activation of the tyrosine kinase inside the cell. Two types of DDR, DDR1 and DDR2, have been related to human cancers because of the discovery of alterations of DDR genes in several human cancers. However, not much is known about DDR behavior at the cell-collagen interface. We are combining biological information and force based microscopy to shed light on how DDRs function in physiological and pathological conditions. We have measured the kinetics, bond lengths and activation energy of DDR-collagen interactions at the single molecular level on live cells, including cells that are deficient in DDR and cells that overexpress DDR, as well as cancer cells. We have developed methods to take multiple attachments into account and obtain clean data. Interactions measured on live cells were compared to measurements between extracted extracellular domains of DDR and collagen plated on a substrate to determine how these interactions are altered by the microenvironment of the cell. The distribution of DDR receptors on live cells was determined by using a combination of fluorescence imaging and AFM-based adhesion mapping.   

Quantitative analysis of mass density fluctuation inside biological cells under the effect of alcohol using light localization properties

Light localization properties can be used to analyze the nanoscale level alterations inside in the biological cells. We present study of mass density fluctuation in the nuclei of colon cells, under the effect of alcohol, by quantifying the degree of structural disorder, of nanoscale, from their transmission electron microscopy (TEM) images. The light localization properties of the disordered optical lattice system, created using the TEM image data, were studied by statistically analyzing the inverse participation ratio (IPR) of the localized eigenfunctions of the optical lattices. The study, conducted on rat model, shows that nanoscale morphology of the colon cells with symptoms of carcinogenesis increases further under the effect of alcohol (ethanol). The quantified structural disorder strength, measured in the length scale 12.5 -- 75 nm, for the cells under the effect of ethanol was noted to be significantly higher in comparison to the cells not under the influence of ethanol. This study is first of its kind where the effect of alcohol on the biological cells has been studied by quantifying the nanoscale level of mass density fluctuation inside the cells, using the mesoscopic physics approach.   

Arterial mechanobiology: The interrelation of elastin, collagen, and GAGs

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load-bearing component in the arterial wall. Pathogenesis of many cardiovascular diseases is associated with loss of organization and function of the ECM. However the interrelation of the function of collagen and elastin and the effect of ECM structural changes on vascular mechanics are not well understood. This talk will focus on our recent study on the interrelations of ECM constituents and how they contribute to the mechanical function of the arterial wall. Our recent study coupling mechanical loading and multi-photon imaging demonstrates an interesting sequential engagement of elastin and collagen fibers in response to mechanical loading. Our study also suggests that the elastin fibers are under tension and impart an intrinsic compressive stress on collagen. Such delicate interrelation between elastin and collagen is essential for an artery to function normally. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen while glycosaminoglycans (GAGs) are often neglected, most likely because of the relatively low content in arterial tissue. Our study shows that GAGs play a role in engaging the elastin and collagen fibers in the arterial wall and thus indirectly affect the biomechanical function of arteries. Together these results provide a more comprehensive understanding of the mechanobiology of arteries with the goal of incorporating such information in understanding disease progressions and structurally based constitutive models.   

Density fluctuations and intercellular fluid flow in epithelial monolayers

Number density plays a key role in influencing collective migration in cell layers, which exhibit a transition from fluid-like to solid-like motion as cells become increasingly packed. In confluent monolayers of MDCK cells, we observe multi-cellular patterns of alternating high-density and low-density regions. Since there is no free space in these layers, changes in cell density correspond to changes in projected cell area. With confocal microscopy we find that cell area fluctuations are approximately the same as cell volume fluctuations. Thus, multi-cellular density fluctuations in monolayers involve significant levels of fluid transport in and out of cells to accommodate their volume changes. To elucidate the relationship between density fluctuations and fluid transport we monitor cell layers in time-lapse, performing multiple simultaneous measurements. We study cell density dynamics by analyzing divergence in the migration velocity field. We also dye the cells with a cytosol dye incapable of traveling between cells, using dye dilution as a marker for fluid flow. We find that diverging/converging regions of cells contain about 15 cells and oscillate like 2D standing waves with a period of a few hours. We find that fluid waves propagate through the cell layer over multicellular length scales, accommodating these collective cell volume fluctuations.   

The effect of antiarrhythmic drugs on rate-dependent cardiac behavior

While there are many studies examining the biomolecular effects of antiarrhythmic drugs and many studies examining their clinical effect, their effect on cardiac dynamics at the cellular and tissue levels is not well understood. We use a mathematical model of a human ventricular cell to study the effect of antiarrhythmic drugs on the appearance of alternans and 2:1 behavior over a range of doses. We study three different classes of drugs, calcium channel blockers, potassium channel blockers and sodium channel blockers, and compare their effects on cellular dynamics.