Nikola Kojic

Ex vivo Rheology of Spider Silk

SUMMARY We investigate the rheological properties of microliter quantities of the spinning material extracted ex vivo from the major ampullate gland of a Nephila clavipes spider using two new micro-rheometric devices. A sliding plate micro-rheometer is employed to measure the steady-state shear viscosity of ∼1 μl samples of silk dope from individual biological specimens. The steady shear viscosity of the spinning solution is found to be highly shear-thinning, with a power-law index consistent with values expected for liquid crystalline solutions. Calculations show that the viscosity of the fluid decreases 10-fold as it flows through the narrow spinning canals of the spider. By contrast, measurements in a microcapillary extensional rheometer show that the transient extensional viscosity (i.e. the viscoelastic resistance to stretching) of the spinning fluid increases more than 100-fold during the spinning process. Quantifying the properties of native spinning solutions provides new guidance for adjusting the spinning processes of synthetic or genetically engineered silks to match those of the spider.

An EGFR autocrine loop encodes a slow-reacting but dominant mode of mechanotransduction in a polarized epithelium

The mechanical landscape in biological systems can be complex and dynamic, with contrasting sustained and fluctuating loads regularly superposed within the same tissue. How resident cells discriminate between these scenarios to respond accordingly remains largely unknown. Here, we show that a step increase in compressive stress of physiological magnitude shrinks the lateral intercellular space between bronchial epithelial cells, but does so with strikingly slow exponential kinetics (time constant ~110 s). We confirm that epidermal growth factor (EGF)‐family ligands are constitutively shed into the intercellular space and demonstrate that a step increase in compressive stress enhances EGF receptor (EGFR) phosphorylation with magnitude and onset kinetics closely matching those predicted by constant‐rate ligand shedding in a slowly shrinking intercellular geometry. Despite the modest degree and slow nature of EGFR activation evoked by compressive stress, we find that the majority of transcriptomic responses to sustained mechanical loading require ongoing activity of this autocrine loop, indicating a dominant role for mechanotransduction through autocrine EGFR signaling in this context. A slow deformation response to a step increase in loading, accompanied by synchronous increases in ligand concentration and EGFR activation, provides one means for cells to mount a selective and context‐appropriate response to a sustained change in mechanical environment.—Kojic, N., Chung, E., Kho, A. T., Park, J.‐A., Huang, A., So, P. T. C., Tschumperlin, D. J. An EGFR autocrine loop encodes a slow‐reacting but dominant mode of mechano‐transduction in a polarized epithelium. FASEB J. 24, 1604‐1615 (2010). www.fasebj.org

A 3-D model of ligand transport in a deforming extracellular space

Cells communicate through shed or secreted ligands that traffic through the interstitium. Force-induced changes in interstitial geometry can initiate mechanotransduction responses through changes in local ligand concentrations. To gain insight into the temporal and spatial evolution of such mechanotransduction responses, we developed a 3-D computational model that couples geometric changes observed in the lateral intercellular space (LIS) of mechanically loaded airway epithelial cells to the diffusion-convection equations that govern ligand transport. By solving the 3-D fluid field under changing boundary geometries, and then coupling the fluid velocities to the ligand transport equations, we calculated the temporal changes in the 3-D ligand concentration field. Our results illustrate the steady-state heterogeneities in ligand distribution that arise from local variations in interstitial geometry, and demonstrate that highly localized changes in ligand concentration can be induced by mechanical loading, depending on both local deformations and ligand convection effects. The occurrence of inhomogeneities at steady state and in response to mechanical loading suggest that local variations in ligand concentration may have important effects on cell-to-cell variations in basal signaling state and localized mechanotransduction responses.

Quantification of three-dimensional dynamics of intercellular geometry under mechanical loading using a weighted directional adaptive-threshold method

Capturing and quantifying dynamic changes in three-dimensional cellular geometries on fast time scales is a challenge because of mechanical limitations of imaging systems as well as of the inherent tradeoffs between temporal resolution and image quality. We have combined a custom high-speed two-photon microscopy approach with a novel image segmentation method, the weighted directional adaptive-threshold (WDAT), to quantify the dimensions of intercellular spaces of cells under compressive stress on timescales previously inaccessible. The adaptation of a high-speed two-photon microscope addressed the need to capture events occurring on short timescales, while the WDAT method was developed to address artifacts of standard intensity-based analysis methods when applied to this system. Our novel approach is demonstrated by the enhanced temporal analysis of the three-dimensional cellular and extracellular deformations that accompany compressive loading of airway epithelial cells.

MULTISCALE MODELING OF MOLECULAR DIFFUSION IN TISSUE

Tissue can be considered as a composite medium through which occurs transport of molecules. Transport of matter by diffusion within this medium is affected not only by internal microstructural geometry, but also by physico-chemical interactions between solid phase (proteins, fibers) and transported molecules or particles. We implement a new hierarchical multiscale microstructural model [1], [2] for simulation of transport of molecules through tissue. Our model is based on a novel numerical homogenization procedure. The equivalent diffusion parameters of the continuum model consist of equivalent bulk commonly used diffusion coefficients and new equivalent distances from the solid surface. Numerical examples include, among others, a model of diffusion within tissue in the vicinity of a capillary through which molecules are transported by convection. A study of the effects of collagen mesh density within tissue on transported molecule concentration profiles is presented.