Richard Arevalo

Cyclic hardening in bundled actin networks

Nonlinear deformations can irreversibly alter the mechanical properties of materials. Most soft materials, such as rubber and living tissues, display pronounced softening when cyclically deformed. Here we show that, in contrast, reconstituted networks of crosslinked, bundled actin filaments harden when subject to cyclical shear. As a consequence, they exhibit a mechano-memory where a significant stress barrier is generated at the maximum of the cyclic shear strain. This unique response is crucially determined by the network architecture: at lower crosslinker concentrations networks do not harden, but soften showing the classic Mullins effect known from rubber-like materials. By simultaneously performing macrorheology and confocal microscopy, we show that cyclic shearing results in structural reorganization of the network constituents such that the maximum applied strain is encoded into the network architecture.

Size-Dependent Rheology of Type-I Collagen Networks

We investigate the system size-dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by the ratio of the gel thickness to the mesh size. These findings have broad implications for cell-matrix interactions, the interpretation of rheological tissue data, and the engineering of biomimetic scaffolds.

Development of a confocal rheometer for soft and biological materials

We discuss the design and operation of a confocal rheometer, formed by integrating an Anton Paar MCR301 stress-controlled rheometer with a Leica SP5 laser scanning confocal microscope. Combining two commercial instruments results in a system which is straightforward to assemble that preserves the performance of each component with virtually no impact on the precision of either device. The instruments are configured so that the microscope can acquire time-resolved, three-dimensional volumes of a sample whose bulk viscoelastic properties are being measured simultaneously. We describe several aspects of the design and, to demonstrate the systems capabilities, present the results of a few common measurements in the study of soft materials.

Stress Heterogeneities in Sheared Type-I Collagen Networks Revealed by Boundary Stress Microscopy

Disordered fiber networks provide structural support to a wide range of important materials, and the combination of spatial and dynamic complexity may produce large inhomogeneities in mechanical properties, an effect that is largely unexplored experimentally. In this work, we introduce Boundary Stress Microscopy to quantify the non-uniform surface stresses in sheared collagen gels. We find local stresses exceeding average stresses by an order of magnitude, with variations over length scales much larger than the network mesh size. The strain stiffening behavior observed over a wide range of network mesh sizes can be parameterized by a single characteristic strain and associated stress, which describes both the strain stiffening regime and network yielding. The characteristic stress is approximately proportional to network density, but the peak boundary stress at both the characteristic strain and at yielding are remarkably insensitive to concentration.

A Novel 3D Fibril Force Assay Implicates Src in Tumor Cell Force Generation in Collagen Networks

New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

Four-dimensional structural dynamics of sheared collagen networks

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