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We report the engineering of a protein-based dynamic hydrogel that can be reversibly tuned between stiff and soft states via a redox reaction. When cultured on this hydrogel surface, human lung fibroblasts can dynamically and reversibly change their morphology in response to changes in hydrogel stiffness.

Melanoblasts disperse throughout the skin and populate hair follicles through long-range cell migration. During migration, cells undergo cycles of coordinated attachment and detachment from the Extra-Cellular Matrix (ECM). Embryonic migration processes that require cell-ECM attachment are dependent on the integrin family of adhesion receptors. Precise regulation of integrin-mediated adhesion is important for many developmental migration events. However, the mechanisms that regulate integrin-mediated adhesion in vivo in melanoblasts are not well understood. Here, we show that autoinhibitory regulation of the integrin-associated adapter protein talin coordinates cell-ECM adhesion during melanoblast migration in vivo. Specifically, an autoinhibition-defective talin mutant strengthens and stabilizes integrin-based adhesions in melanocytes, which impinges on their ability to migrate. Mice with defective talin autoinhibition exhibit delays in melanoblast migration and pigmentation defects. Our results show that coordinated integrin-mediated cell-ECM attachment is essential for melanoblast migration and that talin autoinhibition is an important mechanism for fine-tuning cell-ECM adhesion during cell migration in development.

usc Cells in multicellular organisms are arranged in complex three-dimensional patterns. This requires both transient and stable adhesions with the extracellular matrix (ECM). Integrin adhesion receptors bind ECM ligands outside the cell and then, by binding the protein talin inside the cell, assemble an adhesion complex connecting to the cytoskeleton. The activity of talin is controlled by several mechanisms, but these have not been well studied in vivo. By generating mice containing the activating point mutation E1770A in talin (Tln1), which disrupts autoinhibition, we show that talin autoinhibition controls cell-ECM adhesion, cell migration, and wound healing in vivo. In particular, blocking autoinhibition gives rise to more mature, stable focal adhesions that exhibit increased integrin activation. Mutant cells also show stronger attachment to ECM and decreased traction force. Overall, these results demonstrate that modulating talin function via autoinhibition is an important mechanism for regulating multiple aspects of integrin-mediated cell-ECM adhesion in vivo. Refereed/Peer-reviewed

Attachment of cells to the extracellular matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin (encoded by

Attachment of cells to the extracellular matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin (encoded by rhea in flies) is necessary for linking integrins to the cytoskeleton, and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. Here, we show that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies that seen with null alleles of Talin. Moreover, we show that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.

The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.