Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin.

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ∼4.2 µm) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

A guide to mechanobiology: Where biology and physics meet.

Cells actively sense and process mechanical information that is provided by the extracellular environment to make decisions about growth, motility and differentiation. It is important to understand the underlying mechanisms given that deregulation of the mechanical properties of the extracellular matrix (ECM) is implicated in various diseases, such as cancer and fibrosis. Moreover, matrix mechanics can be exploited to program stem cell differentiation for organ-on-chip and regenerative medicine applications. Mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology, bioengineering and biophysics. Here we provide an introductory overview of the key players important to cellular mechanobiology, taking a biophysical perspective and focusing on a comparison between flat versus three dimensional substrates. This article is part of a Special Issue entitled: Mechanobiology.

The integrin expression profile modulates orientation and dynamics of force transmission at cell-matrix adhesions.

Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins α5ß1 and αvß3, changes, thus altering the integrin composition of cell-matrix adhesions. Here, we show that enhanced αvß3 expression can fully compensate for loss of α5ß1 and other ß1 integrins to support outside-in and inside-out force transmission. α5ß1 and αvß3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, α5ß1 and αvß3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using αvß3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by α5ß1. Thus, altering the relative abundance of fibronectin-binding integrins in cell-matrix adhesions affects the spatiotemporal organization of force transmission.

P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration.

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas -/- mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.

Dynamics and mechanism of p130Cas localization to focal adhesions.

The docking protein p130Cas is a major Src substrate involved in integrin signaling and mechanotransduction. Tyrosine phosphorylation of p130Cas in focal adhesions (FAs) has been linked to enhanced cell migration, invasion, proliferation, and survival. However, the mechanism of p130Cas targeting to FAs is uncertain, and dynamic aspects of its localization have not been explored. Using live cell microscopy, we show that fluorophore-tagged p130Cas is a component of FAs throughout the FA assembly and disassembly stages, although it resides transiently in FAs with a high mobile fraction. Deletion of either the N-terminal Src homology 3 (SH3) domain or the Cas-family C-terminal homology (CCH) domain significantly impaired p130Cas FA localization, and deletion of both domains resulted in full exclusion. Focal adhesion kinase was implicated in the FA targeting function of the p130Cas SH3 domain. Consistent with their roles in FA targeting, both the SH3 and CCH domains were found necessary for p130Cas to fully undergo tyrosine phosphorylation and promote cell migration. By revealing the capacity of p130Cas to function in FAs throughout their lifetime, clarifying FA targeting mechanism, and demonstrating the functional importance of the highly conserved CCH domain, our results advance the understanding of an important aspect of integrin signaling.