Abstract
Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.
Highlights
In multi-cellular organisms, cell-to-cell junctions are force-bearing and highly dynamic, both critical functional requirements for embryogenesis and tissue homeostasis
Previous studies have shown that the absence of myosin IIA disorganizes cadherin mediated cell-cell adhesion [25], and cadherins are at least partially required for recruitment of myosin IIA to cellcell adhesion [26,27]
Substrate stretching induces actin bundles to orient perpendicular to the sites of cell-cell adhesion (Fig 3) and myosin IIA accumulates along cell-cell contacts where these actin bundles terminated (Fig 4C)
Summary
In multi-cellular organisms, cell-to-cell junctions are force-bearing and highly dynamic, both critical functional requirements for embryogenesis and tissue homeostasis. The actin-myosin contractile network exerts force on the sites of cell-cell adhesion, and is an integral component in strengthening adhesive structures. Force Dependent Biotinylation of Myosin IIA actin-myosin generated forces alter the protein organization at cell-cell contacts is an important detail in the regulation of cell-cell adhesion. The role of the actin cytoskeleton in cadherin-mediated cell-cell adhesion has been extensively studied. The cadherins, a family of calcium-dependent cell-cell adhesion proteins, play fundamental roles in cell organization during physiological and pathological processes in multi-cellular organisms. While β-catenin is a well-known component of Wnt pathway, α-catenin recently emerged as a critical player in regulating the actin network at the sites of cadherin mediated cell-cell adhesion
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