Adherens junctions are specialized sites of strong cell-cell adhesion built around cadherin adhesion molecules that organize an actin cytoskeleton to mechanically couple adjacent cells. Adherens junctions can therefore harness actomyosin generated forces and integrate them across entire sheets of interconnected cells to drive the morphogenetic movements that fashion tissues, organs, and entire early embryos. Despite its importance, little is known as to how actin is assembled at adherens junctions. A previous model proposed a linkage between cadherins and actin via the catenins, which would have provided a simple mechanism for recruiting actin to adherens junctions. This model was seriously challenged however when it was demonstrated that ?-catenin couldn't bind simultaneously to both ?-catenin and F-actin. Therefore, additional factors peripheral to the core cadherin-catenin complex are necessary to organize the actin cytoskeleton at adherens junctions. We developed an in vitro system to reconstitute actin assembly at adherens junctions to identify such factors and have already used this system to identify ?-actinin as an essential factor necessary for arp2/3 mediated junctional actin assembly. ?-Actinin and arp2/3 alone however cannot account for actin organization at adherens junctions. We reasoned that junctional proteins in close proximity to ?-actinin would also be involved in junctional actin assembly and used photoactivatable crosslinking to identify six factors in close proximity to ?-actinin in cadherin enriched plasma membrane sheets. We have confirmed that all six of these proteins localize to adherens junctions, all of them are thought to regulate the actin cytoskeleton, and mutations in two of them result in the same inherited kidney disease as mutant forms of ?-actinin known as FSGS suggesting a common cell biological function. We therefore hypothesize that these new factors are important for organizing actin at adherens junctions. In the first aim we determine which of ?-actinin's neighbors at the junction are necessary for recruiting it to adherens junctions and use biochemical methods to identify direct binding interactions between these factors and ?-actinin. In the second aim, we determine the roles of these newly identified factors in organizing actin at adherens junctions in cells and use biochemical methods to determine the effects of these factors on actin assembly in vitro. In the third aim we determine the role of ?-actinin and its junctional neighbors in cadherin mediated cell adhesion and adherens junction assembly and maturation.
TO HUMAN HEALTH: Cadherins and adherens junctions are implicated in numerous developmental defects and diseases, and a number of the factors that we have identified in our biochemical reconstitution are implicated in the inherited kidney disease, Focal Segmental Glomerulosclereosis. Defining the molecular mechanisms underlying actin assembly at adherens junctions will not only reveal core mechanisms necessary for human development but might also shed light on the pathology of certain diseases in adults.
|Yu-Kemp, Hui-Chia; Brieher, William M (2016) Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly. J Biol Chem 291:658-64|
|Nadkarni, Ambika V; Brieher, William M (2014) Aip1 destabilizes cofilin-saturated actin filaments by severing and accelerating monomer dissociation from ends. Curr Biol 24:2749-57|
|Brieher, William (2013) Mechanisms of actin disassembly. Mol Biol Cell 24:2299-302|
|Tang, Vivian W; Brieher, William M (2013) FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions. J Cell Biol 203:815-33|