The coordination of cell adhesion and cytoskeletal regulation is critical for tissue and organ assembly during embryogenesis, and also during wound repair and tissue homeostasis in adults. Disruption of cell adhesion is a critical step in tumor metastasis, and plays a role in skin and heart diseases. We have developed a model system to study coupling of cell adhesion and regulation of the actin cytoskeleton, using the fruit fly Drosophila. Tools available in this system allow us to combine very powerful genetic approaches with the ability to study cell biological events in the context of intact animals, often in real time. Work in many labs provided a static """"""""textbook"""""""" model for how the core cadherin:catenin complex at adherens junctions (AJs) mediates adhesion and links adhesive junctions to actin. However, cells in embryos and adults are far from static. Further, certain key features of the textbook model have been called into question. Our current challenge is to revise this model, revealing how adhesion and cytoskeletal regulation are coordinated and regulated to enable the remarkably diverse cell behaviors found in developing embryos. Here we address two broad unanswered questions in this area, each providing the basis for one of our Specific Aims.
Aim 1 Define mechanisms by which AJs are connected to the actin cytoskeleton and explore how these affect apical constriction, AJ remodeling and other processes. The canonical model suggests that there is a direct mechanical connection between AJs and actin, mediated by linked interactions between cadherins, ?-catenin, ??catenin, and actin. However, recent work called this into question. Defining whether and how AJs are linked to the actin cytoskeleton is a key question for our field. We hypothesize that a direct mechanical connection is critical in robust cell shape changes like those of apical constriction. Based on our preliminary data, we further hypothesize that Canoe and Rap1 help mediate this link. We will test this hypothesis, examining mechanisms by which Cno and Rap1 act, and exploring whether they play a more general role in apical constriction and AJ remodeling events.
Aim 2 Define mechanisms by which adhesion and actin dynamics are coordinately regulated. To accomplish the complex cell behaviors of morphogenesis, cells must closely coordinate adhesion and actin dynamics. Defining mechanisms by which this occurs is a key task for the field. We identified critical roles for the tyrosine kinase Abl and the actin regulator Ena in morphogenesis. We use them as a model for understanding mechanisms coordinating cell adhesion and cytoskeletal regulation. We hypothesize actin regulators like Ena are stored in an inactive state at AJs. We hypothesize that Abl regulates actin assembly at AJs and nearby by regulating Ena localization and/or function, acting as a scaffold, and influencing actin directly. We hypothesize that Ena helps generate distinct actin structures using different domains and partners, and that Ena is integrated with other actin regulators in filopodia. We will test these hypotheses.
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